Proefschrift Borgdorff

T H E E L U S I V E H E A R T the right ventricle in chronic abnormal loading conditions

M.A.J. Borgdorff

THE EL

USIVE H

EART

M.A.J. BorgdorFF

‘ t h e r i g h t v e n t r i c l e i s a n e l u s i v e h e a r t ; i t s f u n c t i o n

a n d f o r m a r e o n l y r e c e n t l y b e g i n n i n g t o b e u n c o v e r e d .

Y e t i t s i m p o r t a n c e i n t h e p h y s i o l o g y o f c o n g e n i t a l

h e a r t d i s e a s e s a n d p u l m o n a r y a r t e r i a l h y p e r t e n s i o n c a n

h a r d l y b e u n d e r e s t i m a t e d . ’

adjective \ē-ˈlü-siv, -ˈlü-ziv\

: hard to find or capture, understand, define, isolate or identify

: tending to evade grasp or pursuit

Example: the blue whale (Balaenoptera musculus) is one of the ocean's most elusive inhabitants

elu·sive

ISBN 978-90-367-7172-6 Also available

as an ebook www.theelusiveheart.wordpress.com

T H E E L U S I V E H E A R T

the right ventricle in chronic abnormal loading conditions

door Marinus A.J. Borgdorff, op maandag 27 oktober 2014 om 12.45 uur precies in het Academiegebouw van de Rijksuniversiteit Groningen, Broerstraat 5, Groningen

Direct aansluitend aan de promotie in het Academiegebouw

Vanaf 21.00 uur bent van harte welkom in Land van Kokanje, Oude Boteringestraat 9, Groningen

Uitnodiging voor het bijwonen van de openbare verdediging van het proefschrift

RECEPTIE

FEEST

PARANimfen

Reinout Borgdorff Leisteenstraat 13 9743 VA Groningen [email protected]

Michael G. Dickinson Joel M. Harding [email protected]

T H E E L U S I V E T H E E L U S I V E T H E E L U S I V E T H E E L U S I V E

H E A R TH E A R TH E A R TH E A R T

the right ventricle in chronic abnormal loading

conditions

door Marinus A.J. Borgdorff, op maandag 27oktober 2014 om 12.45 uur precies in hetAcademiegebouw van de RijksuniversiteitGroningen, Broerstraat 5, Groningen

Direct aansluitend aan de promotie in hetAcademiegebouw

Vanaf 21.00 uur bent u van harte welkom inLand van Kokanje, Oude Boteringestraat 9,Groningen

Uitnodiging voor het bijwonen van de openbareverdediging van het proefschrift

RECEPTIE

FEEST

PARANimfen

Reinout Borgdorff Leisteenstraat 139743 VA [email protected]

Michael G. DickinsonJoel M. Harding

[email protected]

THE EL

USIVE H

EART

M.A.J. BorgdorFF

THE ELUSIVE HEART

The right ventricle in chronic abnormal loading conditions

Marinus A.J. Borgdorff

Borgdorff, Marinus A.J.

The elusive heart. The right ventricle in chronic abnormal loading conditions

Proefschrift Groningen

ISBN: 978-90-367-7172-6 (book) 978-90-367-7171-9 (E-book)

© Copyright 2014 M.A.J. Borgdorff

All rights reserved. No part of this publication may be reproduced, stored in

a retrieval system, or transmitted in any form or by any meands, without

permission of the author, and, when appropriate, the publisher holding the

copyrights of the published articles.

Coverphoto: Regent st/Vigo st. London, England.

Back cover definition adapted from: elusive. 2014. In Merriam-Webster.com.

Retrieved July 6, 2014, from http://www.merriam-webster.com/dictionary/

elusive

Layout and printing by Gildeprint - www.gildeprint.nl

The elusive heart


Proefschrift

Ter verkrijging van de graad van doctor aan de

Rijksuniversiteit Groningen

op gezag van de

rector magnificus, prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

maandag 27 oktober 2014 om 12:45 uur.

door

Marinus Alexander Jacobus Borgdorff

geboren op 15 februari 1982

te Zeist

Promotor prof. dr. R.M.F. Berger

Copromotor dr. B. Bartelds

Beoordelingscommissie prof. dr. W.H. van Gilst

prof. dr. T. Ebels

prof. dr. R. Naeije

Financial support by the Dutch Heart Foundation for publication of this thesis

is gratefully acknowledged. Part of the research described in this thesis was

supported by grants of the Dutch Heart Foundation (DHF-2006T038 and DHF-

2007T068).

Paranimfen Michael G. Dickinson

Joel M. Harding

My heart may fail,

but You are the strength of my heart

and my portion forever.

Psalm 73:26

1 The elusive heart: an introduction 11

PART I

2 Distinct loading conditions reveal various patterns of right ventricular 27

adaptation

Am J Physiol Heart Circ Physiol. 2013 Aug 1;305(3):H354-64

3 Differential responses of the right ventricle to abnormal loading 55

conditions in mice: pressure vs. volume load

Eur J Heart Fail. 2011 Dec;13(12):1275-82

4 Characterization of right ventricular failure in chronic experimental 77

pressure load

Under review

PART II

5 Sildenafil enhances systolic adaptation, but does not prevent diastolic 111

dysfunction in the pressure loaded right ventricle

Eur J Heart Fail. 2012 Sep;14(9):1067-74

6 Sildenafil treatment in established right ventricular dysfunction 135

improves diastolic function and attenuates interstitial fibrosis independent

from afterload

Am J Physiol Heart Circ Physiol. 2014 May 30. pii: ajpheart.00843.2013.

[Epub ahead of print]

7 A cornerstone of heart failure treatment is not effective in experimental 155

right ventricular failure

Int J Cardiol 2013 Nov 5;169(3):183-9

PART III

8 Right ventricular failure due to chronic abnormal loading conditions; 179

What have we learned of preclinical research?

Under review

CONTENTS

9 General discussion and future prospects 219

Appendices

Summary in Dutch 243

Acknowledgements 249

Bibliography 255

About the author 261

THEELUSIVEHEAR T:ANINTRODUCTI ON

MAJ Borgdorff

1

Chapter 112

‘The heart is an amazing organ. From a distance, its appearance and function

may seem simple. This muscular organ just pumps blood. It receives blood from

the body on one side and pumps it back to the body on the other. Yet beyond

this splendid simplicity lies a complexity that is hardly matched by any other

organ in the body. The heart is composed of muscle-layers, blood vessels and

valves, which in itself are composed of networks of specialized cells, fibers and

compartments. Then there are sub cellular structures measuring only a few

micrometers, nanometers across and then there are billions of tiny molecules,

which cannot be seen by even the most powerful microscopes. They are roaming

solitary or swarming like shoals of herring or tightly organized like the ranks of a

Roman legion. They zip across membranes in milliseconds, they tirelessly latch on

and let go of each other, and they withstand enormous pressures sustaining the

heart’s architecture. All these countless, widely variant components have their

own unique and wonderful three-dimensional shape and rhythm, meticulously

fashioned to their function, and yet they act all in harmonious choreography to

produce the simple and singular pulse of life. Oh yes, the heart is an amazing

organ.’

The elusive heart: an introduction 13

Embryology

The heart is the first organ to be formed during human development. Fifteen days

into gestation, two horseshoe-shaped clusters of cells lie on the ventral surface of

the embryo in an anterior-posterior relationship; the heart fields. Cells from the

first heart field form an arched tube that is subsequently invaded by cells from

the second heart field(1). These second heart field derived cells predominately

home at the superior terminus of the tube and develop into the conal trunk and

right ventricle. As the tube loops and twists, the cells from the first heart field

form both atria and the left ventricle(2). A third group of cells from the neural

crest joins with second heart field-derived cells to form the multiple aortic

arches that predominate at early gestational stages. Intermediate structures

that delineate these compartments such as the interventricular septum and the

atrioventricular valves seem to consist of cells from both first-, and second heart

field. At day 55, hardly 8 weeks into gestation, the heart has started beating and

the layout of the mature heart (two atria receiving the venous return, functional

atrioventricular valves, two separate ventricles and semilunar valves connecting

the heart to the systemic and pulmonary circulations) is formed.

Congenital heart defects

This basic building plan adequately describes the normal heart, but about

1 out of 100 children is born with an abnormal heart: they have a congenital

heart defect (CHD)(3). The group of congenital heart defects comprises a wide

spectrum of abnormalities ranging from lesions with hardly any consequences

for normal physiology, which do not require any treatment and may go unnoticed

throughout life to lesions with major consequences for physiology that, left

untreated, are not compatible with life. Examples include mild narrowing

of valves and small defects in septa for the former category and complete

abnormal architecture with abnormal or absent connections and basically one

functional ventricle for the latter. Congenital heart defects have long been a

leading cause death in children. However, in recent decades enormous progress

has been made in the management of congenital heart defects(4). Whereas

only a few decades ago a high percentage of children with a CHD would die

at a very young age, currently most of the patients survive through childhood,

into adolescence and adulthood(5). They form a new and quickly expanding

group of patients with unique characteristics and challenges(6): their heart

Chapter 114

and vessels are repaired, but rarely completely normal. Consequently, there

are oftentimes chronic changes in the physiology of their heart and circulation.

The cardiac ventricles may face longstanding abnormal loading conditions, such

as an increased volume load requiring the ventricle to pump more blood per

minute that normal, or increased pressure load, in which a normal volume of

blood must be pumped against an abnormally high resistance. In other patients

the ventricles are challenged by a combination of volume- and pressure load.


Follow-up studies have shown that whether the left or right ventricle is

abnormally loaded plays a crucial role in determining the outcome of these

patients. Patients with a chronically loaded right ventricle are much more likely

to develop early heart failure(7). That means that already at young age, they are

more likely to experience symptoms because of insufficient pump function of

the right heart (RV failure). The right ventricle thus, appears to be particularly

vulnerable to chronic abnormal loading. More than in the left ventricle,

abnormal loading conditions (especially pressure load) on the RV are thought to

impede function, which causes symptoms like fatigue, exercise intolerance and

edema and may ultimately threaten survival(8).

An archetype-disease of the overloaded RV is pulmonary hypertension (PH). PH

is characterized by excessive remodeling of the pulmonary vasculature, resulting

in increased pulmonary vascular resistance and RV pressure load, which leads

to RV failure and premature death(9). Even in the setting of maximal treatment,

the prognosis of PH-patients is very poor, with a 5-year survival of about 50%(10-

13). Interestingly, it is the severity of RV dysfunction rather than the severity of

the pulmonary vascular disease that appears to determine prognosis in these

patients(14,15).

However, the importance of the RV is not limited to the fields of congenital heart

defects and pulmonary hypertension: in recent years RV function has emerged

as a pivotal determinant of outcome, also in different forms of left sided heart

disease(16-18).

Collectively, these progressive insights have sparked increasing interest in the

right ventricle over the last few decades. In 1982, my birth year, 1,471 scientific

papers on right ventricular failure were published; in 1992, 2,765; in 2002, 4,994;


and in 2012 nearly 10,000! (trumping for instance ‘myocardial infarction’, which

was at 7,933 papers in 2012) (source: US National Library of Medicine, National

Institutes of Health at www.pubmed.gov, using queries ‘right ventricular failure’

and ‘myocardial infarction’). The shift of attention towards the right ventricle

is truly remarkable: currently regarded as a central factor in a broad range of

cardiovascular diseases, 50 years ago it was the subject of debates whether or

not the RV had any hemodynamic significance at all(19-21).

The elusive heart

However, despite the expansion of scientific literature on right ventricular

failure, it remains largely unknown how chronic abnormal loading conditions

physiologically lead to RV failure, and which biological mechanisms govern the

adaptive and maladaptive processes associated with the changed physiology

(9,22). To further complicate this field of research, the more became known

about the RV, the more it became apparent that the RV differed markedly from

the LV and thus LV derived knowledge could not necessarily be applied in RV

diseases. Indeed, major anatomical, functional and embryological differences

exist between the RV and LV. Most importantly, the RV is morphologically

and functionally adapted for the generation of low pressure perfusion of

the pulmonary vascular bed(23). It is thin-walled, compliant, has a complex

triangular-crescent shape and a peristaltic contraction pattern. The coronary

perfusion is different than for the LV, there is a different oxygen/energy demand

and the coupling between the ventricle and the vascular bed is different(9,24).

Intriguingly, the RV is derived from different embryological precursor cells

than the left ventricle(25). However, it is largely unknown to what extent RV

cardiomyocytes differ from LV cardiomyocytes and whether the embryological

difference translates in a distinct response to chronic abnormal loading

conditions(26).

In summary, there is a paucity of knowledge at all levels about the normal and

pathological function of the RV(22); the right ventricle is the elusive heart. As

a consequence, there are currently no clinically established treatments for RV

failure.

Chapter 116

Treating RV failure

However, today one could suggest three possible routes towards the treatment

of RV failure, and these are all three well worth pursuing. Firstly, treatment

strategies that have proven invaluable for left ventricular failure(27,28) (e.g.

beta adrenergic blockade, inhibition of the renin-angiotensin-aldosterone

system) could be of benefit in right ventricular failure(15). Although –as pointed

are before- there are important embryological, functional and morphological

differences between the LV and RV, and the etiology of left and right ventricular

failure are very different (ischemic heart disease vs. chronic abnormal loading),

it might be that there are common pathways and adverse changes that can

effectively blocked in RV failure as much as in LV failure.

A second route towards the identification of successful RV treatment relates

to drugs used to treat pulmonary hypertension. These drugs directly target

the pulmonary vascular disease (e.g. phosphodiesterase type 5A(PDE5A)-

inhibitors, endothelin antagonists), but have been suggested to have also direct

beneficial effects on the RV(29,30). Treatment of RV failure with PDE5A-inhibitor

Sildenafil for instance, might be attractive as it would kill two birds with one

stone, simultaneously reducing pulmonary vascular resistance and sustaining RV

function. However, also some authors do warn for adverse effects on the RV of

effective PAH-targeted drugs (30).

The third route is the most challenging and requires identification of the (mal)

adaptive mechanisms involved in RV failure. Such mechanisms may provide new

therapeutic targets of which the value can be asserted in preclinical proof-of-

concept studies.

The studies presented in this thesis represent steps on all three routes.

Animal models of RV failure

Unfortunately, answering the here studied research questions required animal

experiments. This puts both the researcher and society as a whole in the dilemma

of weighing the ethical and emotional burden of using animals to a scientific

end against the importance of developing treatments for a disease that causes

significant suffering in the lives of (young) patients and their families. While

animals should never be used for research when alternative methods exist,

animal models of RV failure are of paramount importance in the current stage

of RV research for myriad reasons. Firstly, they allow for highly controlled and


detailed studying of the pathophysiology and pathobiology of the RV subjected

to abnormal loading conditions and its responses to therapeutic interventions.

The severity of pressure load or volume load can be precisely titrated and the

RV response can be measured at any chosen moment thereafter. This avoids

the heterogeneity that complicates the interpretation of findings in clinical

studies, in which the mechanism, severity and duration of abnormal loading

vary from patient to patient leaving ample room for debate where to place the

observed changes in the spectrum from adaptation to failure. This becomes

especially apparent in studies of therapeutic interventions. Animal studies allow

for specific timing in starting or withdrawing treatment, which is important

because the effect of therapeutic intervention may to a large extent depend on

its timeliness. On one hand the intervention may be too late, when the harm

is already done, on the other hand the intervention may be too early, having

no (or even adverse) effects in early stages of disease, while being beneficial

in more advanced stages of the disease. In clinical studies, the stage of disease

is determined by using secondary parameters such as echocardiographic

measurements or the presence of (subjective) symptoms. In animal studies the

pathophysiology of RV overload can be studied from day to day and therapeutic

intervention can be commenced at any given moment; elucidating its effects in

a time-, and severity specific manner.

Secondly, in animal models assessment of RV function by pressure-volume

analysis is feasible. Pressure-volume analysis is the gold-standard of assessing

ventricular function, but requires an invasive procedure to catheterize the RV,

which limits its applicability in the clinical setting. Echocardiography is a practical

and easy alternative (both in clinical and animal studies) but provides only limited

information on RV function, most importantly because echo-derived function

parameters are highly load dependent and the complex RV anatomy precludes

accurate measurement of RV volumes. Cardiac magnetic resonance imaging

does allow RV volume measurements but is time consuming and lacks pressure

data. Pressure-volume analysis provides simultaneous pressure and volume

data in real-time, of which load-independent parameters of RV contractility,

compliance, muscle energetics and other important quantitative measures of

function can be derived. Especially diastolic function can hardly be assessed

by any other technique than pressure-volume analysis, which may explain why

the role of diastolic function in RV failure has received little attention so far.

Chapter 118

AIM AND OUTLINE OF THIS THESIS

We therefore used pressure-volume analysis for functional RV assessment and

evaluation of treatment effects in all the studies in rat models.

The third reason why animal models are a necessary evil in RV research, is the

possibility to collect tissue samples to study biomolecular and histopathological

changes. In clinical studies, sources of tissue samples are limited to surgery

specimens and material from autopsies. Obviously, these represent a very

narrow segment of the spectrum of RV disease and may not adequately reflect

the pathobiological changes that determine disease progression in early stages

of the disease, particularly when the patient may be amenable to treatment.

Finally, animal models are also a safe platform to assess the putative beneficial

and adverse effects of therapeutic interventions without putting patients at risk.

Historically, many models of RV failure (31)(32)came from the field of PH-

research. Although much of the current knowledge about the pressure

loaded RV is obtained in these models, they have limitations that require the

(additional) development of other models. Direct effects of the agents used to

induce PH (monocrotaline, SUGEN, hypoxia) on the RV limits their use to study

the pathobiology of RVF. Additionally, in PH models, direct effects of drugs on

the RV are difficult to distinguish from indirect effects caused by reduction of

pressure load (e.g. due to pulmonary vasodilatation). The pulmonary artery

banding (PAB) model circumvents these limitations and some reports suggest

that PAB may be a valid model of RV failure (33, 34). However, extensive

functional and pathobiological characterization of a PAB model displaying RV

failure is lacking in literature.

Compared to the pressure loaded RV, the volume loaded RV has received

very little attention so far, also in terms of animal models, despite its clinical

significance (35,36).

In this thesis, we aimed to expand the current knowledge of right ventricular

failure due to chronic abnormal loading conditions, using preclinical models, by

describing in detail the physiological and biological consequences of different

types of abnormal loading and by studying the effects of therapeutic agents with

a putative beneficial effect on the RV.


Specifically, we aimed to:

1. Characterize the clinical, physiological and biological RV response in rodent

models of chronic pressure load, volume load and combined pressure-volume

load.

To this end, we tested whether the adaptive RV response depends on the ‘type’

of loading. Do different loading conditions elicit a common or distinct adaptive

response?

In chapter 2, we compared RV hemodynamics, voluntary exercise and

hypertrophy in rat models of pressure overload due to pulmonary artery

banding (PAB), pressure overload due to experimental PH, combined pressure-

and volume overload and isolated volume load.

A similar comparison was made in the studies described in chapter 3, where we

tested whether the RV response to pressure load is different than the response

to volume load, in terms of ventricular function (measure by cardiac MRI),

voluntary exercise tolerance and activation of hypertrophy pathways.

2. Identify biological processes that play a role in right ventricular failure due to

fixed chronic RV pressure load.

To this end, we characterized advanced right ventricular failure in a model of

severe pressure load (PAB) (chapter 4). To identify physiological and biological

processes involved with the RV response to pressure load and the transition

from subclinical to clinical RV failure, we compared healthy rats to rats with

‘clinical’ RV failure and rats with ‘subclinical’ RV failure using echocardiography,

pressure-volume analysis, histological techniques and transcriptome-wide

expression profiling.

3. Study the effects phosphodiesterase type 5A-inhibiting therapy on fixed

chronic RV pressure load and chronic RV volume load.

Does PDE5A-inhibition have a beneficial effect on the RV, apart from its

vasodilatory effects on the pulmonary vasculature? If so, is this effect specific

for the pressure loaded RV or does it also benefit the (hypertrophied) volume

overloaded RV? And how important is the timing of the start of treatment? To

Chapter 120

answer these questions, we studied the effects of phosphodiesterase 5-blocker

Sildenafil in models of abnormal loading. We assessed preventive effects of

PDE5-inhibition by starting Sildenafil treatment from day 1 in both volume- and

pressure load (chapter 5). We followed-up the preventive study in pressure load

by a therapeutic study (chapter 6), where Sildenafil treatment was not started

until RV dysfunction had developed.

4). Study the effects of renin-angiotensin-aldosterone-system inhibiting therapy

on fixed chronic RV pressure load.

Does a cornerstone of left ventricular failure treatment also work in the RV?

In chapter 7, we studied RAAS-inhibiting therapy by angiotensine II-receptor

blocker Losartan and mineralo-corticoid-receptor blocker Eplerenone. Based

on its effects in the left ventricle we hypothesized that this intervention would

prevent pressure load-induced RV dysfunction, particularly disturbances of

diastolic function.

In chapter 8, we integrated our findings in a review of the current literature

on animal models of right ventricular failure due to chronic abnormal loading

conditions.

Finally in chapter 9, specifically the results presented in this thesis are placed in

a broader perspective and directions for future research are indicated.


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PART I

DISTINCTLOADIN GCONDITIONSRE VEALVARIOUSPA TTERNSOFRIGHT VENTRICULARAD APTATION

MAJ Borgdorff, B Bartelds, MG Dickinson, P Steendijk, M de Vroomen,

RMF Berger

Am J Physiol Heart Circ Physiol. 2013 Aug 1;305(3):H354-64.

doi: 10.1152/ajpheart.00180.2013. Epub 2013 May 31.

2

Chapter 228

ABSTRACTRight ventricular (RV) failure due to chronically abnormal loading is a main

determinant of outcome in pulmonary hypertension (PH) and congenital heart

disease. However, distinct types of RV loading have been associated with

different outcomes. To determine whether the adaptive RV response depends on

loading type, we compared hemodynamics, exercise and hypertrophy in models

of pressure overload due to pulmonary artery banding (PAB), pressure overload

due to PH, combined pressure- and volume overload and isolated volume load.

Ninety-four rats were subjected to either PAB, monocrotaline-induced PH (PH),

aorto-caval shunt (shunt) or combined monocrotaline and aorto-caval shunt

(PH+shunt). We performed pressure-volume analysis and voluntary exercise

measurements at 4 weeks. We compared PAB to PH (Part I) and PH+shunt to

either isolated PH or shunt (Part II). Part I: Enhanced contractility (end systolic

elastance and preload recruitable strokework) was present in PH and PAB, but

strongest in PAB. Frank-Starling mechanism was active in both PAB and PH. In

PAB this was accompanied by diastolic dysfunction (increased end diastolic

elastance, relaxation constant), clinical signs of RV failure and reduced exercise.

These distinct responses were not attributable to differences in hypertrophy.

Part II: in PH+shunt the contractility response was blunted compared to PH,

which caused pseudo-normalization of parameters. Additional volume overload

strongly enhanced hypertrophy in PH. We conclude that different types of

loading result in distinct patterns of RV adaptation. This is of importance for the

approach to patients with chronically increased RV load and for experimental

studies in various types of RV failure.

Distinct loading conditions reveal various patterns of RV adaptation 29

INTRODUCTIONRight ventricular failure is a detrimental condition that is associated with

significant morbidity and mortality in patients with congenital heart disease

and/or pulmonary hypertension (PH) (11, 19, 34, 38). In these conditions,

persistent abnormal loading of the right ventricle (RV) leads to RV failure in

the long term(17, 50). However, physiological and molecular mechanisms of

RV adaptation to these abnormal loading conditions and its derailment into RV

failure are largely unknown (3, 50). As a consequence, no heart failure therapy

exists that specifically targets the RV.

Different animal models have been used to study the abnormally loaded RV,

but interpretation of data and translation to the clinical setting is hampered by

conceptual concerns.

Firstly, distinct types of RV overload are used in experimental models. These

include the induction of PH, where the RV interacts with an increased dynamic load

due to a high resistance pulmonary vascular bed (i.e. peripheral-type pressure

overload), and pulmonary arterial banding (PAB) resulting in an increased load

with an absolute, fixed uncoupling of the ventriculo-vascular interplay (i.e.

proximal-type pressure overload). It is unknown whether these distinct types

of pressure overload result in common or distinct adaptive responses. Clinical

observations suggest that the RV tolerates congenital pulmonary valve stenosis

(a proximal-type pressure overload) better than PH (a peripheral-type pressure

overload)(24). This notion is supported by experimental data(6) and requires

comparison of functional adaptation of the RV in models of either type.

Secondly, it is unknown how additional increased volume overload impacts

the ability of the RV to adapt to increased pressure overload. This situation is

clinically encountered in patients with congenital heart disease associated with

systemic-to-pulmonary shunts who develop PH and appear to have a very poor

prognosis(32).

Finally, most animal studies lack a ‘clinical’ indicator of the severity of RV

dysfunction and provide limited characterization of RV function, which

complicates translation to the clinical setting.

We therefore aimed to characterize the clinical and functional RV response to

distinct types of abnormal RV loading using voluntary exercise measurement and

pressure-volume analysis. We focused on two clinically relevant perspectives:

1. proximal-type vs. peripheral-type RV pressure overload (PAB vs. PH) and

Chapter 230

MATERIALS AND

METHODS

2. Isolated peripheral-type pressure overload vs. peripheral-type pressure

overload combined with volume overload (PH vs. PH+shunt).

We hypothesized that the response patterns of the RV, in terms of hemodynamics,

clinical symptoms and hypertrophy, would depend of the type of abnormal

loading. This would have major consequences for both the interpretation of

experimental research and its translation to clinical practice.

Animal models

Animal care and experiments were conducted according to the Dutch Animal

Experimental Act; the investigation conforms to the Guide for the Care and Use

of Laboratory Animals published by the US National Institutes of Health (NIH

Publication No. 85-23, revised 1996). The Animal Experiments Committee of the

University of Groningen, the Netherlands approved the experimental protocol.

The rats were individually housed with a 12:12-h light-dark cycle and fed ad

libitum.

Ninety-four Wistar rats (male; 160-230g; Harlan, Horst, the Netherlands) were

assigned to one of the following experimental groups; 1) proximal type pressure

load via pulmonary artery banding (PAB, n=10), 2) peripheral type pressure-load

via monocrotaline-induced PH (PH, n=16 + n=17; low and high dose, see below),

3) PH combined with volume overload (PH+Shunt, n=14), 4) volume overload

only (shunt, n=10), 5) and 6) sham –operated controls (CON, n=9 + n=18). Since

not all experiments could be performed concurrently two separate control

groups were created. Because of subtle differences in baseline hemodynamics

between both control groups, relative changes of the abnormal loading groups

(versus the corresponding control group) were compared, rather than absolute

values for hemodynamic parameters. Number of rats used per analysis is also

indicated in the figure legends.

PAB procedure as described previously (7): Pulmonary artery banding (PAB) was

performed via a left lateral thoracotomy, using an 18G needle to standardize

the degree of stenosis. For PAB surgery, rats were anesthetized with isoflurane/

air mixture (5% for induction, 2-3% maintainance); analgesia was ensured by

Buprenorphine (0.01-0.05 mg/kg sc. during surgery and the two following days).

PH was induced via subcutaneous injection of Monocrotaline (MCT) (Sigma-

Aldrich Chemie B.V., Zwijndrecht, the Netherlands).


Figure 1 Schematic overview of experimental design

PAB= pulmonary artery banding, PH= pulmonary hypertension, CON= control, PH+shunt= pulmonary hypertension+ aorto-caval shunt, shunt= aorto-caval shunt model, SHAM= thoracotomy, laparotomy or subcutaneous injection as performed in PAB, Shunt or monocrotaline injection respectively, but without the model-inducing step. Exercise tolerance was measured during five days prior to model induction and five days prior to termination. All other analyses were performed at 4 weeks.

To create a group of rats with a range of PH-pressures, we used both

monocrotaline 30 mg/kg bodyweight and 80 mg/kg bodyweight (see below). A

monocrotaline dosage of 60mg/kg bodyweight was used in the PH+shunt group,

because 80mg/kg with additional aorto-caval shunt led to rapid deterioration of

the rats (<2 weeks) and did not allow comparison to PH at a relevant time point.

Aorto-caval shunt surgery was performed via a laparotomy as described

previously, where standardization of the shunt-size was reached by using an 18G

needle(4, 7, 13, 48). For shunt surgery rats were anesthetized with isoflurane/

air mixture (5% for induction, 2-3% maintainance); analgesia was ensured by

Buprenorphine (0.01-0.05 mg/kg sc. during surgery and the two following days).

Sham-operated controls: Control groups received sham-surgeries (thoracotomy

and laparotomy) to control for PAB and aorto-caval shunt procedures or saline-

injection and laparotomy to control for monocrotaline injection and aorto-caval

shunt procedure.

Chapter 232

Two animals (1 in shunt, 1 in a control group) died in the first week after surgery

due to abdominal complications of the laparotomy. Additionally, two animals

in the PH+shunt group died prematurely during echocardiography (before

pressure-volume measurements could be performed). Their echocardiograms

showed very poor RV function.

We made two comparisons in this study: Part I of this study compared peripheral-

type pressure overload (PH) to proximal-type pressure overload (PAB) using 3

groups: peripheral-type pressure load (PH, monocrotaline sc. 80mg/kg body

wt); proximal-type pressure load (PAB, pulmonary artery banding); and a sham-

operated control group. To obtain a range of pressures, an additional group

was treated with less monocrotaline (30mg/kg body wt); data obtained in this

group were added to the PH group only in linear regression analysis. Part II of

the study compared isolated peripheral-type pressure overload to peripheral-

type pressure overload combined with volume overload, and for this purpose

4 groups were used: PH (same group as in part I); PH+shunt (monocrotaline

60mg/kg bodyweight + surgical aorto-caval shunt); and a sham-operated control

group. A group with isolated volume overload (aorto-caval shunt) was used as

additional control.

For a timeline of all measurements as well as an overview of the groups, we

refer to Fig 1.

Voluntary exercise measurements and signs of failure

To measure voluntary exercise (7), running wheels were mounted in the rat

cages. Because of the large inter-individual variation, rats were measured

before and 4 weeks after surgery/monocrotaline injection. Five days before

surgery and 5 days before sacrifice, animals were allowed to run in the cage

wheel. Running distance and time spend in the wheel were recorded daily using

a digital magnetic counter (Commodoor Cycle Odometer, Commodoor, the

Netherlands). The change in running distance at 4 weeks vs. baseline was used

as primary parameter of exercise.

Throughout the experiment, rats were daily examined for clinical signs of

right ventricular failure. Clinical signs of heart failure were defined according

to previously described ‘ABCDE-criteria’(7). A: appearance and activity, B:

bodyweight, C: cyanosis and circulation, D: dyspnea and tachypnea, E: edema

and effusion.


Right ventricular hemodynamics

Hemodynamic characterization of the RV was performed by pressure-volume

studies, obtained by right heart catheterization four weeks after surgery as

described previously (7). Rats were anesthetized with isoflurane (5% induction;

2-3% maintenance), intubated and ventilated. The right jugular vein was

dissected and cannulated facilitating hypertonic saline infusions. Following

bilateral thoracotomy and pericardiotomy a combined pressure-conductance

catheter (SPR-869, Millar Instruments Inc., Houston, TX, USA) was introduced

via the apex into the RV and positioned in the RV outflow tract. RV pressures

and conductance were recorded using a MPVS 400 processor at a sample rate

of 1.000 Hz with Chart 5 (Millar Instruments Inc., Houston, TX, USA). Analyses

were performed offline using custom-made software (CircLab 2009/2010,

P. Steendijk). The volume signal of the conductance catheter was calibrated

for parallel conductance and slope factor (alpha) in order to obtain absolute

volumetric values. The parallel conductance was estimated by infusing 10μL

of hypertonic (10%) saline via the jugular vein cannula(2). Slope factor was

calculated as uncalibrated conductance catheter cardiac output divided by

LV cardiac output, measured by echocardiography. Heart rate, pressures,

maximal and minimal speed of pressure change (dPdtmax and dPdtmin, volumes

and relaxation constant tau were derived from steady state measurements.

End systolic pressure-volume relations (ESPVR), end diastolic pressure-volume

relations (EDPVR) and Preload Recruitable Strokework (PRSW) were determined

from measurements obtained during transient progressive constriction of the

vena cava inferior. The slopes of ESPVR and PRSW were used as measures

of systolic function, the slope of EDPVR (end diastolic elastance, which can

be understood as stiffness) was used as a measure of diastolic function: we

found consistently linear –not mono-exponential- EDPVRs. Stroke volume-End

diastolic volume diagrams of the different groups were made using steady state

measurements from the individual animals.

Echocardiography

Echocardiography was performed on the day before sacrifice as described

before(7). Rats were anesthetized with isoflurane (5% induction; 2-3%

maintenance). Echocardiography was performed using a Vivid Dimension 7

system and 10S-transducer (GE Healthcare, Waukesha, WI, USA). Systolic aorta

Chapter 234

diameter was measured in parasternal long axis view (in triplo), pulsed wave

Doppler of aorta flow was obtained in the 5-chamber view. Stroke volume was

calculated as (aorta diameter)2 × 3.14 × velocity time integral (VTI). To obtain

cardiac output, stroke volume was multiplied by the heart rate, calculated from

the duration of the heart beat in the Doppler signal. The mean of measurements

from 6-12 consecutive beats with a proper signal was taken to average out beat-

to-beat variation.

Termination, organ weights and hypertrophy

After heart catheterization, the rats were terminated by removing the heart

from the thorax. Heart, lungs and liver were dissected. RV, interventricular

septum, left ventricle and atria were separated and weighed.

qRT-PCR, Western Blot, Immunohistochemistry

To further analyze the remodeling response of the RV, we performed qRT-PCR,

Western blotting and immunohistochemistry. For assessment of the activity

of key regulating pathways of hypertrophy, we measured 1/2Erk (MAPKinase

pathway), Akt (PI3K pathway) and regulator of calcineurin type 1 (RCAN1;

calcineurin-NFAT pathway). To see whether distinct loading conditions elicited

differences in fetal reprogramming we measured natriuretic propeptide type A

(NPPA) and myosin heavy chain isoforms beta and alpha (MYH7 and 6). qRT-PCR:

RV (free wall) tissue was snap-frozen in liquid nitrogen. Total RNA was extracted

using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA); high quality

was confirmed (RQI 9.3) using Experion (Bio-Rad, Veenendaal, the Netherlands).

Conversion to cDNA by QuantiTect Reverse Transcription (Qiagen, Venlo, the

Netherlands). Gene expression was measured with Absolute QPCR SYBR Green

ROX mix (Abgene, Epsom, UK) in the presence of 7.5ng cDNA and 200nM forward

and reverse primers. qRT-PCR was carried out on the Biorad CFX384 (Bio-Rad,

Veenendaal, the Netherlands) using a standard protocol for the following

genes: NPPA, RCAN1,MYH7 and MYH6. Primer sequences are available upon

request. mRNA levels are expressed in relative units based on a standard curve

obtained by a calibrator cDNA mixture. All measured mRNA expression levels

were corrected for 18S reference gene expression. Western blotting: Protein

was extracted from snap-frozen RV tissue using RIPA buffer. We used antibodies

to phosphorylated and total Akt (1:1000; Cell Signalling Technology, Danvers,


MA, USA) and phosphorylated and total MAPkinase ERK1/2 (1:1000; Santa Cruz

Biotechnology, Santa Cruz, CA, USA)(47). Primary antibody binding was visualized

by horseradish peroxidase–conjugated secondary antibodies and enhanced

chemiluminescence (Perkin-Elmer, Waltham, MA, USA). Immunohistochemistry:

transversal midventricular RV tissue-slices were fixated using 4% formalin

and embedded in paraffin. Four-µm-thick sections were cut, deparaffinized

and rehydrated in decreasing graded alcohol and xylene. For determination

of cardiomyocyte surface area, these were stained using Gomori’s reticulin

silver staining and photographed using a camera fitted on a microscope (Zeiss

Benelux, Sliedrecht, the Netherlands) at 40x magnification and analyzed using

Image-Pro software (MediaCybernetics, Bethesda, MD, USA). Only transversally

cut myocytes were included; per section measurements were averaged from 60

cells in 4 different fields.

Fibrosis, PDE5A-axis, PKG-1 activity

For determination of the amount of fibrosis, RV sections were stained using

NovaUltra™ Masson Trichrome Stain kit (IHC World, Woodstock, MD, USA) and

photographed using a digital slide scanner (NanoZoomer 2.0-HT, Hamamatsu

Photonics Nederland, Almere, the Netherlands) at 20x magnification and

analyzed using Image Scope 11 (Aperio Technologies, Inc. Vista, CA, USA). The

extent of fibrosis was quantified as the blue-stained percentage of the total

tissue area, measured per whole section. The edges of the tissue and major

vessels including perivascular fibrosis were excluded from analysis to obtain

purely myocardial interstitial fibrosis. The phosphodiesterase type 5A-protein

kinase G1 (PDE5A-PKG1) -axis, is suggested to be involved in the RV response to

abnormal loading (37) and might have specific relevance to diastolic function(5,

27). We analyzed the activity of this pathway by measuring PDE5A mRNA and

protein expression, myocardial cGMP levels and PKG-1 activity as described

previously (7, 37).

Statistical analysis

Quantitative data are expressed as mean± standard error of the mean (SEM)

for the control group and percentage change versus matched control group

for all other groups, unless mentioned otherwise. All variables were tested for

normality. Differences between groups were evaluated using one-way ANOVA

Chapter 236

RESULTS

followed by post hoc analysis (protected Fisher’s least significant difference

method) or Kruskal-Wallis-, and Mann-Whitney U-test, as appropriate. To

examine the interaction between RV pressure and various parameters and

evaluate differences between groups we performed linear regressions and

2-way ANOVA. P<0.05 was considered significant (PASW Statistics 18 for

Windows, SPSS, Chicago, Illinois).

PART I: RV pressure overload induced by Pulmonary Hypertension vs.

Pulmonary Artery Banding

PH and PAB elicit distinct hemodynamic responses in the RV

The RV responded to pressure overload with enhancing contractility (measured

by heart catheterization, representative PV-loops in Fig 2A): end systolic

elastance (Ees) and preload recruitable stroke work (PRSW) were significantly

increased in both PH and PAB (Fig 2B). However, the degree of increase was

lower in PH. In PH, PRSW was correlated to peak pressure (Fig 2C), in contrast

to PAB (Fig 2C). Ees was not correlated with increasing pressure in either of the

models.

Diastolic function (as measured by heart catheterization) deteriorated in PAB,

but not in PH. End diastolic elastance (Eed, Fig 2D) and relaxation constant tau

(Table 1) were increased in PAB, but not in PH. Active myocardial relaxation was

estimated to be complete at 3.5 times uncorrected tau≈73ms (52). The total

duration of diastole was ≈100-140ms at heart rate 300/min (0.5-0.66*heart

beat duration), so increased end diastolic elastance did not reflect incomplete

relaxation, but increased stiffness.

Ventricular dilatation and a rightward shift of the Frank-Starling curve (derived

from heart catheterization data) were present in both groups. The latter was

more prominent in PAB than in PH (Fig 2E), indicating that, at a similar preload,

output was lower in PAB than in PH.


Figure 2 Right ventricular hemodynamics in PH and PAB

A Representative pressure volume loops of the experimental groups during vena cava occlusion. End systolic pressure volume relations marked by solid lines, end diastolic pressure volume relations marked by dashed lines. CON is shown as reference. B End systolic elastance (*(RVmass/BW) (% increase vs. CON) and Preload Recruitable Stroke Work (% increase vs. CON) (n=7-8). C PRSW vs. RV peak pressure. D End diastolic elastance (% increase vs. CON) (n=6-9) E End diastolic volume vs. Stroke volume (Frank-Starling relations). Absolute volumes (μL). All hemodynamic parameters were obtained from pressure-volume analysis. Mean±SEM, * p<0.05 vs. CON, $ p<0.05 vs. PH

Figure 3 Voluntary exercise and RV failure symptoms in PH and PAB

Exercise vs. RV peak pressure. Exercise is expressed as relative change in distance run before sacrifice vs. baseline (n=6-15). Mean±SEM for the groups is indicated by the left-hand (CON), middle (PH) and right-hand (PAB) line with error bars. Significance is vs. CON B Percentage of rats presenting clinical signs of right ventricular failure (n=9-16). ABCDE refer to symptom-categories: A activity and appearance, B bodyweight, C cyanosis and/or hampered peripheral circulation, D dyspnoe and/or tachypnoe, E effusions: pleural or ascites.

Chapter 238

CON

PHPA

B p-values

CON

1CO

N 2

Absolute

Relative Δ

(%)

Absolute

Relative Δ

(%)

PH vs. CO

NPA

B vs. CON

PH vs. PA

B

PeakP (mm

Hg)

26±225±1

50±496±16

70±9144±24

0,0000,000

0,041W

allstress Peak (mm

Hg)

156±11151±5

277±2683±17

492±62214±40

0,0150,000

0,001ED

P (mm

Hg)

1±0.32±0.6

5±1199±62

6±1428±119

0,0450,001

0,034H

R (/min)

306±10351±14

326±16-5±1

300±10-2±3

0,2050,784

0,370SV (μL)

269±7290±15

275±20-5±7

227±19-16±7

0,5350,091

0,224CI (m

L/min/g BW

)0.26±0.01

0.28±0.010.26±0.01

-8±50.24±0.02

-5±80,247

0,5300,675

ESV (μL)320±42

287±41431±41

50±14648±29

102±90,010

0,0000,012

EDV (μL)

589±47577±46

706±5730±7

874±3948±6

0,1030,001

0,032EF (%

)47±3

52±440±1

-25±226±1

-45±30,000

0,0020,010

dPdtmax (m

mH

g/s)1315±75

1634±1142030±194

24±122812±471

114±360,324

0,0000,002

dPdtmin (-m

mH

g/s)964±41

1322±1271976±155

50±122491±256

158±270,018

0,0000,000

dPdtmax ind(1/s)

51±361±2

42±2-32±3

40±3-21±6

0,0000,007

0,091dPdtm

in ind (-1/s)37±1

48±141±3

-13±738±3

1±90,138

0,9240,140

Ees (mm

Hg/m

L)59±8

47±6134±40

188±87155±27

164±460,035

0,0490,775

PRSW (m

mH

g)12±2

24±437±8

53±3244±6

283±540,039

0,0000,000

tau (ms)

15.6±1.112.5±0.5

16.4±1.031±8

20.7±1.433±9

0,0110,014

0,862Tau corr (m

s/s)78±4

73±388±8

21±11103±4

31±60,097

0,0320,451

Eed (mm

Hg/m

L)3.6±0.9

5.4±0.75.6±1.2

4±239.3±1.8

158±500,923

0,0040,003

Table 1 RV hemodynam

ics in CON

, PH and PA

B

PeakP= peak pressure, EDP= end diastolic pressure, H

R= heart rate, SV= stroke volume, CI= cardiac index, ESV= end systolic volum

e, EDV= end diastolic volum

e, EF= ejection fraction, dPdtm

ax/min (ind) = indexed for peak pressure (m

ax) and end diastolic pressure (min) respectively, Ees= end systolic elastance, PRSW

= preload recruitable stroke w

ork, tau corr = corrected for heart rate, eed= end diastolic elastance. CON

= control, PH= pulm

onary hypertension, PAB= pulm

onary artery banding. All hem

odynamic

parameters w

ere obtained from pressure-volum

e analysis, except cardiac output, which w

as measured by echocardiography. D

ata are mean±SEM

. Relative ∆ is percentage change versus respective control group. Significance indicated by p-values.


Voluntary exercise and clinical signs of RVF differ between PH and PAB

PAB reduced voluntary exercise and induced clinical signs of RV failure, such

as dyspnea and edema, more than PH (Fig 3A,B). The severity of reduction in

exercise was not related to RV peak pressure, especially beyond 30% increase of

normal RV peak pressure (Fig 3A).

Hypertrophy and regulators are equally induced in PH and PAB

Pressure load induced RV hypertrophy (Fig 4A), regardless whether it resulted

from PH or PAB (Fig 4B), and RV weight correlated with peak pressure (Fig

4C). Upregulation of RCAN1 (Fig 4D) and MHC-isotype switch (Fig 4E) was also

correlated with peak pressure equally in both PH and PAB, although two PAB

animals lacked a strong MHC-isotype switch (arrows in Fig 4E). There was no

significant fibrosis and protein expression and phosphorylation status of Akt

and ERK1/2 were unchanged in both types of RV pressure overload (data not

shown). PDE5A expression (mRNA and protein) was at control level in both PH

and PAB, but PKG-1 activity tended to be higher in PH (2.83±0.37 AU) than in PAB

(1.87±0.26 AU, p=0.07 vs. PH) and CON (1.95±0.13 AU, p=0.11 vs. PH).

Part II: Pulmonary hypertension vs. pulmonary hypertension + shunt

Combined overload blunts the contractility response, pseudo-normalizing

hemodynamic parameters

Pressure-volume analysis showed that the addition of volume overload to

peripheral-type pressure overload induced further RV dilatation (Fig 5A) and

resulted in comprised contractility as compared with PH Fig 5B). Ees did not

increase in response to isolated volume load, whereas PRSW did.

The change in PRSW following pressure load depended on the type of load (Fig

5C): in PH+shunt PRSW barely increases in response to pressure load, compared

to PH or Shunt.

PH+shunt had a tendency towards diastolic dysfunction (increased end diastolic

elastance), but this did not reach significance due to large inter-animal variation.

Also the other invasively measured parameters of diastolic function did not

indicate pronounced diastolic dysfunction (Table 2). Further, PH+shunt showed

a leftward shift of the Frank-Starling curve (Fig 5E). This, however, may reflect

the reduced heart rate found in these animals, rather than augmented stroke

volume in response to increased preload (Table 2).

Chapter 240

Figure 4 Hypertrophy profile in PH and PAB

A RV weight normalized for bodyweight (mg/g) (n=9-16). B RV weight corrected for RV peak pressure (n=7-12). C RV weight as a function of RV peak pressure. D RCAN1 mRNA expression as a function of RV peak pressure. E MYH7/MYH6 mRNA ratio as a function of RV peak pressure. All data % increase vs. CON, except for mRNA expression (fold change vs. CON) and B (mg/mmHg). Mean±SEM, * p<0.05 vs. CON, $ p<0.05 vs. PH


CON

PHPH

+shu

ntSh

unt

P-va

lues

CON

1CO

N 2

Abs

olut

eRe

lati

ve

Δ(%

)A

bsol

ute

Rela

tive

Δ

(%)

Abs

olut

eRe

lati

ve

Δ(%

)PH

vs.

CO

NPH

+shu

nt v

s.

CON

shun

t vs.

CO

NPH

vs.

PH+s

hunt

PH v

s.

Shun

tPH

+shu

nt v

s.

Shun

t

Peak

P (m

mH

g)26

±225

±150

±496

±16

52±3

107±

1037

±345

±13

<0.0

01<0

.001

0,00

20,

500,

006

0,00

2

Wal

lstr

ess

Peak

(mm

Hg)

156±

1115

1±5

277±

2683

±17

241±

1859

±12

336±

3911

4±25

0,00

20,

035

<0.0

010,

360,

190,

039

EDP

(mm

Hg)

1±0.

32±

0.6

5±1

199±

625±

0.6

182±

312±

0.8

114±

740,

013

0,02

90,

190,

810,

280,

41

HR

(/m

in)

306±

1035

1±14

326±

16-5

±128

8±14

-21±

230

4±7

-4±8

0,08

0,00

10,

560,

060,

270,

007

SV (μ

L)26

9±7

290±

1527

5±20

-5±7

480±

3166

±11

418±

2455

±90,

96<0

.001

<0.0

01<0

.001

<0.0

010,

40

CI (m

L/m

in/g

BW

)0.

26±0

.01

0.28

±0.0

10.

26±0

.01

-8±5

0.44

±0.0

457

±14

0.39

±0.0

252

±90,

61<0

.001

<0.0

01<0

.001

<0.0

010,

73

ESV

(μL)

320±

4228

7±41

431±

4150

±14

652±

149

127±

5283

9±49

162±

150,

220,

005

0,00

10,

070,

015

0,44

EDV

(μL)

589±

4757

7±46

706±

5730

±798

9±62

71±1

112

57±6

111

3±10

0,07

<0.0

01<0

.001

0,00

1<0

.001

0,00

4

EF (%

)47

±352

±440

±1-2

5±2

46±4

-13±

733

±2-2

9±3

0,00

30,

120,

001

0,12

0,56

0,05

dPdt

max

(mm

Hg/

s)13

15±7

516

34±1

1420

30±1

9424

±12

2470

±125

51±8

1922

±177

46±1

30,

140,

003

0,00

10,

070,

160,

75

dPdt

min

(-m

mH

g/s)

964±

4113

22±1

2719

76±1

5550

±12

2257

±123

71±9

1352

±111

40±1

10,

004

<0.0

010,

003

0,17

0,57

0,08

dPdt

max

ind(

1/s)

51±3

61±2

42±2

-32±

347

±2-2

3±3

53±2

5±4

<0.0

01<0

.001

0,64

0,64

<0.0

01<0

.001

dPdt

min

ind

(-1/

s)37

±148

±141

±3-1

3±7

43±1

-9±3

38±1

1±4

0,04

00,

130,

960,

550,

036

0,12

Ees

(mm

Hg/

mL)

59±8

47±6

134±

4018

8±87

84±3

081

±64

41±4

-31±

70,

030

0,32

0,73

0,19

0,02

00,

21

PRSW

(mm

Hg)

12±2

24±4

37±8

53±3

225

±32±

1123

±610

3±53

0,04

80,

940,

031

0,20

0,25

0,00

3

tau

(ms)

15.6

±1.1

12.5

±0.5

16.4

±1.0

31±8

14.7

±1.1

17±9

16.3

±0.9

4±6

0,00

70,

130,

740,

200,

021

0,25

Tau

corr

(ms/

s)78

±473

±388

±821

±11

72±9

-1±1

282

±45±

50,

190,

770,

930,

100,

240,

71

Eed

(mm

Hg/

mL)

3.6±

0.9

5.4±

0.7

5.6±

1.2

4±23

7.9±

1.9

47±3

53.

9±1.

68±

440,

850,

190,

820,

250,

930,

40

Tabl

e 2

RV h

emod

ynam

ics

in C

ON

, PH

+shu

nt, P

H a

nd s

hunt

Peak

P= p

eak

pres

sure

, ED

P= e

nd d

iast

olic

pre

ssur

e, H

R= h

eart

rat

e, S

V= s

trok

e vo

lum

e, C

I= c

ardi

ac in

dex,

ESV

= en

d sy

stol

ic v

olum

e, E

DV=

end

dia

stol

ic v

olum

e, E

F= e

jecti

on

frac

tion,

dPd

tmax

/min

(in

d) =

inde

xed

for

peak

pre

ssur

e (m

ax)

and

end

dias

tolic

pre

ssur

e (m

in)

resp

ectiv

ely,

Ees

= en

d sy

stol

ic e

last

ance

, PRS

W=

prel

oad

recr

uita

ble

stro

ke

wor

k, t

au c

orr

= co

rrec

ted

for

hear

t ra

te, E

ed=

end

dias

tolic

ela

stan

ce. C

ON

= co

ntro

l, PH

= pu

lmon

ary

hype

rten

sion

, PH

+shu

nt=

pulm

onar

y hy

pert

ensi

on+a

orto

-cav

al s

hunt

, sh

unt=

aor

to-c

aval

shu

nt. A

ll he

mod

ynam

ic p

aram

eter

s w

ere

obta

ined

from

pre

ssur

e-vo

lum

e an

alys

is, e

xcep

t car

diac

out

put,

whi

ch w

as m

easu

red

by e

choc

ardi

ogra

phy.

Dat

a ar

e m

ean±

SEM

. Rel

ative

∆ is

per

cent

age

chan

ge v

ersu

s re

spec

tive

cont

rol g

roup

. Sig

nific

ance

indi

cate

d by

p-v

alue

s.

Chapter 242

Reduced voluntary exercise and prominent clinical signs of RVF in combined

overload

Voluntary exercise was significantly reduced in both PH+shunt and shunt

compared to CON (Fig 6A). However, the majority of rats with PH+shunt

exhibited signs of RVF, in contrast to those with PH or shunt (Fig 6B).

Combined overload induces additional hypertrophy and gene expression

PH+shunt induced more RV hypertrophy than PH or shunt (Fig 7A). The very

prominent RV hypertrophy in PH+shunt seems an addition of pressure- and

volume-induced hypertrophy as the RV weights in these rats equaled the sum

of increases of RV weight in shunt and PH (Fig 7B). Combined overload also had

a strong additive effect on MYH-isoform switch (Fig 7C) and NPPA expression

(Fig 7D), whereas RCAN1 upregulation was similar in PH, PH+shunt and shunt

(Fig 7E). Protein expression and phosphorylation status of Akt and ERK1/2 were

unchanged in these groups (data not shown). In both the combined overload

and isolated volume overload there was no significant fibrosis. PDE5A expression

Figure 5 Right ventricular hemodynamics in PH+shunt, PH and shunt

A Representative pressure volume loops of the experimental groups during vena cava occlusion. End systolic pressure volume relations marked by solid lines, end diastolic pressure volume relations marked by dashed lines. CON is shown as reference. B End systolic elastance (% increase vs. CON) and Preload Recruitable Stroke Work (% increase vs. CON) (n=5-9). C PRSW vs. RV peak pressure. D End diastolic elastance (% increase vs. CON) (n=4-10) E End diastolic volume vs. Stroke volume (Frank-Starling relations). Absolute volumes (μL). All hemodynamic parameters were obtained from pressure-volume analysis. Mean±SEM, * p<0.05 vs. CON, # p<0.05 vs. CON and PH+shunt, p=0.19 vs. CON (D)


DISCUSSION

(mRNA and protein) was unchanged (data not shown. Also, PKG-1 activity was

similar in PH, PH+shunt and shunt (data not shown).

This study characterizes right ventricular responses to distinct types of chronic

RV overload and demonstrates that the pattern of response depends on the

type of loading. We found experimental proximal type pressure load (PAB) to

induce significant diastolic dysfunction after 4 weeks, whereas peripheral type

pressure load (PH) did not, independent of severity of RV hypertrophy. Also,

PAB was associated with more prominent RV dilatation and clinical signs of RV

failure compared to PH. The combination of volume load and PH resulted in

additional RV hypertrophy and a pattern of pseudo-normalization with blunted

contractility response.

These different responses to distinct loading conditions imply that experimental

studies on RV failure should use models that match the loading condition of

the clinical disease under study. Moreover, it may be of clinical relevance in the

therapeutic approach of patients with different types of RV loading.

Figure 6 Voluntary exercise and RV failure symptoms in PH+shunt, PH and shunt

A Relative change in distance run during voluntary exercise before sacrifice versus baseline (n=8-15). Mean±SEM, * p<0.05 vs. CON B Percentage of animals presenting clinical signs of right ventricular failure (n=9-16). ABCDE refer to symptom-categories: A activity and appearance, B bodyweight, C cyanosis and/or hampered peripheral circulation, D dyspnoe and/or tachypnoe, E effusions: pleural or ascites.

Chapter 244

The RV responds to chronic pressure overload with both Frank-Starling

mechanism and increased contractility

In chronic pressure overload, the left ventricle is thought to first utilize the

Frank-Starling mechanism, increasing preload to maintain output, and once its

limit has been reached, to increase contractility to compensate for the ‘afterload

mismatch’(42). This concept has been confirmed in aortic banding models(9,

36), but it is unclear whether it is applicable to the RV. Previous studies, both

experimental and clinical, suggested that in the proximal-type pressure overload

increased contractility rather than Frank-Starling mechanism characterizes the

Figure 7 Hypertrophy profile in PH+shunt, PH and shunt

A RV weight normalized for bodyweight (n=10-18). B RV weight as a function of RV peak pressure. C MYH7/MYH6 mRNA ratio as a function of RV peak pressure (n=7-10). D NPPA mRNA expression as a function of RV peak pressure (n=7-10). E RCAN1 mRNA expression as a function of RV peak pressure (n=7-10). All data % increase vs. CON, except for mRNA expression (fold change vs CON). Mean±SEM, * p<0.05 vs. CON, $ p<0.05 vs. all other groups.


RV response (15, 30), whereas in the peripheral-type pressure overload, the

RV response is limited to the Frank-Starling mechanism and does not increase

contractility (21). By direct comparison, however, this study shows that in both

proximal-, and peripheral-type pressure overload the RV response uses the

Frank-Starling mechanism, but additionally depends on increased contractility

for maintaining stroke volume. The LV concept of adaptation to chronic pressure

overload therefore seems to hold also in the RV. However, the proximal-type

pressure overload appears to force the RV to function at the limit of its adaptive

capacity more, or earlier, than peripheral-type pressure overload, illustrated by

the more pronounced right-shift of the Frank-Starling curve and higher Ees and

PRSW in PAB.

Additional volume overload in PH blunts the contractility response, pseudo-

normalizing hemodynamic parameters

Recognizing the importance of preload for the pressure overloaded RV, we

investigated the effect of additional volume overload which might be beneficial

through maintaining stroke volume via the Frank-Starling mechanism. In the

current study the RV indeed reached higher stroke volumes with additional

volume overload. However, in contrast to isolated pressure overload,

contractility failed to increase. This is in line with reports that the chronically

volume overloaded RV cannot increase contractility in response to acute

pressure overload (46). Studies in large animal models of combined volume-

and pressure overload have shown that during disease progression contractility

initially increases, but falls back to pseudo-normal levels in a more advanced

stage of RV dysfunction (28, 40, 41). Causal factors for the blunted contractility

response may include loss of peristaltic contraction pattern (33), disturbed

calcium homeostasis(8, 26) and changes in coronary perfusion(46). Failure to

increase contractility has also been reported in a model of chronic LV volume

overload(25). Also, in LV models, pressure load (aortic banding) and volume

load (aorto-caval shunt) have been shown to induce distinct functional and

molecular responses (e.g. in hypertrophy signaling and calcium handling (35,

47)). Combined pressure-volume load may then lead to adverse, maladaptive

remodeling. Excessive hypertrophy with a strong switch towards the slow-type

beta-myosin heavy chain, associated with poor contractile function(44), found

in the current study, might also play a causal role. The blunted contractility

Chapter 246

response, causing pseudo-normalization of Ees and PRSW, carries the risk of

underestimating RV dysfunction in patients with combined overload of the RV.

Diastolic function in PAB, PH and PH+shunt

Another important observation in this study was the diastolic dysfunction

present in PAB-, but not in PH-rats. Although diastolic dysfunction in PAB has

been previously and consistently reported (7, 18, 30), data in experimental PH

were contradictory (21, 29). The absence of evident diastolic dysfunction in PH

in our study is rather convincingly demonstrated by using a load-independent

parameter (end diastolic elastance), confirmed by secondary parameters

such as relaxation constant tau, maximum speed of pressure decline dP/dtmin

and end diastolic pressure. Nevertheless, PH is thought to also cause diastolic

dysfunction and we propose our data suggest an intrinsic difference in RV

adaptation between PAB and PH.

In the current study, PAB after 4 weeks led to significantly disturbed diastolic

function accompanied by poor exercise and a high proportion of clinical signs

of RVF. Although the increased end diastolic elastance resulted from increased

stiffness (and not incomplete relaxation), these differences were not accounted

for by hypertrophy, which was comparable in both groups, nor by myocardial

fibrosis, which had not yet developed at this time point (7, 21).

The prolonged active relaxation (tau) and increased passive stiffness (end

diastolic elastance) observed, closely resemble the diastolic disturbances seen

in left sided diastolic heart failure(54), where protein kinase G-1 (PKG-1) activity

has been shown to be impaired(49). By phosphorylating specific domains of the

sarcomeric protein titin(27), PKG-1 may improve diastolic properties(5). Indeed,

we found a trend of increased PKG-1 activity in PH, but not in PAB, indicating a

potential protective mechanism against diastolic dysfunction. As the RV might

be particularly vulnerable to diastolic dysfunction(10), these findings identify

new targets for further study on causative mechanisms of RV failure. These

differences in PKG-1 activity suggest that adaptation mechanisms to proximal vs.

peripheral pressure overload are different. PKG-1 activity also differs in distinct

forms of LV disease(49), but its exact role in proximal vs. peripheral LV pressure

overload remains to be elucidated. In general both common and distinct adaptive

mechanisms and differences in vulnerability for diastolic dysfunction have been

suggested in models of LV peripheral pressure load (systemic hypertension) vs.


proximal pressure load (aortic banding) (reviewed in (14, 22). However, as was

the case for the RV, direct comparisons are lacking. Therefore, when comparing

the various studies, it is difficult to rule out confounding effects of time-course,

model inductors (e.g. streptozotocin(1, 16)) or inherent differences in genetic

make-up in different strains (e.g. in spontaneously hypertensive rats(31, 45, 53)).

In PH+shunt no diastolic dysfunction could be demonstrated despite apparent

clinical signs in the majority of animals, including enlarged right atrium, pleural

effusion and ascites. Diastolic dysfunction might have been masked by the

significantly lower heart rates found in this group.

Voluntary exercise in models of abnormal RV loading

Although experimental data cannot be directly translated to clinical patients, the

use of clinically relevant parameters in animal models may be of value. In this

study, voluntary exercise measurement was used because of its analogy with

the 6-minute walk test, a sub maximal exercise test clinically used in PH-patients

to assess functional capacity and efficacy of treatment (43). Although the

optimal definition of clinical heart- or RV-failure is still debated(20), parameters

of functional capacity are usually involved. In general, studies on experimental

RV-adaptation to abnormal loading and RV failure have not provided such

data. We propose exercise data are a valuable adjunct to the interpretation

and clinical relevance of ventricular functional parameters usually measured

in rest conditions. This study demonstrates that different RV response patterns

associated with distinct types of overload were indeed reflected in differential

decreases in voluntary exercise.

Limitations

The use of anesthesia may affect cardiac function, but is inevitable in animal

studies using pressure-volume measurements. Since we used isoflurane, shown

to have only mild negative effects on inotropy and cardiac index in rodents

(23) and since the groups compared were all subjected to identical regimens

of anesthesia, we contend that this did not limit the answers to our research

questions.

We calibrated for slope factor α using echo-measured cardiac output, which

is performed with closed chest, while the catheterization is performed with

open chest. This might have led to a consistent overestimation of the cardiac

Chapter 248

output and, thus, of ventricular volumes. However, the observed ventricular

volumes in this study are in line with previous reports(21, 39). Moreover, the

concomitant use of relative increases in volumes (which is not affected by this

overestimation), discarded this potential limitation.

Assessment of the temporal development of RV responses was not feasible

in our set-up, but would provide additional valuable data and is a worthwhile

objective for future studies.

When studying pathophysiological mechanisms in an animal model, ideally

the model should be representative for the human disease that it mimics(12).

This means that for the pressure and/or volume-loaded RV it ideally should

be characterized by 1) triggers recognized in human disease (e.g. altered

pulmonary blood flow) and 2) a clinical course of progressive RV dysfunction

leading to death. We therefore used pressure load (through PAB) and volume

load (through aortocaval shunt) as clinically recognized inducers of progressive

RV failure. In volume load, we used an additional trigger (monocrotaline) to

induce progressive pulmonary vascular neonitimal lesions to further mimic

the human conditions seen in pulmonary arterial hypertension associated with

congenital heart disease(13). It should be noted that monocrotaline has been

suggested to have direct effects on the RV. Such effects might have contributed

to the observed RV responses to monocrotaline-induced peripheral pressure

overload. Further, the aorto-caval shunt results in biventricular volume overload.

Some of the clinical parameters measured in shunt and PH+shunt could be

influenced LV volume load, but it has been shown that at 4 weeks rats with an

aorto-caval shunt have normal LV function (51). Confirmation of the described

results in other animal models of RV pressure overload, e.g. hypoxia (whether

or not combined with the vascular endothelial growth factor receptor blocker

Sugen 5416) or fawn-hooded rats, may indicate to what extent the conclusions

from this study could be generalized, although these models represent less

recognizable clinical triggers for human RV failure.

As in all preclinical studies, findings in rodent models must not be over-

interpreted and used with caution when translating such findings to human

patients.


ACKNOWLEDGE-MENTS

GRANTS

CONCLUSIONThis study sheds new light on the RV response to different types of abnormal

loading. Firstly, proximal type pressure load (PAB) induced more diastolic

dysfunction than peripheral type pressure load (PH). Secondly, the combination

of PH and volume load resulted in additional RV hypertrophy and a pattern

of pseudo-normalization with blunted contractility response. The distinct

responses imply that experimental studies in various types of RV failure should

use models that match the loading condition of the studied disease. Moreover,

it may be of clinical relevance in the therapeutic approach of patients with

different types of RV loading.

The authors would like to thank A. Smit-van Oosten, M. Weij and A. Zandvoort for

performing the surgeries; J. Takens and B. Boersma for expert technical assistance

and E.R. van den Heuvel for statistical advice. This study was supported by the

Sebald foundation and a grant from the Dutch Heart Foundation (2007T068)

The Netherlands Heart Foundation [grant#: 2007T068]; the Sebald fund

Chapter 250

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DIFFERENTIALRE SPONSESOFTHERI GHTVENTRICLET OABNORMALLOA DINGCONDITION SINMICE:PRESSU REVS.VOLUMELO AD

B Bartelds, MAJ Borgdorff, A Smit-van Oosten, J Takens, B Boersma,

MG Nederhoff, NJ Elzenga, WH van Gilst, LJ De Windt, RMF Berger

Eur J Heart Fail. 2011 Dec;13(12):1275-82.

doi: 10.1093/eurjhf/hfr134. Epub 2011 Oct 24.

3

Chapter 356

ABSTRACTAims

Right ventricular (RV) dysfunction is a major determinant of long-term morbidity

and mortality in congenital heart disease. The right ventricle (RV) is genetically

different from the left ventricle (LV), but it is unknown as to whether this has

consequences for the cellular responses to abnormal loading conditions. In the

LV, calcineurinactivation is a major determinant of pathological hypertrophy and

an important target for therapeutic strategies. We studied the functional and

molecular adaptation of the RV in mouse models of pressure and volume load,

focusing on calcineurin-activation.

Methods and results

Mice were subjected to pulmonary artery banding (PAB), aorto-caval shunt

(Shunt), or sham surgery (Control). Four weeks later, mice were functionally

evaluated with cardiac magnetic resonance imaging, pressure measurements,

and voluntary cage wheel exercise. Right ventricular hypertrophy and calcineurin-

activation were assessed after sacrifice. Mice with increased pressure load (PAB)

or volume load (Shunt) of the RV developed similar degrees of hypertrophy,

yet revealed different functional and molecular adaptation. Pulmonary artery

banding increased expression of Modulatory-Calcineurin-Interacting-Protein 1

(MCIP1), indicating calcineurin-activation, and the ratio of beta/alpha-Myosin

Heavy Chain (MHC). In addition, PAB reduced exercise capacity and induced

moderate RV dilatation with normal RV output at rest. In contrast, Shunt did

not increase MCIP1 expression, and only moderately increased beta/alpha-

MHC ratio. Shunt did not affect exercise capacity, but increased RV volumes and

output at rest.

Conclusions

Pressure and volume load induced different functional and molecular

adaptations in the RV. These results may have important consequences for

therapeutic strategies to prevent RV failure in the growing population of adults

with congenital heart disease.

Differential RV responses in mice: pressure vs. volume load 57

INTRODUCTIONCongenital heart disease has been treated with increasing success rates in

recent years. However, long-term outcome in congenital heart disease is

characterized by increasing rates of morbidity and mortality (1 – 3). Right

ventricular (RV) dysfunction has been shown to be an important determinant

of long-term outcome (2). The RV is frequently subjected to longstanding

abnormal loading conditions as pressure or volume load in congenital heart

disease. Examples of these abnormal loading conditions include: residual lesions

after repair of tetralogy of Fallot (e.g. pulmonary stenosis and/or pulmonary

insufficiency) or the RV as the systemic ventricle in congenitally corrected

transposition or in univentricular circulation such as in hypoplastic left heart

syndrome. Moreover,RV dysfunction predicts mortality in chronic heart failure

(4). The mechanisms of RV dysfunction and RV failure are as yet unknown. In

fact, the mechanisms of RV adaptation to abnormal loading conditions are

poorly understood and have not been extensively studied to date (5). In adults

without congenital heart disease, left ventricular (LV) dysfunction is the most

prominent cardiovascular disease and hence LV responses to abnormal loading

conditions have been extensively studied. In the LV, calcineurin-activation is a

key activator of pathological hypertrophy (6), and interference with calcineurin-

activation has been shown to reduce LV hypertrophy and improve outcome

(7). However, knowledge obtained from studies on LV adaptation cannot be

directly transferred to the RV, since the RV is morphologically (8) and genetically

(9) different from the LV. The RV myocardium is derived from a different set of

precursor cells, the so-called anterior heart field (10). Whether differences in

genetic make-up lead to different responses to common stressors is a matter

of debate (11). Moreover, the RV is functionally different as it is coupled to the

low-resistance pulmonary vasculature (12). Differences in the genetic make-

up, morphology, and functional environment suggest that the RV response

to abnormal loading conditions may differ from that of the LV. In order to

understand RV adaptation, models of RV abnormal loading conditions have been

developed. So far, models of pressure load in the rat and larger animals prevail

(13,14). However, genetically engineered animals will facilitate the study of the

molecular pathways involved in the induction of compensatory RV hypertrophy

as well as in the transition to RV failure. Hence, the need for mouse models of

RV abnormal loading conditions is emerging. In these mouse models molecular

Chapter 358

METHODS

pathways can be coupled to clinically relevant functional adaptation, such as

exercise capacity, haemodynamics, and cardiac magnetic resonance imaging

(MRI). In this study, we compared the functional and molecular adaptation to

abnormal loading conditions of the RV in two mouse models relevant for clinical

lesions seen in congenital heart disease, i.e. increased pressure or volume load.

C57Bl6 mice, purchased from Harlan (Harlan, The Netherlands) were used.


Experimental Act. The investigation conformed to the Guide for Care and Use

of Laboratory Animals published by the US National Institutes of Health (NIH

publication No. 85-23, revised 1996).

Surgery

Mice weighing 18–20 g underwent surgery as described in the online

supplement. In short:

Pulmonary artery banding

Following left lateral thoracotomy a 7-0 suture was placed around the pulmonary

artery and tied over a 23G needle bent into an L-shape (15). The L-shaped

needle was then removed and the thorax and skin were closed in layers. Mice

undergoing sham thoracotomy served as controls (Control).

Aorto-caval shunt (Shunt)

Following mid-line laparotomy, a puncture was made with a 25G needle through

the abdominal aorta towards the inferior caval vein while compressing the

distal vein (16,17). The puncture site in the aorta was closed with tissue glue

(16,17). The abdomen was closed in layers. Mice undergoing sham laparotomy

served as controls. We evaluated 10 mice for pulmonary artery banding (PAB),

12 for Shunt, and 10 sham-operated mice for PAB and 12 sham-operated mice

for Shunt. Since there were no differences between the two groups of sham-

operated animals we grouped these together and referred to them as Control.


Voluntary cage wheel exercise

Exercise capacity was tested with voluntary cage wheel exercise (18) before and

3 weeks after surgery in eight Control mice, five Shunt mice, and four PAB mice

as described in the online supplementary methods.

Cardiac magnetic resonance imaging

Cardiac MRI was performed 4 weeks after surgery using a 9.4 T magnet (Bruker,

Mouse MRI Facility, Interuniversity Cardiology Institute of the Netherlands). We

performed cardiac MRI in five Control mice, six Shunt mice, and six PAB mice.

These mice were not used for exercise studies. A detailed description of the

MRI procedure is given in the online supplementary methods. The acquired

cine MRI data were analysed with Qmass digital imaging software for rodents

(Medis, Leiden, The Netherlands). Right ventricular end-diastolic volume (EDV)

and end-systolic volume (ESV) as well as LV EDV and ESV were measured. From

these volumes, stroke volume (SV), ejection fraction (EF), and RV output were

calculated. Stroke work was calculated as [(RV systolic pressure–RV diastolic

pressure)×SV] and wall stress as (RV systolic pressure×RVEDV)/RV ED mass. The

combined data sets of the pressure measurements and MRI measurements

were used to create virtual pressure–volume loops.

Haemodynamics

Right ventricular pressures were measured invasively under anaesthesia in three

Control, three Shunt, and three PAB mice 4 weeks after surgery with a fluid-filled

polyurethane catheter (outer diameter 1.6, inner diameter 0.3), inserted via the

jugular vein.

Tissue analysis

After the last study, the mice were sacrificed under anaesthesia (with

2–3% isoflurane and oxygen). The right ventricular free wall (RV free wall),

interventricular septum (S), and left ventricular free wall (LV free wall) were

separated, weighed, and stored at 2808C for further analysis. Gene and protein

expression were determined as described in detail in the online supplementary

methods.

Chapter 360

RESULTS

Statistics

Data are presented as mean+standard error of the mean (SEM). Differences

between groups were detected with one-way analysis of variance and post-

hoc least significant difference test (with the use of SPSS 16.0 software). A

P-value <0.05 was considered significant. Sham-operated mice for the PAB and

Shunt procedures were equal and pooled and served as controls for all surgical

procedures.

Functional adaptation

Pressure load, via PAB, induced an increase in peak-systolic RV pressure,

whereas volume load, via Shunt, did not affect RV pressures (Table 1, Figure 1A).

Three weeks after surgery, PAB mice spent significantly less time in the wheel as

compared with Shunt or Control mice (Figure 1B). At baseline, before surgery,

there were no significant differences in time spent in the wheel between PAB,

Shunt, and Control mice. Wheel speed did not change in the different groups

between baseline and 3 weeks after surgery, and so the time in the wheel was

directly correlated with covered distance. Mice with a pressure-loaded RV (PAB)

had mild RV dilatation as shown by increased end-diastolic and end-systolic

RV volumes (Figure 1C), whereas RV SV, output and EF were preserved when

compared with Control mice (Table 1). Mice with a volume load (Shunt) had

severe RV dilatation (Figure 1C), with increased RV SV and output (Table 1).

Shunt mice had a decreased RV EF (Table 1). In PAB mice, LV volumes and LV

output did not change. In Shunt mice, LV EDV and ESV were increased (Table 1).

There were no significant differences in heart rate during MRI studies between

the three groups (Table 1). Examples of cine loops are presented in the online

data supplement. Right ventricular mass was significantly increased in mice

with a PAB as well as in those with a Shunt (Table 1), which was confirmed at

autopsy. Pressure loading imposed a greater increase in energy demand on the

RV than volume load, since RV stroke work was more increased in PAB mice than

in Shunt mice (Figure 2A). Also, RV wall stress was only significantly increased

in PAB mice (Figure 2B). Virtual pressure-volume loops derived from MRI and

pressure measurements, showed a rightward and upward shift in the PAB mice

but only a rightward shift in the Shunt mice (Figure 2C).

RESULTS


Figure 1 Functional adaptation to pressure or volume load of the right ventricle. (A) Typical examples of right ventricular pressure curves in a mouse without (left, Control) and with a PAB (right). Pressure is recorded in mmHg. (B) Voluntary cage wheel exercise in mice with PAB or Shunt time spent in the wheel before surgery (pre) and 3 weeks after surgery (post) in Control (n=8), PAB (n=5), and Shunt mice (n=5). *,§P< 0.05 vs. Control and vs. Shunt. (C) Magnetic resonance imaging-derived right ventricular volumes: right ventricular end-diastolic (left) and end-systolic (right) volumes in microlitres. Typical short axis frames at the level of the papillary muscles from cardiac magnetic resonance imaging in the three groups are shown at the top. N=5 for Control, N=6 for PAB, and N=6 for Shunt. *P <0.05 vs. Control, †P<0.05 vs. PAB, # t-test Control vs. PAB P= 0.026, and 0.013 for right ventricular end-diastolic and right ventricular end-systolic volume, respectively. PAB, pulmonary artery banding.

Chapter 362

Figure 2 Haemodynamics. Calculated haemodynamics showed a higher increase in energy demand in the pressure-loaded right ventricle than in the volume-loaded right ventricle. (A) Stroke work was increased, more in PAB than in Shunt mice. (B)Wall stress was only increased in PAB mice. (C) Virtual pressure–volume loops derived from magnetic resonance imaging volumes (see methods). N=5 for Control, N=6 for PAB, and N=6 for Shunt. *P<0.05 vs. Control, †P<0.05 vs. PAB, §P<0.05 vs. Shunt. PAB, pulmonary artery banding.

RESULTS


Cardiac remodeling

Both, PAB and Shunt induced similar degrees of RV hypertrophy: RV free wall

weight was increased absolutely and relative to body weight (Figure 3A, Table

2). Shunt mice also showed increased septal weight, whereas PAB mice showed

no change in septal weights (Table 2). Left ventricular-weight was not affected

by PAB, whereas Shunt mice showed also increased LV weights (Table 2). Typical

examples of Gomori-stained sections illustrate the RV hypertrophy in mice with

a PAB or Shunt (Figure 3B). There was no significant increase in the expression

of markers of cardiac fibrosis, i.e. collagen type 1 or 3 (Col1A2, Col3A1 (19),

Table 2). Despite the similar increase in RV hypertrophy in the pressure and

volume-loaded RVs, there were marked differences in molecular responses.

Pulmonary artery banding induced a strong increase in typical markers of cardiac

hypertrophy (Figure 3C), as shown by the increase in expression of natriuretic

peptide A (NPPA), alpha 1 skeletal muscle actin (Acta1), and the decrease in

alpha-Myosin Heavy Chain (MHC, myh6). In contrast, Shunt mice only showed

moderate changes in expression of these markers. The RV hypertrophy in the

PAB mice was accompanied by a significant increase in expression of Modulatory

Control PAB Shunt

pRV systolic (mmHg) 17+2 37+9* 14+1Cardiac MRI

Body weight (g) 22.7+3.2 24.9+1.7 26.4+1.8RV hypertrophy (mg/g) 0.94+0.17 1.48+0.23* 1.56+0.28*Heart rate (b.p.m.) 404+40 376+49 446+58†

RVSV (mL) 20+3 24+5 43+10*,†Output (mL/min) 8.1+1.0 8.9+0.7 20.0+6.2*,†Mass (mg) 34+5 61+9* 62+12*EF (%) 68+4 62+6 58+9*

LVEDV (mL) 45+9 45+7 70+14*,†ESV (mL) 19+5 20+8 29+7*,†SV (mL) 26+5 26+4 41+9*,†Output (mL/min) 10.2+2.2 9.4+0.7 19.4+6.7*,†Mass (mg) 69+8 60+5 98+19*,†EF (%) 57+6 58+12 59+7

Table 1 Haemodynamic and magnetic resonance imaging data. RV, right ventricle; LV, left ventricle; RV hypertrophy was calculated as the ratio of RV free wall weight and RV body weight; EDV, end-diastolic volume; ESV, end-systolic volume; SV, stroke volume; EF, ejection fraction. N = 5 for Control, 6 for Shunt, and 6 for PAB for MRI data; for RV pressures, n = 3 in each group. *P <0.05 vs. Control, †P <0.05 vs. PAB.

Chapter 364

Calcineurin Interacting Protein-1 (MCIP1), indicating calcineurin-activation,

whereas MCIP1 was not significantly elevated in Shunt mice (Figure 4A). In the

left ventricles of the PAB mice and Shunt mice there was no significant change

in MCIP1 activation (Figure 4A). Pulmonary artery banding also induced a

significant increase in beta-MHC (myh 7, Figure 4B) and a switch in beta/alpha-

MHC ratio (Table 2). In the left ventricle, myh7 was also significantly increased

after PAB (Figure 4B). In contrast, in the Shunt mice neitherbeta-MHC expression

(Figure 4B) nor beta/alpha-MHC ratio (Table 2) were significantly increased in

the right ventricle. The amount of phosphorylated extracellular regulated kinase

(ERK1/2), recently described as a switch to induce eccentric vs. concentric

hypertrophy (20), was decreased in the RV and LV of Shunt mice, in line with the

response to increased pre-load. In the PAB mice, the amount of phosphorylated

ERK1/2 was also decreased, possibly associated with the mild RV dilatation. The

amount of phosphorylated Akt, a signalling pathway in physiological forms of

hypertrophy (21), was not significantly changed in the RV of mice with a PAB or

Shunt, but tended to decrease in the LV of Shunt mice.

Figure 3 Morphological and molecular analysis of the right ventricle. (A) Right ventricular hypertrophy, expressed as right ventricular free wall (mg)/bodyweight (g) showed a similar increase in PAB and Shunt mice. N=22 for Control, 12 for Shunt, and 10 for PAB. *P<0.05 vs. Control. (B) Left: typical example of Gomori-stained sections from right ventricular and left ventricular sections were visualized at ×40 magnification. The scale bar represents 75 micrometers. Right: the cross-sectional area of the right ventricular myocytes. (C) Real-time polymerase chain reaction analysis for hypertrophic markers in the right ventricle. Cyclophilin was used as reference gene, expression in the control group was set to 1. N=4 for each group. *P<0.05 vs. Control, §P<0.05 vs. Shunt, 1 P<0.06 vs. Control. PAB, pulmonary artery banding.


Control PAB Shunt

N 22 10 12Body weight (g) 23.6+2.6 24.8+1.4 25.3+2.1Heart weight (mg) 107+11 131+11* 169+29*,†RV (mg) 20+5 40+5* 37+9*Septum (mg) 20+7 22+5 35+14*,†LV (mg) 66+8 70+10 97+15*,†RV/(LV + S) 0.24+0.06 0.44+0.07* 0.28+0.04S/BW 0.91+0.32 0.87+0.22 1.49+0.44*,†HW/BW 4.59+0.51 5.30+0.49* 6.65+0.89*,†RV gene expressionBeta/alpha-MHC 1.0+0.1 19.0+4.9* 4.6+2.0Col1A2 1.0+0.1 1.6+0.3 1.5+0.5Col3A1 1.0+0.1 1.5+0.3 0.7+0.2

Table 2 Cardiac weights and right ventricular gene expression. S, interventricular septum; BW, body weight; H, heart weight, which is the sum of the weights of RV free wall, interventricular septum, and LV free wall. Gene expression levels in the RV free wall were expressed as fold-increase vs. control mice. *P<0.05 vs. Control, †P<0.05 vs. PAB.

Figure 4 Calcineurin-activation and hypertrophy signalling. (A) Gene expression of Modulatory Calcineurin-Interacting-Protein 1, a marker of calcineurin-activation, increased in the right ventricular free wall (RV) with pressure load (PAB), but not significantly with volume load (RV). LV= left ventricular free wall (LV), cyclophilin was used as reference gene. (B) Gene expression of beta-myosin heavy chain (myh7) increased in the right ventricle and left ventricle of PAB mice compared with cyclophilin as reference gene. (C) Protein levels of phosphorylated/total extra-cellular-regulated kinase decreased in the right ventricle and left ventricle. (D) Protein levels of phosphorylated/total Akt did not change in the right ventricle and decreased in the volume-loaded left ventricle. The data of the Control mice were set to 1. N=4 in all groups. *P<0.05 vs. Control mice, §P<0.05 vs. Shunt, 1 P= 0.06 vs. right ventricle. PAB, pulmonary artery banding.

Chapter 366

In this study, we have demonstrated that mice with increased pressure or

volume load of the RV develop similar degrees of RV hypertrophy but show

different patterns of functional and molecular adaptation. Mice with a pressure-

loaded RV (PAB) had reduced exercise capacity with moderately increased RV

volumes, whereas SV was unchanged suggesting a harbinger of decompensating

RV hypertrophy, anticipating clinical failure. The functional adaptation of the

RV was coupled with an increase in MCIP1 expression, indicating calcineurin-

activation, and a switch in MHC isoform ratio. In contrast, mice with a

volume-loaded RV (Shunt) had normal exercise capacity, albeit with higher RV

volumes and RV SVs at rest, indicating a pattern of adequately compensated

RV hypertrophy. Moreover, in the RV of Shunt mice there was no significant

MCIP-activation and only a mild change in MHC isoform ratio. The pressure-

loaded mice already worked at higher RV volumes measured with cardiac MRI,

as was illustrated in the virtual pressure–volume loops. Previous studies with

pulmonary banding in different species showed models in which RV volumes

(assessed with conductance catheters or angiography) either did not change

(14,22) or increased similarly (23,24). However, none of these studies evaluated

functional outcomes. From our studies it may be suggested that the pressure-

loaded RV uses heterometric adaptation (Starling mechanism) to adapt and that

given the reduced exercise tolerance this adaptation fell short in these mice.

In contrast, in the volume-loaded mice, we showed a dramatic increase in RV

volumes with normal functional adaptation. This study showed a difference in

functional adaptation to a volume vs. pressure load that also led to differences

in ‘clinical’ outcome as assessed by exercise. Although nowadays it is recognized

that longstanding volume load for the RV, such as pulmonary insufficiency after

Fallot repair (1) leads to RV failure, pressure load appears to be more harmful

as it leads to RV failure in a shorter time frame, through unknown mechanisms.

It could be due to the higher RV stroke work with pressure load (Figure 2),

leading to a higher metabolic demand and thereby changing mitochondrial

potential which affects stress signaling (25). Another possibility is that the

fixed afterload of the PAB mice made them more dependent upon heart rate

changes for the response to increased demands, as was recently also shown

in patients with RV failure due to pulmonary hypertension (26). The type of

loading may also induce different molecular signalling patterns, which affect

DISCUSSION


the RV response. Indeed, we showed a remarkable difference in calcineurin-

activation, a known pathway of pathological hypertrophy in the left ventricle

(6). This difference was accompanied by a differential response in beta-MHC

expression, part of a generalized hypertrophic response pattern to stress (27),

between volume and pressure load of the RV in mice. Since the calcineurin

pathway has been successfully targeted in experimental and clinical leftsided

heart failure (28), these results suggest new targets for therapies to improve

the pressure-loaded RV. For the volume-loaded RV, it is not yet known what

mechanisms contribute to RV adaptation. Recently, it was suggested that in the

LV with a volume load, early activation of the Akt-pathway may be responsible

for a more physiological form of cell growth (21), although the Aktpathway has

also been implicated in pathological hypertrophy in other studies (29). After 4

weeks, we did not observe activation of the Akt-pathway anymore, which may

be a time-related phenomenon. The eccentric hypertrophy classically described

in volumeloaded hearts was recently suggested to be induced by de-activation

of the ERK1/2 pathway (20). Our study confirms these results in the RV and

LV of volume-loaded mice. Interestingly, in our mildly dilating pressure-loaded

mice, we also observed a decrease in phosphorylated ERK1/2. This may be a

secondary effect after the initial pressure response, possibly due to activation

of dual-specificity phosphatase (6,30) a known inhibitor of ERK-activation. De-

activation of ERK could be a first sign of decompensation in the pressure-loaded

mice (30), and if so, prevention of ERK deactivation may be a strategy to prevent

the pressureloaded RV from dilation.

In this study, we showed a marked difference in cardiac remodeling between

pressure and volume load in the RV. So far, treatment modalities for both the

pressure-loaded RV as well as the volume-loaded RV are lacking. In studies

in pressure-loaded LVs (31), similar response patterns were seen as in the

pressureloaded RVs with respect to calcineurin-activation, suggesting that

despite the difference in genetic make-up between the right and left ventricle

(9), the building blocks of the ventricle may be the same (11). Hence, it may

be inferred that strategies aimed at reducing calcineurin-activation may have a

similar beneficial effect on the pressure-loaded RV as the pressure-loaded LV. In

the volumeloaded RV, however, there appeared to be differences in activation

of Akt and myh7 at 4 weeks, although the pathophysiological significance of

Chapter 368

these differences is as yet unclear. More studies into the cellular response of

the RV to volume load are needed to clarify these issues and develop treatment

strategies. A limitation of this study is that the mice were evaluated at one time-

point in the process of RV adaptation. Coupling of these functional outcomes

to molecular signals at serial time-points after induction of the abnormal

loading conditions may provide further understanding of these mechanisms of

adaptation and dysfunction.

In conclusion, in this study we functionally characterized two mouse models of

abnormal loading conditions of the right ventricle, that is increased pressure

load and increased volume load. We demonstrated that mice with increased

pressure and volume load of the RV developed similar degrees of RV hypertrophy,

but showed differences in functional and molecular adaptation. Pressure load

showed a pattern of adaptation suggesting a harbinger of decompensating

RV hypertrophy accompanied by calcineurin-activation, whereas volume load

induced a pattern of adequately compensated RV hypertrophy and no signs of

calcineurin-activation. These findings may have important consequences for

developing strategies to prevent RV failure in the abnormally loaded RV.

This work was supported by the Netherlands Heart Foundation (NHS2006T038)

and the Sebald Foundation.

FINANCIALSUPPORT


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16 Mori T, Chen YF, Feng JA, Hayashi T, Oparil S, Perry GJ. Volume overload results in exaggerated cardiac hypertrophy in the atrial natriuretic peptide knockout mouse. Cardiovasc Res 2004;61:771–779.

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Chapter 372

SUPPLEMENTARY METHODS Surgery

Mice were operated upon weighing 18-20 g, approximately 6-8 weeks of age.

Mice were anesthetized in a closed box filled with oxygen and 2-3% isoflurane.

Pulmonary artery banding (PAB): The thorax was shaved and the mice were

placed on a heating pad at a temperature of 38˚C. Next, the mice were intubated

with a 20G needle and mechanically ventilated with a Harvard miniventilator

(model 687, HugoSacks, Germany) at a rate of 180 breaths per min and a tidal

volume of 125 µl. A left lateral thoracotomy was performed through the 2nd

intercostal space. The pulmonary artery was exposed and a 7-0 suture was

placed around the pulmonary artery and tied over a 23G needle bent in an

L-shape(1). Thereupon, the L-shaped needle was removed. Next, the thorax and

skin were closed in layers. Mice receiving a sham thoracotomy served as controls

(Control). In order to determine the adequate size of PAB, we tested different

size of needles (21-25G). In our hands, a 23G needle produced a reproducible

degree of RV hypertrophy with an acceptable mortality rate (29%, 4 out of 14

mice), whereas 25G needle induced a similar amount of RV hypertrophy at the

cost of a higher surgical mortality. Therefore, we have chosen to characterize the

PAB induced with a 23G needle.

Aorto-caval Shunt (Shunt): the mice were ventilated using a mask. A midline

laparotomy was performed; the intestines were exteriorized and wrapped in

wet gauze. The abdominal aorta and inferior caval vein were placed in vessel

loops. With a 25G needle, bent in an L-shape, a puncture was made through the

abdominal aorta towards the inferior caval vein while compressing the distal

vein (2, 3). The needle was withdrawn and the puncture site in the aorta was

closed with tissue glue(2, 3). The intestines were replaced and the abdomen

was closed in layers. Mice receiving a sham laparotomy served as controls.

Postoperatively, all mice received buprenorphine s.c. every 12 hours for 2 days.

The mortality rate for the Shunt-mice was 86% (2 out of 14 mice).

We evaluated 10 mice for pulmonary artery PAB (PAB), 12 for Aorto-caval

Shunt (Shunt) and 10 Sham operated mice for PAB and 12 sham-operated mice

for Shunt. Since there were no differences between the two groups of sham-

operated animals we grouped these together and referred to them as Control.


Cardiac MRI

Cardiac MRI was performed 4 weeks after surgery by magnetic cardiac imaging

(9.4T Bruker, Mouse MRI facility, Interuniversity Cardiology Institute of the

Netherlands). We performed cardiac MRI in 5 Control mice, 6 Shunt mice,

and 6 PAB mice. These mice were not used for exercise studies. Mice were

anesthetized with oxygen and 2-3% isoflurane and placed on a pad recording

heart rate and respiration in a cone-shaped box connected to the ventilator. The

cone was placed in the magnet.

At first, the exact position of the heart was determined with a scout scan. This

scout scan yielded three orthogonal views through the thorax, in a transversal,

a coronal and a sagittal slice. The orientation of this scan was adjusted in a way

that perfect axial, two-chamber and four-chamber views of the left ventricle

were obtained. Then, the cine slices were positioned perpendicular to an

imaginary axis between the right outflow tract and the utmost apical part of

the RV. A stack of seven to eight 1 mm cine slices was measured to cover for the

whole top to base RV.

An ECG triggered fast gradient echo cine sequence (FLASH) was used with the

following parameters: repetition time 13 msec, echo time 1.967 msec, field

of view 3.0 cm2, acquisition matrix 256x256, and slice thickness 1.0 mm. At a

heart rate of ~450 bpm, the corresponding cardiac cycle length was 130-140

ms, allowing for approximately 10 frames per heart beat at a repetition time of

13 msec. The acquired cine MRI data were analyzed with Qmass digital imaging

software for rodents (Medis, Leiden, The Netherlands). RV end-diastolic volume

(EDV) and end-systolic volume (ESV) were measured. From these volumes,

stroke volume (SV), ejection fraction (EF) and RV output were calculated. In the

same orientation, left ventricular EDV and ESV were measured. After the final

experiments, all animals were sacrificed.

Hemodynamics

We measured RV pressures invasively under anesthesia in 3 Control, 3 Shunt,

and 3 PAB mice 4 weeks after surgery. The mice were anesthetized with 2-3%

isoflurane and oxygen and ventilated with a mask. Rv pressure was measured

with a fluid-filled polyurethane catheter (OD 1.6, ID 0.3), inserted via the jugular

vein.

Chapter 374

Tissue analysis

After the last study, the mice were sacrificed under anetshesia (with 2-3%

isoflurane and oxygen). At sacrifice, the mice were weighed and the heart was

excised. Right ventricular free wall (RV free wall), interventricular septum (S)

and left ventricular free wall (LV free wall) were separated, weighed and stored

at -80˚C for further analysis. RNA was isolated using Trizol reagent (Invitrogen)

from the RV free wall, LV free wall, and interventricular septum separately. cDNA

was synthesized using Moloney murine leukemia virus reverse transcriptase

and random primers (Invitrogen, Burlington, ON, Canada). Real-time PCR

experiments were performed on a CXF384 real time system C1000 Thermal

cycler (BioRad Laboratories, Veenendaal, The Netherlands) using SYBR Green

PCR Master Mix according to the manufacturer’s instructions (Eurogentec, San

Diego, CA). Primers sequences are available on request. Expression levels were

obtained from a dilution standard curve and compared with cyclophilin in order

to calculate the relative expression levels. Western blots were performed using

the following anitbodies: Akt (Cell Signaling, #4691), phosphorylated Akt (Cell

Signaling, #4060), ERK1/2 (Santa Cruz, sc-135900), and phosphorylated ERK

(Santa Cruz, sc-7383). Expression levels in the Control mice were set to 1.

Statistics

Data are presented as mean and standard deviation of the mean. Differences

between groups were detected with one-way ANOVA and post-hoc LSD test. A

p-value < 0.05 was considered significant. Sham-operated mice for the PAB and

Shunt surgeries were equal and pooled and served as controls for all surgeries.

1 Tarnavski O, McMullen JR, Schinke M, Nie Q, Kong S, Izumo S. Mouse

cardiac surgery: Comprehensive techniques for the generation of

mouse models of human diseases and their application for genomic

studies. Physiol Genomics 2004;16:349-360.

2 Mori T, Chen YF, Feng JA, Hayashi T, Oparil S, Perry GJ. Volume overload

results in exaggerated cardiac hypertrophy in the atrial natriuretic

peptide knockout mouse. Cardiovasc Res 2004;61:771-779.

3 van Albada ME, Schoemaker RG, Kemna MS, Cromme-Dijkhuis AH, van

Veghel R, Berger RM. The role of increased pulmonary blood flow in

pulmonary arterial hypertension. Eur Respir J 2005;26:487-493.


SUPPLEMENTARY VIDEOS

4 Buitrago M, Lorenz K, Maass AH, Oberdorf-Maass S, Keller U,

Schmitteckert EM, Ivashchenko Y, Lohse MJ, Engelhardt S. The

transcriptional repressor nab1 is a specific regulator of pathological

cardiac hypertrophy. Nat Med 2005;11:837-844.

5 Konhilas JP, Maass AH, Luckey SW, Stauffer BL, Olson EN, Leinwand

LA. Sex modifies exercise and cardiac adaptation in mice. Am J Physiol

Heart Circ Physiol 2004;287:H2768-2776.

Supplementary cine-Magnetic Resonance videos available at http://eurjhf.

oxfordjournals.org/content/13/12/1275/suppl/DC1

CHARACTERIZATI ONOFRIGHTVENT RICULARFAILURE INCHRONICEXPE RIMENTALPRESS URELOAD

MAJ Borgdorff, B Bartelds, MG Dickinson, P Steendijk, M de Vroomen,

VW Bloks, AMC Koop, RMF Berger

Under review

4

Chapter 478

ABSTRACTBackground

Right ventricular failure (RVF) due to pressure load is a major cause of death in

congenital heart diseases and pulmonary hypertension. The mechanisms of RVF

are yet unknown. We used an experimental approach based upon clinical signs

of RVF to delineate functional and biological processes associated with RVF.

Methods and Results

Wistar rats were subjected to a pulmonary artery banding (PAB n=12) or sham

surgery (CON, n=7). After 52±5 days, 5/12 PAB rats developed clinical symptoms

of RVF (inactivity, ruffled fur, dyspnea, ascites) necessitating termination

(PAB+CF). We compared these to PAB rats without clinical symptoms of RVF

(PAB-). PAB resulted in reduced cardiac output, RV stroke volume, TAPSE, and

increased end diastolic pressure (all p<0.05 vs. CON) in all rats, but PAB+CF rats

were significantly more affected than PAB-, despite similar pressure load (p=ns

PAB- vs. PAB+CF). Pressure-volume analysis showed enhanced contractility

(end systolic elastance) in PAB- and PAB+CF, but diastolic function (end diastolic

elastance, end diastolic pressure) deteriorated especially in PAB + CF. In

PAB+CF capillary density was lower than in PAB-. Gene-array analysis revealed

downregulation of both fatty acid oxidation and carbohydrate metabolism in

PAB+CF.

Conclusion

In chronic PAB, half of the rats developed clinical symptoms of RVF, which were

associated with progressive deterioration of diastolic function, hypoxia-prone

myocardium, increased response to oxidative stress and suppressed myocardial

metabolism. This model represents clinical RVF and allows for unraveling of

mechanisms involved in the progression from RV adaptation to RV failure and

the effect of intervention on these mechanisms.

Characterization of RV failure in chronic pressure load 79

INTRODUCTIONRight ventricular failure (RVF) due to increased pressure load is a primary risk

factor for morbidity and mortality in patients with congenital heart diseases

as well as in patients with pulmonary hypertension (PH)1, 2. Moreover, RV

dysfunction has also been demonstrated to be an important prognostic

determinant in left ventricular failure3.

Unfortunately, the pathophysiology of RV failure is yet insufficiently understood4,

which precludes the development of RV specific treatments. Research into

these mechanisms is hampered by the lack of a clear definition of RV adaptation

versus RV failure and by the lack of a model reflecting clinical RVF. It is in this

perspective that a National Heart, Lung and Blood Institute working group on

cellular and molecular mechanisms of right heart failure stated that ‘researchers

must develop reliable, reproducible and relevant animal models of RV failure’5.

Since RV function is a critical prognostic determinant in PH2, RV dysfunction

has often been studied in animal models of PH, such as the monocrotaline

rat model6. Although these models have proven to be valuable, they have

two important disadvantages: direct therapeutic effects on the RV cannot be

distinguished from (afterload-reducing) effects on the pulmonary vasculature

and the used ‘hits’ necessary to induce PH may affect the RV7. The use of a

pulmonary artery banding (PAB) to inflict chronic pressure load on the RV

circumvents these limitations. However, it has been debated whether the

phenotype of the chronic PAB model represents compensated adaptation

instead of RV failure8-10, highlighting the need for a clear definition of RV failure.

Previously described PAB models showed features of chronic adaptation and

mild RV dysfunction, e.g. RV dilatation, reduced TAPSE and hypertrophy9-11, but

whether these represent the clinical phenotype of RV failure is unclear. The

clinical phenotype of RV failure consists of signs and symptoms as dyspnea at

rest, hepatomegaly, ascites, pleural effusion and mortality5, 12.

In the current study we aimed to characterize a phenotype of clinical RVF in rats

with a tighter PAB (1.1 mm) than described before9, 10, 13, 14, using clinical symptoms

in the rats (ABCDE-system), as reported previously6, 15. We further aimed to

relate the occurrence of this phenotype with hemodynamic, pathophysiologic

and molecular patterns of RV function, using echocardiography, pressure-

volume analysis and transcriptome-wide expression profiling in the RV.

Chapter 480

METHODSAnimal model and study design

Wistar rats (n=21; male; 160-180g; Charles River, the Netherlands) were

randomized into 2 groups: sham (CON, n=7) or pulmonary artery banding (PAB,

n=14, two of which died during surgery). PAB was performed through a left

lateral thoracotomy as described before14, however, with a tighter constriction

(1.1mm vs. 1.3mm before). Sham surgery was similar to the PAB surgery, with

the exception of the actual banding of the pulmonary artery.

From the moment of PAB/sham surgery onward, the animals were daily checked

for signs of clinical RV failure (see Definition of clinical RV failure)15. When a rat

was identified as developing clinical RV failure, echocardiography and pressure-

volume analysis was performed: these rats are the PAB+Clinical Failure (PAB+CF)

group (n=5). Four rats with a PAB that did not show signs of clinical RVF were

analyzed and terminated at the same follow-up duration (PAB minus Clinical

Failure: PAB- group, n=4). At 11 weeks the remaining rats (7 CON and 3 PAB) were

terminated. See Figure 1A for the experimental set-up. A detailed description of

the methods is provided in the online-only Data Supplement.

Definition of clinical RV failure

Rats were examined daily for clinical signs of RV failure according to a predefined

ABCDE-checklist6, 15, which includes Appearance, Activity, Bodyweight,

Circulation (peripheral), Cyanosis, Dyspnea/tachypnea and Edema, Effusions

(see online-only Data Supplement). Clinical RV failure was defined as presence

of at least: inactivity and ruffled fur and severe dyspnea and palpable ascites.

The presence of ascites and pleural effusion was in all cases confirmed after

termination.

Pressure-volume measurements, echocardiography, exercise

Functional analysis of the RV was performed before termination using invasive

pressure-volume measurements in anesthetized (isoflurane/air mixture, 5%

induction; 2-3% maintenance; analgesia with buprenorphine 0.01 mg/kg s.c.),

ventilated rats following thoracotomy using a conductance catheter as described

before15 and in the online-only Data supplement. Stroke volume as measured by

echocardiography (in mL) was used to calibrate the conductance-derived stroke

volume (in arbitrary units) derived from the conductance signal. Steady-state


pressure-volume loops were used for calculation of all parameters. End systolic

and end diastolic elastance were determined using the single-beat method.

Echocardiography was performed at 5 weeks and at termination as described

before15. Apical 3- and 4-chamber views and parasternal short- and long axis

views were used to measure ventricular and atrial dimensions and tricuspid

annular plane systolic excursion (TAPSE) and to assess tricuspid insufficiency. The

gradient across the PAB was measured using continuous wave Doppler. Cardiac

output was calculated as (aorta diameter)2 * 3.14 * velocity time integral * heart

rate, using systolic aorta diameter and pulsed wave Doppler measurements of

aorta flow. Beat-to-beat variation was accounted for by averaging measurements

from 6 to 12 consecutive beats.

Voluntary exercise was measured using running wheels mounted in the cages

for 5 consecutive days at baseline and after 5 weeks as described previously6, 14,

15 and in the online-only Data Supplement.

Histological analyses

Ventricular remodeling was studied using RV free wall tissue. We used using

standard histological techniques for measurement of RV cardiomyocyte

cross-sectional area, fibrosis, capillary density and macrophage infiltration, as

described in the online-only Data Supplement.

Gene expression

Changes in gene expression in the RV myocardium were investigated using

transcriptome-wide expression profiling and qRT-PCR for specific genes. Total

RNA was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA,

USA); high quality was confirmed (RQI 9.3) using Experion (Bio-Rad, Veenendaal,

the Netherlands). For the array-studies, RNA was purified for individual rats

(n=7/4/5 CON/PAB-/PAB+CF) using the Qiagen RNeasy mini kit (Venlo, The

Netherlands); RNA quality was verified (RIN >9) (Agilent, Amstelveen, the

Netherlands). Biotin-labeling, hybridization, washing, scanning of GeneChip

Rat Gene 1.1 ST arrays (Affymetrics) and processing in the MADMAX

pipeline (Nutrigenomics Consortium, Wageningen, The Netherlands)16 using

Bioconductor software packages were all performed according to standard

Affymetrix protocols at expert labs. Extensive quality control is described in the

online-only Data supplement. Array data are deposited at the Gene Expression

Omnibus (GEO) database (GSE46863).

Chapter 482

RESULTS

Significantly regulated genes were related to biological processes using DAVID17.

Gene set enrichment analysis18, which includes expression of all genes (not just

significantly regulated genes) was performed, allowing detection of significant

regulation of gene sets, even if the expression of individual genes is not

significantly different between groups.


Quantitative data are expressed as mean±standard error of the mean (SEM).

Testing of differences between CON, PAB- and PAB+CF was performed using

ANOVA with Bonferroni post hoc correction for multiple testing. The rats that

survived 11 weeks of PAB (n=3) were not included in the PAB- group so that the

time exposed to PAB was equal in the PAB- and PAB+CF groups and potential

confounding of the results by time-differences was avoided19. Retrospective

PAB- vs. PAB+CF comparisons at the 5 week time point were evaluated using

t-tests. P<0.05 was considered significant (PASW Statistics 20 for Windows,

SPSS, Chicago, Illinois). Statistical analysis of the transcriptome array is described

separately in the online-only Data Supplement.

After a mean period of 52±5 days, 42% of the rats developed clinical RVF (5/12,

Fig 1B). Tightness of PAB was similar in rats with or without signs of clinical RVF,

assessed by peak RV pressure that was equally increased in both groups at 5

weeks (echo-measured PAB gradient (Fig 1C) as well as at termination (invasively

measured RV pressure, Fig 1D).


Figure 1 A Schematic overview of experimental set-up. B Clinical symptoms of RV failure. Solid box = symptoms present. Open box = symptoms absent. Each row represents 1 rat. ABCDE refer to: A activity and appearance, B bodyweight, C cyanosis and/or hampered peripheral circulation, D dyspnea and/or tachypnea, E effusions: pleural or ascites (see supplement for details). C PAB gradient measured by echocardiography at 5 weeks after surgery. D Invasively measured RV peak pressure measured before termination. Mean±SEM. Arrows indicate p<0.05 between respective groups.

Rats with or without clinical signs of failure showed distinct functional,

morphological and pathological profiles

All rats with PAB showed functional and morphological RV-abnormalities,

but these were significantly more pronounced in PAB+CF than in PAB-. At

termination, PAB+CF rats had a lower cardiac index (Fig 2A), RV stroke volume

(Table S1) and TAPSE (Fig 2B). Furthermore, PAB+CF rats had a more enlarged

right atrium (Fig 2C) and pericardial effusion (3/5 of PAB+CF vs. 0/4 of PAB- rats;

Fig 2D). In PAB+CF, the right atrium was also more hypertrophic (Fig 2E) and

the liver wet/dry-weight ratio was increased signifying congestion (Fig 2F). In

line with this, postmortem examination revealed macroscopic liver congestion

(so-called ‘nutmeg liver’) and ascites in PAB+CF, but not in PAB- (representative

images in Fig 2G).

PAB consistently induced RV dilatation and tricuspid insufficiency, but no

differences were found between PAB- and PAB+CF (Table S1).

Chapter 484

Pulmonary artery banding resulted in enhanced systolic function, while the

occurrence of clinical symptoms is associated with worse diastolic function

Pressure-volume analysis revealed hemodynamic distinctions between PAB-

and PAB+CF (representative pressure-volume loops in Fig 3A-C). End systolic

elastance (a measure of contractility) showed a trend to be higher in PAB+CF

(Fig 3E). However, the performed stroke work in PAB+CF was significantly lower

than in PAB- (Fig 3F), reflecting decreased stroke volume despite enhanced end

systolic elastance.

Advanced diastolic dysfunction hallmarked PAB+CF compared to PAB-: both

end diastolic elastance (Fig 3C, p=0.06) and end diastolic pressure (Fig 3D) were

higher in PAB+CF. This was due to increased stiffness rather than to incomplete

active relaxation as tau did not differ between PAB- and PAB+CF (Table S1).

Figure 2 Echocardiographic and pathological confirmation of clinical RVF

PAB+CF was distinct from PAB- with regard to cardiac index (A), TAPSE (B), RA diameter (C), presence of pericardial effusion (example echo-image in D), RA weight (E) and liver wet-to-dry ratio (F). Representative images of liver congestion (left-hand panel in G) and ascites (right-hand panel in G). Mean±SEM. Arrows indicate p<0.05 between respective groups. TAPSE= tricuspid annular plane systolic excursion, RA= right atrium, TV= tricuspid valve


Figure 3 Pressure-volume analysis

Representative pressure-volume loops of CON, PAB- and PAB+CF (A-C), end systolic pressure volume relations indicated by solid lines, end diastolic pressure volume relations indicated by dashed lines. Indices of diastolic function; end diastolic elastance (C) and end diastolic pressure (D). End systolic elastance (E) and stroke work (F). Mean±SEM. Arrows indicate p<0.05 between respective groups. Eed= end diastolic elastance, Ped= end diastolic pressure, Ees = end systolic pressure

Association of fibrosis, hypertrophy and capillary growth to RVF with clinical

symptoms

Diastolic dysfunction has been associated with increased stiffness due to

interstitial fibrosis and/or hypertrophy. However, fibrosis was lower in PAB+CF

than in PAB- (Fig 4A,E). This was not accompanied by blunted signaling in the

pro-fibrotic pathways or attenuated expression of collagen-isoforms (Fig 4F).

Fibrosis has been suggested to result from deficient hemoxigenase-1 (HO-1)

activation against oxidative stress10, and in line with this we found that HO-1

was specifically upregulated in PAB+CF (Fig 4F). We could not demonstrate

differences in NOX-4 activation or macrophage infiltration between PAB- and

PAB+CF (data not shown).

The amount of RV hypertrophy was equal in PAB- and PAB+CF, whether expressed

as (normalized) RV weight or cardiomyocyte cross sectional surface area (Fig

Chapter 486

4B,C,E). Also mRNA expression of genes involved in hypertrophy (myosin heavy

chain-isoforms, regulator of calcineurin 1) and the natriuretic peptides type

A and B were increased equally in PAB- and PAB+CF (Fig 4F). However, mRNA

expression of the two isoforms of actin was different in PAB- and PAB+CF. The

predominant isoform (ACTC) was normal in PAB-, but downregulated in PAB+CF

(Fig 4F). The fetal isoform (ACTA) which is known to be upregulated in response

to stress was indeed upregulated in pressure load, but ~50% less in PAB+CF than

in PAB- (Fig 4F).

Insufficient capillary growth to supply the hypertrophic myocardium is suggested

to be a main cause of pressure-load induced heart failure10. In both groups

capillary density was decreased (Fig 4E), accompanied by a reduction in VEGF-A

and VEGF receptor type 2 expression (data not shown). However, in PAB+CF the

number of capillaries per cardiomyocyte was significantly less than in PAB- (Fig

4D).

Cardiac index and voluntary exercise predict the onset of RVF with clinical

symptoms

We performed echocardiography and voluntary exercise measurements at

35 days after PAB surgery, well before the onset of clinical RVF (52±5 days).

Retrospectively comparing PAB- and PAB+CF rats, we found that voluntarily

run distance, RV stroke volume, TAPSE and cardiac index were all significantly

lower at 5 weeks in the rats that later developed clinical RVF as compared with

those that did not develop symptoms of RVF (Fig 5A-D). In contrast, there were

no differences between PAB+CF and PAB- with regard to ventricular dilatation

and tricuspid insufficiency at 5 weeks (Table S1). While significantly different

between PAB+CF and PAB-, cardiac index (Fig. 5E) and TAPSE (Table S1) were

stable from 5 weeks onward, also in rats that developed clinical RVF.

The hemodynamic data of the PAB- group (which was prematurely terminated

concurrently with the PAB+CF group) and the PAB rats that survived 11 weeks

were similar (Table S2).


Figure 4 Histology and gene expression

RV fibrosis (A, E: two top rows are representative images of Masson-Trichrome stained RVs, ruler is 500μm, black box with 1mm). RV hypertrophy (RV free wall weight normalized for tibia length, B) and RV cardiomyocyte cross-sectional area (C, E third row are representative images of RV sections stained with a membrane marker (wheat germ agglutinin, green), ruler is 75μm. RV capillary density, expressed as capillary-to-cardiomyocyte ratio (D, E bottom row are representative images of RV sections stained with capillary-marker lectin, ruler is 130μm). mRNA expression of genes related to hypertrophy, fetal gene program, oxidative stress and fibrogenesis (CON =1, relative to 36B4 reference gene expression). Mean±SEM. Arrows indicate p<0.05 between respective groups. CCSA= cardiomyocyte cross-sectional area, cap= capillary, MYH7/6= β/α-myosin heavy chain, RCAN1= regulator of calcineurin 1, NPPA/B= natriuretic pro-peptides type A/B, ACTC= α-cardiac actin, ACTA= α-skeletal actin, HO-1= hemoxygenase-1, TGFβ-1= transforming growth factor- β-1, OPN-1= osteopontin-1, COL1A2/3A1= collagen subunits 1A2 and 3A1.

Chapter 488

Changes in gene expression following PAB

PAB induced significant changes in expression of >3000 genes. 1,437 of these

(Table S3) and 263 of the significantly regulated gene sets were present in

both PAB- and PAB+CF (Fig 6, S1). As expected, up regulation in the common

gene pattern predominantly involved cardiac/cellular growth, and multiple

interwoven and well known signaling pathways (MAPK-ERK1/2, PI3K-Akt-

NFkappaBeta-mTOR, Integrins, TGF-beta, endothelin etc.). Down regulation was

seen in specific gene sets related to metabolism, for example down regulation

of PPAR alpha signaling, intermediary enzymes in fatty acid metabolism, PGC1

alpha signaling, mitochondrial gene expression and oxidative phosphorylation

(Fig 6, S1).

Figure 5 Comparison of PAB- and PAB+CF at 5 weeks

PAB- and PAB+CF were significantly different at 5 weeks with regard to run distance (A, percentage change in run distance at 5 weeks vs. baseline), RV stroke volume (B), TAPSE (C) and cardiac index (D, in mL/min/g bodyweight). Cardiac index was stable in all groups at termination vs. 5 weeks (E). Mean±SEM. Arrows indicate p<0.05 between respective groups. PAB(11w)= PAB rats terminated at 11wks, TAPSE= tricuspid annular plane systolic excursion.


Figure 6 Significantly regulated gene sets in CON, PAB- and PAB+CF

Gene set enrichment analysis. Number of differentially enriched gene sets vs. CON in PAB-(blue) and PAB+CF (red) is indicated in the circles, separately for positive/negative enrichment (up regulation/down regulation). Commonly enriched gene sets in purple. Pie-charts show distribution of the gene sets in categories for PAB-, PAB+CF and COMMON, again separately for positive/negative enrichment. N=7/4/5 for CON/PAB-/PAB+CF. False discovery rate (FDR) <15%, nominal P value <0.05 and normalized enrichment score >1.3 was considered significant.

Changes in gene expression specifically associated with either PAB+CF or PAB-

1,666 genes and 104 gene sets were specifically regulated in PAB+CF, many of which

related to cellular (energy) metabolism (Fig 6, S1, Table S4). Most prominently,

there was a significant down regulation of glycolysis/gluconeogenesis related

gene sets in PAB+CF, in contrast to PAB- (Fig 7). In addition, while the changes in

fatty acid metabolism and beta-oxidation were comparable between PAB- and

PAB+CF, the down regulation of oxidative phosphorylation, the tricarboxylic acid

Chapter 490

cycle and amino acid metabolism appeared to be more pronounced in PAB-CF

than in PAB- (Fig 7).

In contrast to the PAB+CF group, only a few additional genes (215) and gene

sets (20) were specifically regulated in PAB- (Fig 6, S1). The 215 genes were not

significantly related to a specific biological process. Likewise, the found gene

sets were aspecific. No gene sets were regulated in opposite directions in PAB-

vs. PAB+CF.

Figure 7 Enrichment of gene sets associated with energy metabolism

Normalized enrichment scores (NES) of PAB- and PAB+CF vs. CON. N=7/4/5 for CON/PAB-/PAB+CF. * indicates false discovery rate (FDR) <15%, nominal P value <0.05 and normalized enrichment score >1.3 for respective gene set vs. CON.


DISCUSSIONIn this study, the rat model of chronic ‘tight’ pulmonary artery banding leads

to a morphological and functional RV response, characterized by hypertrophy,

dilatation, enhanced contractility and diastolic dysfunction. Clinical symptoms

of RVF occurred in half of the rats. These symptoms were associated with

progressive deterioration of diastolic function, a hypoxia-prone cellular

environment in the RV myocardium, increased intrinsic protective response to

oxidative stress and suppressed myocardial metabolism. The here described

model represents clinical RVF due to increased pressure load and can be used to

unravel the mechanisms involved in the progression from RV adaptation to RV

failure and to assess the effect of interventions on these mechanisms.

PAB as a relevant model of RVF

A clinical relevant model of RVF is invaluable for basic research and urgently

needed given the growing population of patients with a chronic pressure loaded

RV5, 20. PAB avoids the disadvantages of PH models, but divergent reports on

long-term preservation of RV function in PAB8, 11, 21 have led to debates whether

RV adaptation to PAB should be considered either compensated adaptation

or RV failure. Important aspects of the current study are the use of a tighter

PAB (19G: 1.1mm) than previous studies (18G-16G; 1.3-1.7mm)10, 13, 22 and the

use of clinical signs to define RVF. In this model of fixed afterload, clinical RVF

developed in up to 50% of rats within 11 weeks. Extensive evaluation of the

RV revealed both functional and morphological differences between the rats

with and those without clinical RVF. The findings suggest that the occurrence

of clinical RVF in the setting of pressure overload is associated with progressive

deterioration of diastolic function, less increase in ventricular fibrosis, decreased

capillary growth and altered ventricular energy metabolism. Interestingly, in

retrospect echocardiographic findings and voluntary exercise performance at 5

weeks (when no clinical signs of RVF were present yet), were predictive for the

occurrence of RVF at a later stage (Fig 5). This allows studying the effects of early

interventions that may be targeted at diastolic dysfunction or ventricular energy

metabolism.

Chapter 492

Clinical RVF is characterized by diastolic dysfunction with enhanced contractility

Deteriorating diastolic function appears to be part of the pathophysiology of RV

failure. From previous experimental studies, as well as from a recent study in

PAH patients23 it is known that chronic RV pressure overload induces diastolic

dysfunction14, 24. This study now adds the observation that the occurrence of

clinical RVF is associated with deteriorating diastolic function. Diastolic function

is the resultant of both active relaxation, which depends on Ca2+ re-uptake by

phospholamban-modulated SERCA2a and sodium-calcium exchanger NCX, and

passive chamber properties. Active relaxation of the RV is thought to be disturbed

in acute pressure overload25. In the current study however, active relaxation

(tau) was only mildly prolonged and did not further increase with clinical RVF. In

contrast, passive chamber properties (Eed, EDP) revealed progressive stiffening.

Interstitial fibrosis contributes to myocardial stiffening in heart failure. However,

we found interstitial fibrosis to be less increased in the PAB-rats with clinical RVF

and deteriorated diastolic dysfunction, as compared to those without clinical RVF

and less diastolic dysfunction. The pro-fibrotic signaling (TGFβ1, osteopontin)

appeared similarly activated in both groups, suggesting a higher degradation

of collagen in the PAB-rats with RVF. In other words, the increased myocardial

stiffening in PAB-rats with clinical RVF cannot be explained by increased

interstitial fibrosis. Stiffening of sarcomeres might be another explanation for

the deterioration of diastolic function, due to reduced phosphorylation of the

protein titin, the ‘molecular spring’ of the sarcomere, as recently suggested23.

Enhanced contractility in RVF might seem paradoxical, but it is a consistent

finding in the chronic pressure overloaded RV14, 24, 26 and in PAH patients23.

Therefore, the key to effective treatment of RVF may not be found in further

enhancing contractility, but rather in interventions preserving RV diastolic

function.

Hypertrophy, capillary formation, oxidative stress

Pathological hypertrophy, characterized by activation of e.g. the calcineurin-

NFAT signaling system27 was similarly present in both PAB-groups. However,

capillary-myocyte ratio was reduced in PAB-rats with clinical RVF. This finding is

in line with previous studies suggesting that insufficient capillary formation to

supply the hypertrophic myocardium contributes to the development clinical

RVF10.


Myocyte hypoxia may also lead to RV failure via increased oxidative stress28,

which has been linked to cardiac stiffness29. We found circumstantial evidence

of increased oxidative stress, increased heme oxygenase-1 mRNA expression

a powerful anti-oxidant enzyme in PAB-rats with clinical RVF, but not in those

without clinical RVF. So, in this PAB model, the development of clinical signs

of RVF was associated with a state of hypoxia-prone cellular environment and

increased intrinsic protective response to oxidative stress.

Downregulation of energy metabolism in RVF

Relative hypoxia and altered states of oxidative stress of the hypertrophic

myocardium have been suggested to cause metabolic changes in the RV30. RV

pressure load leads to changes in fatty acid oxidation (FAO) and uncoupling of

glycolysis from glucose-oxidation (GO), but so far it is unclear whether these

changes are adaptive or, in contrast, contribute to failure28, 31, 32. In compensated

PAB models FAO has been described to be enhanced31, 32 and pharmacological

inhibition of FAO improved cardiac output31. However, in our failing PAB model

there was marked down regulation of the FAO related gene program which

suggests that this therapeutic approach might be detrimental in advanced

clinical RVF. Although downregulation of genes expressing FAO enzymes not

necessarily implies reduced protein, Faber et al previously showed reduced

levels of proteins involved in FAO in a model of PAB21. The concomitant down

regulation of gene sets involved in carbohydrate metabolism, tricarboxylic acid

cycle and oxidative phosphorylation in PAB+cF may reflect energy deprivation

of the myocardium which is thought to be a final common pathway in heart

failure33. Indeed, in models of compensated RV pressure load, transcriptome

and protemic studies show upregulation of carbohydrate and oxidative

phosphorylation pathways 21, 34, which may be a prelude to the transition to

failure. Taken together, these findings suggest that multi-level disruption of the

myocardial energy metabolism may be pivotal in the pathobiology of RV failure.

Limitations

This study comes with some limitations that should be discussed. The definition

of RVF is a recurring subject of debate. The lack of a generally accepted definition

for RVF is hampering studies addressing the progression from beneficial RV-

adaptation to maladaptive RV-responses or RVF. We sought for a clinical relevant

Chapter 494

ACKNOWLEDGE-MENTS

FINANCIAL SUPPORT

definition of RVF and used clinical signs of heart failure in the PAB rats. However,

since RV failure is not a distinct entity but rather a continuum of progressive

disease states, our clinical definition of RV failure, dichotomizing rats in a group

with or without clinical RV failure in an effort to study differences throughout

the process of RV failure, may be artificial. However, because of the analogy

with the clinical presentation in patients, we considered this approach as a

clinical relevant definition of RVF. Secondly, the observational approach of this

study yielded several associations between clinical RVF and biological/genetic

changes, but obviously, does not prove causal relationships. Nevertheless, the

identification of these associations allows for focused studies to delineate the

mechanistic role of these changes in RVF.

Conclusion

The rat model of chronic ‘tight’ pulmonary artery banding leads to a

morphological and functional RV response, characterized by hypertrophy,

dilatation, enhanced contractility and diastolic dysfunction. Clinical symptoms

of RVF, that occur in half of the animals in this model, were associated with

progressive deterioration of diastolic function, a hypoxia-prone cellular

environment in the RV myocardium, increased intrinsic protective response to

oxidative stress and suppressed myocardial metabolism. This model represents

clinical RVF due to increased pressure load and allows for unraveling of the

mechanisms involved in the progression from RV adaptation to RV failure and

the effect of intervention on these mechanisms.

The authors are greatly indebted to Michel Weij, who performed the pulmonary

artery banding surgeries. We would also like to thank Bibiche Boersma and

Martin Dokter for excellent technical assistance and Andre Zandvoort and

Annemieke Smit-van Oosten for valuable help with the animal experiments.

This study was supported by the Sebald foundation and the Netherlands Heart

Foundation [grant#: 2007T068]


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Chapter 498

SUPPLEMENTARY MATERIAL Methods


Experimental Act and conform to the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health (NIH Publication No.

85-23, revised 1996). The Animal Experiments Committee of the University of

Groningen, the Netherlands approved the experimental protocol.

Symptoms and signs of clinical RV failure (ABCDE system)

Rats were daily checked for symptoms of RV failure, as described before1, 2. The

A-symptoms were considered present when the animal had a ruffled fur, red

discoloration of head and neck (due to decreased cleaning-behaviour) or was

less active than previously, despite stimulation. Bodyweight in RVF can either

decrease due to low intake or steeply increase due fluid retention in chest and

abdomen. The bodyweight-symptom was therefore considered present if there

was a change in bodyweight of more than 15 grams in <48 hours. Cyanosis

was checked at exposed skin on head, paws and tail. Hampered peripheral

circulation was considered present if both front paws and hind legs/tail were

pale and markedly colder than normally. Dyspnea and tachypnea were defined

as markedly increased breathing-effort and, -frequency, respectively. Edema and

effusions were defined as fluid collection in thorax and/or abdomen, palpable

(ascites) and confirmed at termination (pleural/pericardial effusion and ascites).

Voluntary exercise

To measure voluntary exercise3, 4, running wheels were mounted in the rat

cages. Five days before PAB/sham surgery, 5 days before the 5-wks time point

and 5 days before sacrifice (for those that reached the 11-wks time point; due to

the sudden onset of clinical RVF (<48u), exercise testing could not be performed

in PAB- and PAB+CF rats at end point), rats were allowed to run in the cage

wheel. Running distance was recorded daily using a digital magnetic counter

(Commodoor Cycle Odometer, Commodoor, the Netherlands) and used as a

measure of voluntary exercise. Because of large inter-individual variation, the

percentage change in run distance versus baseline was used as outcome.


Echocardiography

Echocardiography was performed at 5 weeks and at termination in all animals.

Echocardiography was performed as described previously 4 using a Vivid

Dimension 7 system and 10S-transducer (GE Healthcare, Waukesha, WI, USA).

Rats were anesthetized with isoflurane (5% induction; 2-3% maintenance;

a pulse-oxymeter (Nonin Medical, Plymouth, MN, USA) was used to monitor

adequacy of anesthesia). We used apical 3- and 4- chamber views and parasternal

short and long axis views to measure RV dimensions, tricuspid insufficiency,

TAPSE, and gradient across the PAB. Cardiac output was calculated using systolic

aorta diameter and pulsed wave Doppler of aorta flow as (aorta diameter)2 ×

3.14 × velocity time integral (VTI) x heart rate. The mean of measurements from

6-12 consecutive beats with a proper signal was taken to average out beat-to-

beat variation.

Pressure-volume analysis

At termination, hemodynamic characterization of the RV was performed

by pressure-volume analysis, obtained by RV catheterization according to a

previously described protocol4.

Rats were anesthetized with isoflurane (5% induction; 2-3% maintenance;

a pulse-oxymeter (Nonin Medical, Plymouth, MN, USA) was used to monitor

adequacy of anesthesia), intubated and ventilated. Analgesia was applied using

buprenorphine 0.01 mg/kg s.c. at the start of the procedure. Subsequently the

rat was positioned supine under a stereomicroscope (Zeiss, Hamburg, Germany)

and fixated on a temperature-controlled warming pad. The right jugular vein

was dissected and cannulated facilitating hypertonic saline infusions. Following

bilateral thoracotomy and pericardiotomy a combined pressure-conductance

catheter (SPR-869, Millar Instruments Inc., Houston, TX, USA) was introduced

via the apex into the RV and positioned in the RV outflow tract. RV pressures and

conductance were recorded using a MPVS 400 processor at a sample rate of 1.000

Hz with Chart 5 (Millar Instruments Inc., Houston, TX, USA). Subsequently, via

the dissection in the neck, the right carotid artery was exposed and the catheter

was introduced via the right carotid artery and ascending aorta into the LV to

measure LV pressures. Blood loss during the procedure was minimal (<0.5mL).

Analyses were performed offline using custom-made software (CircLab 2012, P.

Steendijk). Steady-state pressure-conductance data were obtained by averaging

the values of 3 steady-state recordings (at least 7 loops each).

Chapter 4100

Parameters obtained from pressure-volume loops included heart rate, peak

pressure, end diastolic pressure and maximal and minimal first time-derivative

of pressure (dP/dtmax and dP/dtmin). The relaxation time constant (tau) was

calculated as the time constant of monoexponential decay of RV pressure during

isovolumic relaxation.

Stroke volume (in arbitrary units) derived from the conductance signal was

calibrated, using stroke volume (in mL) measured by echocardiography. End

systolic and end diastolic elastance were determined using the single-beat

method56.

Organ weights, staining

After heart catheterization, the rats were euthanized by removing the heart from

the thorax. Heart, lungs and liver were dissected. RV, interventricular septum,

LV and both atria were separated and weighed. The liver lobe and lung lobe

were weighed, dried overnight at 65°C and weighed again to determine wet

weight/dry weight ratio. Midventricular RV sections were fixated (formalin) and

stained to assess cardiomyocyte cross-sectional area (wheat germ agglutinin),

fibrosis (Masson Tri-chrome), capillary density (lectin) and macrophages (CD68)

as described previously3, 4, 7 8. Microscopy-imaging was performed at the UMCG

Imaging Center (UMIC), which is supported by the Netherlands Organization for

Health Research and Development (ZonMW grant 40-00506-98-9021).

qRT-PCR

To characterize the hypertrophy response and study the regulation fibrosis and

capillary growth, expression of the fetal gene program (myosin heavy chain

isoforms, natriuretic pro peptides type A and B) and markers of hypertrophy

(ACTA, ACTC, RCAN1), fibrosis (TGFβ-1, OPN-1, Col1A2, Col3A1), capillary

growth (VEGF-A, VEGF-R1, VEGF-R2), and oxidative stress (HO-1, NOX-4) were

measured. RV (free wall) tissue was snap-frozen in liquid nitrogen. Total RNA

was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA);

high quality was confirmed (RQI 9.3) using Experion (Bio-Rad, Veenendaal, the

Netherlands), before conversion to cDNA by QuantiTect Reverse Transcription

(Qiagen, Venlo, the Netherlands). Gene expression was measured with Absolute

QPCR SYBR Green ROX mix (Abgene, Epsom, UK) in the presence of 7.5ng cDNA

and 200nM forward and reverse primers. qRT-PCR was carried out on the Biorad


CFX384 (Bio-Rad, Veenendaal, the Netherlands) using a standard protocol.

Primer sequences are available upon request. mRNA levels are expressed in

relative units based on a standard curve obtained by a calibrator cDNA mixture.

All measured mRNA expression levels were corrected for 36B4 reference gene

expression.

Transcriptome-wide expression profiling

Total RNA was isolated from the right ventricular free wall using TRI reagent

(Sigma, St. Louis, MO) according to the manufacturer’s protocol. RNA was

purified for individual rats (n=7/4/5 CON/PAB-/PAB+CF) using the Qiagen

RNeasy mini kit (Venlo, The Netherlands); RNA quality was verified (RIN >9)

(Agilent, Amstelveen, the Netherlands). Biotin-labeling, hybridization, washing

and scanning of GeneChip Rat Gene 1.1 ST arrays (Affymetrics) were performed

according to standard Affymetrix protocols.

Quality control and normalization

Scans of the Affymetrix arrays were processed in the MADMAX pipeline

(Nutrigenomics Consortium, Wageningen, The Netherlands)9 using Bioconductor

software packages. Quality control was carried out by visual inspection of the

heat map, Affymetrics Quality Control metrics, Relative Log Expression-plot,

Normalized Unscaled Standard Error-plot and hierarchical clustering. Expression

levels of probe sets were normalized using the robust multi-array average

algorithm10 with 19239 transcripts passing the filter. Probe sets were assigned

to genes using the custom CDF library version 15.1.1. Array data are deposited

at the Gene Expression Omnibus (GEO) database (GSE46863).

Differential expression of individual genes

Differentially expressed probe sets were identified using an IBMT regularized

t-test11. P values were corrected for multiple testing using a false discovery rate

method. Probe sets that satisfied the criterion of a false discovery rate <1% were

considered significantly regulated.

Gene set enrichment analysis

Gene set enrichment analysis (GSEA, version 3.1)12 was used to explore changes

in the global gene expression pattern. Out of 899 predefined gene sets (gene

Chapter 4102

set size set to min=50, max=500), those passing the criteria false discovery

rate (FDR) <15%, nominal P value <0.05 and normalized enrichment score >1.3

were considered significant. All gene sets available were obtained from the C2-

curated Molecular Signatures Database.

DAVID

Database for Annotation, Visualization and Integrated Discovery (DAVID)

software was used to categorize genes into biological processes13, 14. In DAVID,

statistical significance of differential expression of a biological process was

assessed using moderated t-tests; p-values were adjusted for multiple testing to

control false discovery rate using the Benjamini method. P<0.01 was considered

significant.

Comparisons in transcriptome array

Within the mentioned significance criteria, the PAB- vs. CON and PAB+CF vs.

CON comparisons were sufficiently powered. The third comparison (PAB- vs.

PAB+CF), however, only yielded significantly regulated genes with additional

filtering (fold change>1.3). We therefore described the differences between

PAB- and PAB+CF by contrasting the PAB-/CON and PAB+CF/CON comparisons.


REFERENCESBorgdorff MA, Bartelds B, Dickinson MG, Steendijk P, Berger RM. A cornerstone of heart failure treatment is not effective in experimental right ventricular failure. Int J Cardiol. 2013; 169:183-189.


Bartelds B, Borgdorff MA, Smit-van Oosten A, Takens J, Boersma B, Nederhoff MG, Elzenga NJ, van Gilst WH, De Windt LJ, Berger RM. Differential responses of the right ventricle to abnormal loading conditions in mice: pressure vs. volume load. Eur J Heart Fail. 2011; 13: 1275-1282.

Borgdorff MA, Bartelds B, Dickinson MG, Boersma B, Weij M, Zandvoort A, Sillje HH, Steendijk P, de Vroomen M, Berger RM. Sildenafil enhances systolic adaptation, but does not prevent diastolic dysfunction, in the pressure-loaded right ventricle. Eur J Heart Fail. 2012; 14: 1067-1074.

Brimioulle S, Wauthy P, Ewalenko P, Rondelet B, Vermeulen F, Kerbaul F, Naeije R. Single-beat estimation of right ventricular end-systolic pressure-volume relationship. Am J Physiol Heart Circ Physiol. 2003; 284: H1625-30.

Rain S, Handoko ML, Trip P, Gan TJ, Westerhof N, Stienen G, Paulus WJ, Ottenheijm C, Marcus JT, Dorfmuller P, Guignabert C, Humbert M, Macdonald P, Dos Remedios C, Postmus PE, Saripalli C, Hidalgo CG, Granzier HL, Vonk-Noordegraaf A, van der Velden J, de Man FS. Right Ventricular Diastolic Impairment in Patients with Pulmonary Arterial Hypertension. Circulation. 2013; 128:2016-2025.

van Albada ME, du Marchie Sarvaas GJ, Koster J, Houwertjes MC, Berger RM, Schoemaker RG. Effects of erythropoietin on advanced pulmonary vascular remodelling. Eur Respir J. 2008; 31: 126-134.

Yu L, Ruifrok WP, Meissner M, Bos EM, van Goor H, Sanjabi B, van der Harst P, Pitt B, Goldstein IJ, Koerts JA, van Veldhuisen DJ, Bank RA, van Gilst WH, Sillje HH, de Boer RA. Genetic and pharmacological inhibition of galectin-3 prevents cardiac remodeling by interfering with myocardial fibrogenesis. Circ Heart Fail. 2013; 6: 107-117.

Lin K, Kools H, de Groot PJ, Gavai AK, Basnet RK, Cheng F, Wu J, Wang X, Lommen A, Hooiveld GJ, Bonnema G, Visser RG, Muller MR, Leunissen JA. MADMAX - Management and analysis database for multiple ~omics experiments. J Integr Bioinform. 2011; 8: 160-jib-2011-160.

Allison DB, Cui X, Page GP, Sabripour M. Microarray data analysis: from disarray to consolidation and consensus. Nat Rev Genet. 2006; 7: 55-65.

Sartor MA, Tomlinson CR, Wesselkamper SC, Sivaganesan S, Leikauf GD, Medvedovic M. Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments. BMC Bioinformatics. 2006; 7: 538.

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102: 15545-15550.

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Chapter 4104

13 Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4: 44-57.

14 Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009; 37: 1-13.


Figure S1 Significantly regulated genes in PAB- and PAB+CF

Analysis of gene expression. Number of differentially expressed genes vs. CON in PAB- (blue) and PAB+CF (red) is indicated in the circles. Commonly regulated genes in purple. Number of significantly related biological processes (DAVID) between brackets. Pie-chart shows distribution of the related biological processes in categories for PAB+CF and COMMON. N=7/4/5 for CON/PAB-/PAB+CF. Individual genes: Limma P controlled with false discovery rate <1% was considered significant. DAVID: P<0.01 and Benjamini <0.01 was considered significant.

Chapter 4106

Figure S2 A Heat map display of mRNA expressed in CON, PAB- and PAB+CF. B Dendrogram using Ward hierarchical clustering on the Pearson distance measure (RMA).


CON PAB- PAB+CF

Number of rats 7 4 5WeightsBodyweight at termination (g) 488±23 431±28* 456±21*RV free wall weight (mg) 272±18 624±47* 582±41*LV+IVS weight (mg) 883±48 846±92 823±54Heartcath parametersHR (/min) 308±5 258±24 264±15dP/dtmax corr 49.8±2.2 32.3±3.7* 32.6±1.7*dP/dtmin corr (*-1) 47.7±1.8 32.7±2.8* 23.9±4.0*Tau (ms) 17.6±0.5 27.3±3.9* 23.1±2.5Tau/cyclelength (ms/s) 90±2 114±9* 100±8Echocardiographic parameters 5wkTricuspid insufficiency (present) 0% 100%* 100%*Pericardial effusion (present) 0% 0% 40%*†RVEDD (mm) 3.0±0.2 5.3±0.4* 6.0±0.1*PAB gradient (mmHg) 4±1 74±5* 64±9*RA diameter (mm) 3.5±0.1 5.2±0.3* 7.2±1.0*TAPSE (mm) 2.9±0.1 1.7±0.2* 1.3±0.2*†HR (/min) 368±8 315±18 279±20*SV (uL) 295±13 205±20* 153±17*†Echocardiographic parameters endpointTricuspid insufficiency (present) 0% 100%* 100%*Pericardial effusion (present) 0% 0% 60%*†RVEDD (mm) 4.5±0.1 6.2±0.2* 6.5±0.5*PAB gradient (mmHg) 3±1 72±11* 62±10*RA diameter (mm) 4.5±0.1 6.0±0.1* 7.7±0.6*†TAPSE (mm) 3.4±0.2 2.0±0.2* 1.2±0.1*†HR (/min) 368±6 304±11* 301±13*SV (uL) 426±30 271±24* 152±21*†

Supplemental table 1. Additional heartcatheterization and echocardiographic data in CON, PAB- and PAB+CF.

LV= left ventricle, IVS= interventricular septum, HR= heart rate, dP/dt max corr= dP/dt max normalized for RV peak pressure, dP/dt min corr= dP/dt min normalized for RV end systolic pressure, RVEDD= right ventricular enddiastolic volume, PAB= pulmonary artery banding, RA= right atrium, TAPSE= tricuspid annular plane systolic excursion, SV= stroke volume. Values are mean ± SEM. *= p<0.05 vs. CON, † vs. PAB-

Chapter 4108

PAB- PAB (11w) p-value

Number of rats 4 3Heartcath parametersHR (/min) 258±24 272±6 0.65dP/dtmax corr 32.3±3.7 32.7±2.0 0.94dP/dtmin corr (*-1) 32.7±2.8 32.1±3.7 0.89Tau (ms) 27.3±3.9 25.4±0.7 0.70Tau/cyclelength (ms/s) 114±9 115±4 0.91Echocardiographic parameters 5wkTricuspid insufficiency (%) 100% 100% 1.00Pericardial effusion (%) 0% 0% 1.00RVEDD (mm) 5.3±0.4 5.8±0.3 0.36PAB gradient (mmHg) 74±5 63±4 0.17RA diameter (mm) 5.2±0.3 3.5±0.4 0.19TAPSE (mm) 1.7±0.2 1.9±0.4 0.52HR (/min) 315±18 329±9 0.56SV (uL) 205±20 233±6 0.29

Echocardiographic parameters endpointTricuspid insufficiency (%) 100% 100% 1.00Pericardial effusion (%) 0% 0% 1.00RVEDD (mm) 6.2±0.2 7.0±0.4 0.10PAB gradient (mmHg) 72±11 113±12 0.06RA diameter (mm) 6.0±0.1 6.5±0.3 0.13TAPSE (mm) 2.0±0.2 2.0±0.1 0.96HR (/min) 304±11 328±14 0.23SV (uL) 271±24 292±33 0.63

Due to space restrictions these lengthy tables (combined >40 pages) are not available in the print version.

Supplemental table 3. List of genes commonly up-, or down regulated in PAB- and PAB+CF when compared to CON. FDR= false discovery rate.

Supplemental table 4. Gene Sets significantly downregulated in PAB+CF vs. CON, but not in PAB- vs. CON. SIZE= number of genes in respective GSEA set, NES= normalized enrichment score (analogous to fold change), NOM= nominal, FDR= false discovery rate.

Supplemental table 2. Heartcatherization and echocardiographic data in PAB- and PAB (11w). HR= heart rate, dP/dt max corr= dP/dt max normalized for RV peak pressure, dP/dt min corr= dP/dt min normalized for RV end systolic pressure, RVEDD= right ventricular enddiastolic volume, PAB= pulmonary artery banding, RA= right atrium, TAPSE= tricuspid annular plane systolic excursion, SV= stroke volume.Values are mean ± SEM. Significance indicated by p-values from t-test PAB- vs. PAB(11w).

PART II

5SILDENAFILENHA NCESSYSTOLICAD APTATIONBUTDO ESNOTPREVENTD IASTOLICDYSFUN CTIONINTHEPRES SURE-LOADEDRIG HTVENTRICLE

MAJ Borgdorff, B Bartelds, MG Dickinson, B Boersma, M Weij, A Zandvoort,

HHW Silljé, P Steendijk, M de Vroomen, RMF Berger

Eur J Heart Fail. 2012 Sep;14(9):1067-74.

doi: 10.1093/eurjhf/hfs094. Epub 2012 Jun 22.

Chapter 5112

ABSTRACTAim

Right ventricular (RV) failure due to pressure- or volume overload is a major

risk factor for early mortality in congenital heart disease and pulmonary

hypertension but currently treatments are lacking. We aimed to demonstrate

that phosphodiesterase 5A-inhibitor Sildenafil can prevent adverse remodeling

and improve function in chronic abnormal RV overload, independent from

effects on the pulmonary vasculature.

Methods and results

In rat models of either pressure- or volume overload, we performed pressure-

volume studies to measure hemodynamic effects and voluntary exercise testing

as clinical outcome after 4 weeks of Sildenafil (or vehicle) administration. In

the pressure loaded RV, Sildenafil enhanced contractility (endsystolic elastance

(mmHg/ mL):247±68 vs. 155±71, Sildenafil vs. Vehicle, p<0.05), prevented

RV dilatation (enddiastolic volume (μL): 733±50 vs. 874±39, p<0.05), reduced

wall stress (peak wall stress (mmHg): 323±46 vs. 492±62, p<0.05) and partially

preserved exercise tolerance (running distance (%) -33±15 vs. -62±12, p<0.05).

Protein kinase A was not activated by Sildenafil and thus did not mediate the

observed effects. In contrast, protein kinase G-1 was activated by Sildenafil, but

hypertrophy was not inhibited. Importantly, Sildenafil did not prevent diastolic

dysfunction, whereas RV fibrosis appeared increased in Sildenafil-treated rats. In

the volume loaded RV, Sildenafil treatment did not show any beneficial effects.

Conclusion

We demonstrate Sildenafil to have beneficial, afterload-independent effects

on the pressure loaded RV, but not on the volume loaded RV. These results

indicate that Sildenafil may offer a specific treatment for the pressure loaded RV,

although persistent diastolic dysfunction and RV fibrosis could be of concern.

Preventive Sildenafil treatment in the pressure-, or volume loaded RV 113

INTRODUCTIONRight ventricular (RV) failure is a pivotal determinant of outcome in the

growing population of patients with congenital heart disease(1,2), as well as in

patients with pulmonary arterial hypertension (PAH)(3,4) and patients with left

ventricular (LV) failure(5). RV failure frequently results from chronic pressure

overload, but may also be due to chronic volume overload, e.g. due to pre-

tricuspid shunts or valve insufficiency(6). The RV is understudied compared

to the LV(7) and the mechanisms of RV failure are unknown(8). Consequently,

there are no RV specific treatments(9).

The RV differs from the LV, not only anatomically or because it is coupled to

the low resistance pulmonary circulation, but also because it is derived from

a different set of precursor cells, the so-called secondary heart field(10,11).

Whether the differences in embryologic origin affect RV responses to abnormal

loading conditions is a matter of debate (12), (13).

Most knowledge on the ventricular response to chronic pressure overload is

derived from studies in the LV(14). In murine models of LV pressure overload, it

has been shown that inhibition of phosphodiesterase type 5A (PDE5A) prevents

LV failure via inhibition of adverse hypertrophic signaling(15). The proposed

mechanism is that PDE5A-inhibition activates protein kinase G-1 (PKG-1), an

inhibitor of pathological remodeling. These findings have prompted the start of

the RELAX trial, which studies the effects of Sildenafil in diastolic LV failure, and

is ongoing.

Also in the RV, PDE5A has been shown to be expressed in response to pressure

load(16). Moreover, in isolated heart studies of the hypertrophied RV, PDE5A-

inhibition has been shown to induce a positive inotropic response via activation

of protein kinase A (PKA)(16). PDE5A-inhibitor Sildenafil is widely used in the

treatment of PAH because of its effects on the pulmonary vasculature. However,

direct effects of Sildenafil on RV myocardial function remain largely unknown,

since most studies were performed in animal models of pulmonary hypertension

in which such direct effects on RV myocardium cannot be discerned from the

effects secondary to a decreased RV afterload. Moreover, it is unknown if

Sildenafil could be of benefit for the chronic volume overloaded RV.

We hypothesized that chronic Sildenafil treatment would improve RV adaptation

to abnormal loading conditions responsible for RV failure. We used a rat model

of pressure overload with fixed RV afterload via pulmonary artery banding and a

rat model of RV volume overload by abdominal aorto-caval shunt.

Chapter 5114

MATERIALS AND

METHODSAn expanded methods section can be found in the Supplementary material.

Animal model and study design






Wistar rats (n=60; male; 160-180g; Harlan, Horst, the Netherlands) were

randomly assigned to one of six experimental groups (10/group): pulmonary

artery banding + Sildenafil (PAB+), pulmonary artery banding + placebo (PAB-),

aorto-caval shunt + Sildenafil (ACS+), aorto-caval shunt + placebo (ACS-), sham-

operated + Sildenafil (CON+) and sham-operated + placebo (CON-). Treatment

was given for 4 weeks, then echocardiography and RV catheterization were

performed and the animals were euthanized.

PDE5A-inhibitor Sildenafil (100mg/kg/day) (Pfizer Inc, Capelle aan den IJssel,

the Netherlands) was given in the drinking water from the day of surgery in a

dose that specifically inhibits PDE5A. To ensure adequate PDE5A-inhibition, we

tested this method of Sildenafil administration in an additional group of rats

by measuring plasma levels after 10 days, which ranged from 23-216 nM, well

above the effective minimum plasma level(17).

RV pressure overload was induced by surgical pulmonary artery banding (PAB)

via a left lateral thoracotomy, using an 18G needle to standardize the degree of

stenosis. RV volume overload was induced by surgical aorto-caval shunt (ACS)

via a laparotomy. Sham-operated rats served as control (CON).

Two animals (1 ACS, 1 CON) died in the first week after surgery due to

complications of the laparotomy.



studies, obtained by right heart catheterization four weeks after surgery as

described previously(18).


Exercise tolerance and clinical signs of failure

To measure voluntary exercise tolerance(19), running wheels were mounted in

the rat cages. Five days before surgery and 5 days before sacrifice (4 wks after

surgery), animals were allowed to run in the cage wheel. Running distance was

recorded daily using a digital magnetic counter (Commodoor Cycle Odometer,

Commodoor, the Netherlands)(12).

Rats were daily examined for clinical signs of RV failure according to a predefined

checklist (e-Fig. 1).

Echocardiography

Echocardiography was performed on the day before sacrifice as described

previously (20,21) using a Vivid Dimension 7 system and 10S-transducer (GE

Healthcare, Waukesha, WI, USA).

Organ weights, hypertrophy and fibrosis

After heart catheterization, the rats were euthanized by removing the heart


septum, LV and both atria were separated and weighed. Tissue fixation and

staining to assess cardiomyocyte cross-sectional area and fibrosis were done

using standard methods.

qtr-PCR, Western Blotting, PKA-assay, PKG-1-assay

To characterize the hypertrophy response and study the mechanism of action of

Sildenafil, expression of the fetal gene program (myosin heavy chain isoforms,

natriuretic pro peptides type A and B), key pathways of hypertrophy (NFAT/

Calcineurin, Akt, Erk1/2) and activity of protein kinases type G-1 and A were

measured. Standard methodology was applied; see supplemental material for

extensive descriptions.



Differences between models were evaluated using one-way ANOVA followed

by post hoc Bonferroni analysis or Kruskall-Wallis tests. Sildenafil versus

placebo differences were evaluated using Students t-test or Mann-Whitney U

test as appropriate. Group size is 7-10, unless specified otherwise. P<0.05 was

considered significant (PASW Statistics 18 for Windows, SPSS, Chicago, Illinois).

Chapter 5116

RESULTSPulmonary artery banding and aorto-caval shunt induce distinctive phenotypes

of RV adaptation

Pulmonary artery banding resulted in marked RV pressure load as shown by

increased RV peak pressure (70±9 mmHg) (Table 1) and the pressure-volume

loops in Fig. 1A. Cardiac output (70±4 vs. 82±2 mL/min PAB vs. CON, p<0.05)

and ejection fraction (27±2 vs. 47±3 %, p<0.05) were decreased compared to

control animals (Fig. 1B), despite increased contractility (endsystolic elastance

155±27 vs. 59±8 mmHg/mL, p<0.001) (Fig. 1C). Additionally there was diastolic

dysfunction marked by elevated enddiastolic elastance (9±2 vs. 4±1 mmHg/mL,

p<0.01) (Fig. 1D) and enddiastolic pressure, and prolonged tau (Table 1). Right

ventricular dilatation is shown in pressure-volume loops and by echo (Fig. 1A,

Table 1), which, in combination with increased pressures, resulted in high wall

stress (Fig. 1E).

Figure 1. Hemodynamic and functional characteristics of the right ventricle after PAB or ACS. A Representative pressure volume loops of the 3 experimental groups during vena cava occlusion. Endsystolic pressure volume relations marked by solid black lines, enddiastolic pressure volume relations marked by dashed black lines. B Cardiac output (mL/min) and ejection fraction (%). C Endsystolic elastance (mmHg/mL) D Enddiastolic elastance (mmHg/mL) E Peak wall stress (mmHg). F Relative change in distance run during voluntary exercise before sacrifice versus baseline. Mean±SEM, * p<0.05


CON- PAB- ACS-

Echo parameters PAB gradient (mmHg) 4±1 76±22*† 7±1RVEDD (mm) 3.5±0.1 4.3±0.7* 5.0±1.6*$RAD (mm) 3.3±0.1 5.0±1.8* 6.4±0.9*LVEDD (mm) 4.8±0.1 4.4±0.9 7.5±0.6*$TAPSE (mm) 2.9±0.1 2.4±0.6 3.9±0.2*$Heartcath parametersHR (/min) 306±10 300±10 304±7CO (mL/min) 82±2 70±4* 126±8*$EF (%) 47±3 27±2* 34±2*SV (μL) 269±7 227±19 418±24*$Ppeak (mmHg) 26±1 70±9*† 37±3Ees (mmHg/μL) •1000 59±8 155±27*† 41±4PRSW (mmHg) 11±2 44±6*† 23±6Eed (mmHg/μL) •1000 4±1 9±2* 4±2Ped (mmHg) 1±0.3 6±1*† 2±1Tau/cyclelength (ms/s) 78±4 103±5*† 82±4SW (mmHg •μL) 5,231±331 15,084±1,796* 13,019±997*

These hemodynamic changes were accompanied by 62% reduction in running

distance (Fig. 1F) and 8 out of 10 animals showed clinical signs of RV failure such

as inactivity, weight loss and tachypnea: 2 animals developed overt RV failure

with severely depressed cardiac output (below 75% of CON), severe tricuspid

insufficiency, widely dilated right atrium, ascites, pleural effusion, pale, cold

extremities and severely reduced TAPSE.

Aorto-caval shunt resulted in a substantial volume load reflected by dilatation of

RV, LV and both atria (Fig. 1A, Table 1), increased cardiac output (+55% vs. CON)

(Fig. 1B), and increased TAPSE (Table 1). Except from lower ejection fraction,

systolic and diastolic parameters showed no differences compared to CON

(Table 1). However, running distances decreased by 54% (p<0.05) indicating that

exercise tolerance was reduced compared to CON (Fig. 1F).

Table 1. Main echocardiographic and heartcatheterization data of control (CON-), pulmonary artery banding (PAB-) and aorto-caval shunt (ACS-), all untreated. RVEDD= right ventricle end diastolic diameter, RAD= right atrial diameter, LVEDD= left ventricle end diastolic diameter, TAPSE= tricuspid annular plane systolic excursion, HR= heart rate, CO= cardiac output, EF= ejection fraction, SV= stroke volume, PPeak= maximum pressure, Ees= Endsystolic elastance, PRSW= preload recruitable stroke work, Eed= enddiastolic elastance, Ped= End diastolic pressure, SW= stroke work. Values are mean ± SEM. * p<0.05 vs CON; † p<0.05 vs ACS; $ p<0.05 vs PAB.

Chapter 5118

Sildenafil prevents deterioration of RV hemodynamics in pressure load by

enhancing systolic function

Sildenafil had several beneficial effects on RV hemodynamics after PAB (Fig.

2A-F). Firstly, Sildenafil treatment prevented the decrease in cardiac output

(+20% vs. PAB-, p<0.05) and ejection fraction (+54% vs. PAB-, p<0.05) (Fig. 2B)

by enhancing contractility as reflected by increased endsystolic elastance (+59%

vs. PAB-, p<0.05) (Fig. 2C). Secondly, Sildenafil attenuated ventricular dilatation:

endsystolic volume: Ves (-33% vs. PAB-, p<0.05) (Fig. 2F), thereby significantly

reducing wall stress (WSes -52% vs. PAB-, p<0.05). However, diastolic function

parameters did not change significantly with Sildenafil treatment (Fig. 2D). The

preventive effects of Sildenafil treatment attenuated the decrease in exercise-

tolerance: running distance was reduced by 33% vs. 62% in the untreated group

(p<0.05) (Fig. 2G). Additionally, less animals developed clinical symptoms of RV

failure after Sildenafil treatment (80% vs. 40%) and the only symptom present

in the PAB+ was tachypnea; none of the treated rats developed overt RV failure.

Sildenafil does not affect RV hemodynamics in volume load or normal load

In contrast to the significant, positive effects of Sildenafil treatment after PAB,

Sildenafil did not affect hemodynamics in rats with an ACS (Fig 3A): cardiac

output and ejection fraction were unchanged (Fig 3B), as were endsystolic/-

diastolic elastance (Fig 3C, D), RV wallstress (Fig 3E), ventricular dimensions (Fig

3F) or running distances (Fig 3G).

Similarly in sham operated animals (CON), Sildenafil treatment did not have

hemodynamic or functional effects (Fig 3B-G).

Sildenafil did not prevent adverse RV remodeling in the pressure or volume

loaded RV

Both PAB and ACS resulted in RV hypertrophy as shown by changes in RV weight,

cardiomyocyte cross-sectional area, myosin heavy chain (MHC) – isoform switch

and type B natriuretic peptide (NPPB) expression (Table 2). Also, the calcineurin/

NFAT pathway, involved in the induction of pathological hypertrophy, was

activated in both PAB and ACS, as shown by the upregulation of MCIP (Table 2).


CON- PAB- ACS-

RV weight/BW (mg/g) 0.6±0.01 1.2±0.1* 1.1±0.1*RV weight/ LV+IVS weight (mg/mg) 0.27±0.01 0.54±0.04*† 0.35±0.01LV+IVS weight/BW (mg/g) 2.1±0.06 2.3±0.06 3.1±0.09*$RV cardiomyocyte CSA (AU) 1.00±0.02 1.64±0.01* 1.48±0.16*Gene expressionbetaMHC-alphaMHC ratio 1.0±0.4 10.9±3.2* 10.3±5.6*NPPB/18S 1.0±0.3 22.3±6.0* 16.2±4.2*MCIP/18S 1.0±0.2 7.0±1.2* 5.1±0.9*

Figure 2. Hemodynamic and functional effects of Sildenafil treatment after PAB. A Representative pressure volume loops of PAB untreated (dark grey) and treated (light grey) during vena cava occlusion. Untreated CON shown as reference. Endsystolic pressure volume relations marked by solid black lines, enddiastolic pressure volume relations marked by dashed black lines. B Cardiac output (mL/min) and ejection fraction (%). C Endsystolic elastance (mmHg/mL). D Enddiastolic elastance (mmHg/mL). E Peak wall stress (mmHg). F Comparison of combined endsystolic (left end of bar) and enddiastolic (right end of bar) volumes of untreated CON and PAB, with or without SIL treatment. G Relative change distance run during voluntary exercise at sacrifice versus baseline after PAB. Mean±SEM, * p<0.05, ns=not significant, CON- =untreated control, PAB- = untreated PAB, PAB+ = SIL treated PAB

Table 2. Hypertrophy profile of control (CON-), pulmonary artery banding (PAB-) and aorto-caval shunt (ACS-), all untreated. Gene expression RV, fold increase, normalized with CON- = 1, using 18S as reference gene. BW= bodyweight, CSA= cross-sectional area, MHC= myosin heavy chain, NPPB= natriuretic propeptide type B, MCIP=modulatory calcineurin-interacting protein. Values are mean ± SEM. * p<0.05 vs CON; † p<0.05 vs ACS; $ p<0.05 vs PAB. (n=6-10 for gene expression)

Chapter 5120

Figure 3. Hemodynamic and functional effects of Sildenafil treatment after ACS and in CON. A Representative pressure volume loops of ACS+ and ACS- during vena cava occlusion. Untreated CON shown as reference. Endsystolic pressure volume relations marked by solid black lines, enddiastolic pressure volume relations marked by dashed black lines. B Cardiac output (mL/min) and ejection fraction (%). C Endsystolic elastance (mmHg/mL). D Enddiastolic elastance (mmHg/mL). E Peak wall stress (mmHg). F Comparison of combined endsystolic (left border of bar) and enddiastolic (right border of bar) volumes of CON and ACS, with or without SIL. G Relative change distance run during voluntary exercise at sacrifice vs baseline after CON and ACS. Mean±SEM, ns= not significant

In the pressure loaded RV, treatment with Sildenafil did not prevent pathological

RV remodeling as shown by unchanged RV weight (1.3±0.1 vs. 1.2±0.1 mg/g,

PAB+ vs. PAB-, ns) and expression of MHC-isoforms (beta-MHC to alpha-MHC

ratio 10±2 vs. 11±3-fold change (PAB+ vs. PAB-, ns), NPPB (27±4 vs. 22±6-fold

increase, PAB+ vs. PAB-, ns) and MCIP (8±1 vs. 7±1-fold increase, PAB+ vs. PAB-

, ns). Neither ERK1/2 and Akt, two other main hypertrophy pathways were

influenced by Sildenafil (data not shown). Importantly, Sildenafil did increase

the amount of cardiac fibrosis in the pressure loaded RV 4-fold (Fig. 4A).

Sildenafil treatment increased PKG-1 activity as expected in the pressure

loaded RV (Fig. 4B), suggesting that PKG-1 could be responsible for the positive

inotropic effects. Interestingly, in our model chronic PDE5A-inhibition did not

activate PKA (Fig. 4C). This is in contrast to previous suggestions that activated

PKA would induce the beneficial hemodynamic effects of Sildenafil(16). Lack

of PKA activation was further confirmed by unchanged (PKA-specific) serine

16-phosphorylation of phospholamban (e-Fig. 2).


In the volume loaded RV, Sildenafil exerted opposite – inhibitory- effects on

PKG-1 activity (0.90±0.04 vs. 0.62±0.11 AU, ACS- vs. ACS+, p<0.05), in line with

the lack of hemodynamic effect. Further, hypertrophy, expression of remodeling

pathways and fibrosis were unchanged in the treated volume loaded RV (data

not shown).

Figure 4. A Representative images of Masson-Trichrome-stained right ventricles of untreated CON (n=3) and (un)treated PAB (PAB- (n=2) and PAB+ (n=5)). Ruler is 75μm. Bottom graph shows fold change fibrosis percentage per surface area vs. untreated CON. B Protein kinase G-1 activity, relative (CON=1) (n=6-8/group). C Protein kinase A activity, relative (CON=1) (N=5-6/group). Mean±SEM, * p<0.05

Chapter 5122

In this study, we showed that chronic Sildenafil treatment has direct beneficial

effects on the pressure loaded, but not on the volume loaded RV. In the

pressure loaded RV, chronic Sildenafil treatment enhances systolic RV function,

preventing RV dilatation, reducing wall stress and improving exercise tolerance.

This would endorse Sildenafil as a specific therapy for the pressure loaded

RV. However, Sildenafil did not prevent diastolic dysfunction and increased

myocardial fibrosis, which could have unfavorable consequences for long term

Sildenafil treatment in patients with a pressure loaded RV.

These positive effects of chronic Sildenafil on the pressure loaded RV (independent

of the effects on afterload) have not been described before. Previous

experimental studies have failed to show improvement(22,23), but these were

limited by restricted functional assessment. In these studies RV assessment was

either restricted to measuring cardiac output by thermodilution or RV function

was assessed by echocardiography, which is limited due to the complex RV

morphology(24). This may explain why these studies could not demonstrate

beneficial effects of Sildenafil treatment in PAB. In this study however, we used

invasive pressure-volume (PV) measurements to assess ‘intrinsic’ ventricular

function (independent of loading conditions) and demonstrated positive effects

on end systolic elastance and instantaneous wallstress. PV measurements in the

RV are validated in physiological and pathophysiological states and provide in-

depth information on ventricular and circulatory function(25,26).

The beneficial effects on systolic RV function, found in the current study, also

translated into an improvement of exercise tolerance, a clinically relevant

outcome parameter. The voluntary cage wheel exercise test compares best to the

6-minute walking distance, rather than the maximal exercise capacity(12). These

beneficial findings suggest that there may be a place for Sildenafil therapy in

patients with congenital heart disease and RV dysfunction due to fixed pressure

load as is seen in for instance systemic right ventricle (e.g. in hypoplastic left

heart syndrome), or with residual pulmonary stenosis after surgical treatment.

However, it should be considered whether ‘boosting’ of already increased

contractility -whereas is beneficial in the short term-, will be either beneficial or

detrimental in the long term. Indeed, chronic PDE3-inhibition has been shown

to worsen LV heart failure and increase mortality(27), despite its acute beneficial

effects on hemodynamics(28).

DISCUSSION


The rats with a PAB had clear diastolic dysfunction as shown by increased end

diastolic elastance, end diastolic pressure and prolonged tau. In contrast to

previous studies in the LV(15), we did not observe an improvement in diastolic

function with Sildenafil treatment. The difference with the LV may be due to

distinct embryologic origin(10), because although the core regulatory systems

are identical in both ventricles, stress (e.g. pressure load) induces upstream

activators in a chamber-specific manner(13).

Alternatively, a beneficial effect on diastolic function may have been offset

by the increased myocardial fibrosis. Expression of genes involved in fibrosis

(Collagen-chains 1A2, -3A1 and Osteopontin) has been reported to increase

in Sildenafil treated pressure overloaded RVs(22). We speculate that chronic

stimulation of contractility by Sildenafil could lead to increased fibrosis similarly

to isoproterenol and dobutamine(29,30). This might warrant serious concerns

regarding adverse effects of long term Sildenafil treatment on RV function.

At present Sildenafil is an accepted therapy for patients with PAH, but not

reaching optimal clinical treatment targets since a significant proportion of

patients clinically deteriorates during treatment and require additional PAH

therapies (31). Very recently, an important safety announcement has come out

from Pfizer Inc regarding the use of higher than registered sildenafil doses after

an increased mortality had been observed during a pediatric study (Important

Safety Announcement, issued by Pfizer Inc, 20th September 2011).

It is not clear whether these clinical observations are associated with increased

RV fibrosis and diastolic dysfunction, but specific attention for this potential

relation is warranted.

The precise effects of PDE5A-inhibition seem to depend on the balance of

stimulation/inhibition in a wide array of pathways and it is possible that the

balance between these systems changes in chronic abnormal loading and

PDE5A-inhibition. Indeed, we show that chronic Sildenafil does not lead to

overstimulation of the PKA-pathway. Finally, in this study Sildenafil showed no

beneficial effects on the volume loaded RV. It should be noticed, however, that

rats in our study had, despite a significant volume load, an increased cardiac

output and no RV failure yet. As the volume loaded RV is an increasing problem

in the growing population of survivors of congenital heart disease, there is an

urgent need for studies exploring the mechanisms of RV adaptation to volume

load.

Chapter 5124

ACKNOWLEDGE-MENTS

FUNDING

Limitations

The current study was a prevention study and, consequently, the effects of

Sildenafil in established RV pressure load have to be studied in order to assess

the value of Sildenafil in a clinical setting. Further, longer term studies will be

necessary to assess the effect of increased myocardial fibrosis, but also to assess

the outcome of the increased systolic function in the setting of a fixed afterload.

In conclusion, we showed that chronic Sildenafil treatment has direct beneficial

effects on the pressure loaded, not the volume loaded RV. Chronic Sildenafil

treatment enhances systolic RV function, preventing RV dilatation, reducing

wall stress and improving exercise tolerance. This would endorse Sildenafil as a

specific therapy for the pressure loaded RV. However, Sildenafil did not prevent

diastolic dysfunction and increased myocardial fibrosis, which could have

consequences for long term Sildenafil treatment of patients with a pressure

loaded RV.

We thank Janny Takens, Marnix R van der Tuin and Susanna F Gunnink for

excellent technical assistance and Annemieke Smit-van Oosten, Inge Vreeswijk-

Baudoin and Irma Kuipers for valuable help with the animal experiments.

This study was supported by the Netherlands Heart Foundation [grant#:

2007T068] and by the Sebald fund.


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Chapter 5128

SUPPLE-MENTARY METHODS

All animals were individually housed with a 12:12-h light-dark cycle and fed ad

libitum.

Analgesia: Buprenorphine subcutaneously 0.01 mg/kg at the start of surgery

and 1h, 12h and 1d after surgery.

PAB/ACS/SHAM procedure

The rats were anesthetized with isoflurane (2-3%), intubated and ventilated with

a rodent ventilator (Inspira ASV rodent ventilator, Harvard Apparatus, Holliston,

MA, USA). PAB: Following lateral thoracotomy, a suture was tightened around

the pulmonary artery and an 18-gauge needle. The needle was swiftly removed,

resulting in a 1.2 =mm PAB. ACS: Following midline laparotomy, an aorta-caval

anastomosis was made by an 18-gauge needle. Drops of tissue-glue were used

to fixate the anastomosis. SHAM: Sham surgeries were identical to respective

model-surgery except for the model-inducing step. PAB surgery survival was

100%. ACS surgery survival was 100%, but a few days after surgery one animal

died from abdominal wound dehiscence. SHAM surgery survival was 100%, but

a few days after surgery one animal died from abdominal wound infection.

Sildenafil treatment

In a separate experiment we tested the effectiveness of Sildenafil-administration

via the drinking water using the identical protocol of the main study. Nine rats (5

Controls, 4 with ACS) were administered Sildenafil via the drinking water for 10

days at a dose of 100mg/kg rat/day. After 9 days venous samples were taken from

all animals. Plasma-levels of Sildenafil were measured chromatographically and

ranged from 23-216 nM which exceeds the IC 50 of PDE5A-cGMP-hydrolyzing

activity (1.9nM) but not those of PDE1C (422nM) or PDE2 (15μM). Sildenafil

levels in the rats that received ACS were equal to those in controls.

Pressure-volume studies

Rats were anesthetized with isoflurane, intubated and ventilated. The right

jugular vein was cannulated facilitating hypertonic saline infusions. Following

bilateral thoracotomy a combined pressure-conductance catheter (SPR-869,

Millar Instruments Inc., Houston, TX, USA) was introduced via the apex into the

RV and positioned in the RV outflow tract. RV pressures and conductance were


recorded using a MPVS 400 processor at a sample rate of 1.000 Hz with Chart

5 (Millar Instruments Inc., Houston, TX, USA). Analyses were performed offline

using custom-made software (CircLab 2009/2010, P. Steendijk).

The volume signal of the conductance catheter was calibrated for parallel

conductance and slope factor (alpha) in order to obtain absolute volumetric

values. The parallel conductance was estimated by infusing 10uL of hypertonic

(20%) saline via the jugular vein cannula. Slope factor was calculated as

uncalibrated conductance catheter cardiac output divided by LV cardiac output,

as measured by echocardiography.

Steady-state pressure and volume data were obtained by averaging the values

of 3 steady-state recordings (at least 7 loops each), captured just before vena

cava occlusion.

Endsystolic and enddiastolic pressure-volume relations were determined from

measurements obtained during transient constriction of the vena cava inferior.

Histology

Heart tissue was fixated using 4% formalin and embedded in paraffin. Four-µm-

thick sections were cut, deparaffinized and rehydrated in decreasing graded

alcohol and xylene.

Gomori: For determination of cardiomyocyte surface area, RV (free wall) sections

were stained using Gomori’s reticulin silver staining and photographed using a

camera fitted on a microscope (Zeiss Benelux, Sliedrecht, the Netherlands) at

40x and analyzed using Image-Pro software (MediaCybernetics, Bethesda, MD,

USA). Only transversally cut myocytes were included; per section measurements

were averaged from 60 cells in 4 different fields.

Masson Trichrome: For determination of the amount of fibrosis, RV (free

wall) sections were stained using NovaUltra™ Masson Trichrome Stain kit

(IHC World, Woodstock, MD, USA) and photographed using a digital slide

scanner (NanoZoomer 2.0-HT, Hamamatsu Photonics Nederland, Almere, the

Netherlands) at 20x and analyzed using Image Scope 11 (Aperio Technologies,

Inc. Vista, CA, USA). Fibrosis was quantified as the blue-stained % of the total

tissue area, measured per whole ventricle. The edges of the tissue and major

vessels including perivascular fibrosis were excluded to obtain purely myocardial

interstitial fibrosis.

Chapter 5130

Quantitative PCR

RV (free wall) tissue was snap-frozen in liquid nitrogen. Total RNA was extracted

using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA). Quantitative

real-time Reverse-Transcriptase PCR was carried out using standard methods on

the following genes: MCIP, NPPA, NPPB, beta- and alpha-MHC, phospholamban,

SERCA, HIF1alpha, PDE5. Primer sequences are available on request. mRNA

levels were corrected for 18S mRNA expression.

Western blotting

Protein was extracted from snap-frozen RV (free wall) tissue using RIPA buffer.

We used antibodies to p/t Akt (1:1000; Cell Signalling Technology, Danvers,

MA, USA), p/t ERK1/2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA),

Serine 16-phosphorylated phospholamban (1:1000; Abcam, Cambridge, UK),

and PKG-1 (1:1000; StressGen, Brussels, Belgium). Primary antibody binding

was visualized by horseradish peroxidase–conjugated secondary antibodies

and enhanced chemiluminescence (Perkin-Elmer, Waltham, MA, USA). Protein

expression of beta-actin (1:20000; Sigma-Aldrich Chemie BV, Zwijndrecht,

the Netherlands) was used as loading control for Ser-16 P-phospholamban.

GeneTools software (GeneGnome, Cambridge, UK) was used for densitometry.

PKG-1 assay

PKG-1 activity was assayed by colorimetric analysis (CycLex,Ina, Nagano,

Japan) from RV (free wall) lysates, according to manufacturer’s protocol. Snap-

frozen RV tissue was homogenized in extraction buffer. The supernatant was

diluted with potassium phosphate buffer. The precipitate was dissolved in DE-

buffer. A PKG-1-phospho-threonine 68/119 specific antibody with horseradish

peroxide detection body was added to each well. After incubation and washing

steps, tetra-methylbenzidine was added. The reaction was terminated using

1 M sulfuric acid. Photospectrometry was performed at 450nm. Positive and

negative controls were included for each experimental group.

PKA assay

PKA activity was assayed by colorimetric analysis (MESACUP Protein Kinase

Assay, MBL CO., Ltd, Nagoya, Japan) according to manufacturer’s protocol. Fresh

RV tissue was homogenized in extraction buffer. The supernatant was diluted


with component mixture. 100 μL of each diluted sample was added to each PS-

peptide coated well. After multiple washing steps, 100 μL biotinylated antibody

2B9 was added to each well. After incubation and multiple washing steps, 100

μL POD-conjugated streptavidin was added to each well. After incubation and

multiple washing steps, 100 μL substrate solution was added to each well. After

incubation, stop solution was added and photospectrometry was performed at

492nm. Concurrently a standard curve was made using increasing concentrations

of catalytic subunit of PKA and appropriate positive and negative controls were

included.

e-Figure 1. A Presence of clinical signs of right ventricular failure. Filled square = symptoms present. White square = symptoms absent. Each row represents 1 animal. ABCDE refer to symptom-categories: A activity and appearance, B bodyweight, C cyanosis and/or hampered peripheral circulation, D dyspnoe and/or tachypnoe, E effusions: pleural or ascites. B Example echocardiographic images of advanced RV failure after PAB (RV is on the right side in all images). Top: four-chamber view during diastole, note the dilatation of both right ventricle and right atrium. Center: parasternal short axis view, note the septal bowing towards the LV, and RV hypertrophy. Bottom: 4-chamber view during systole, note the tricuspid insufficiency.

Chapter 5132

e-Figure 2. Protein levels of Serine 16-phosphorylated (active)-phospholamban, beta-actin was used as loading control. Western Blot shown in right panel. CON- = untreated CON (n=3), PAB- = untreated PAB (n=5), PAB+ = SIL treated PAB (n=6). Mean±SEM,* p<0.05.

RESULTS


CON- PAB- PAB+ ACS- ACS+

Heartcath parametersHR (/min) 306±10 300±10 299±10 304±7 304±9EF (%) 47±3 27±2* 38±4# 34±2* 33±3SV (μL) 269±7 227±19 287±11# 418±24*$ 475±43Systolic parametersEes (mmHg/μL) •1000 59±8 155±27*† 247±26# 41±4 55±16PRSW (mmHg) 11±2 44±6*† 59±9 23±6 25±4dP/dt max -Ved (mmHg/s/μL) 2±0.4 6±2*† 6±1 1±0.3 4±1#Diastolic parametersEed (mmHg/μL) •1000 4±1 9±2* 13±2 4±2 6±2Ped (mmHg) 1±0.3 6±1*† 5±1 2±1 3±1Tau/cyclelength (ms/s) 78±4 103±5*† 108±4 82±4 69±4#WallstressWSmax (mmHg) 156±11 492±62*† 323±46# 336±39* 317±40Echocardiographic parametersPAB gradient (mmHg) 4±1 76±22*† 79±16 7±1 7±2TAPSE (mm) 2.9±0.1 2.4±0.6 2.6±0.3 3.9±0.2*$ 4.1±0.2RVEDD (mm) 3.5±0.1 4.3±0.7* 4.2±0.3 5.0±1.6*$ 5.5±0.6LVEDD (mm) 4.8±0.1 4.4±0.9 4.5±0.6 7.5±0.6*$ 6.8±1.2RVEDD/LVEDD 0.73±0.02 1.04±0.36* 0.94±0.14 0.66±0.17 0.83±0.17RA diameter (mm) 3.3±0.1 5.0±1.8* 4.3±0.5 6.4±0.9* 6.0±0.6LA diameter (mm) 3.7±0.1 4.1±0.8 4.5±0.3 6.7±0.7*$ 5.7±0.8GeneralBodyweight (g) 323±10 286±8*† 306±7 332±7 329±11Organ weightsRV weight/BW (mg/g) 0.6±0.01 1.2±0.1* 1.3±0.1 1.1±0.1* 1.2±0.1LV+IVS weight/BW (mg/g) 2.1±0.06 2.3±0.06 2.3±0.05 3.1±0.09*$ 3.3±0.10RA weight/BW (mg/g) 0.11±0.01 0.20±0.05 0.16±0.02 0.33±0.03*$ 0.38±0.02LA weight/BW (mg/g) 0.07±0.01 0.09±0.02 0.11±0.01 0.18±0.01*$ 0.19±0.01Lunglobe wet/dry (mg/mg) 5.4±0.1 5.4±0.1 5.3±0.1 5.3±0.1 5.3±0.1Liverlobe wet/dry (mg/mg) 2.9±0.04 3.0±0.05 3.0±0.03 3.1±0.05* 3.0±0.06

e-Table 1. Additional hemodynamic, echocardiographic and biometric data

Additional hemodynamic, echocardiographic and biometric data. HR= heart rate, EF= ejection fraction, SV= stroke volume, Ees= end systolic elastance, PRSW= preload recruitable stroke work, dP/dt max-Ved= slope of dPdt max- Ved relation, Eed= end diastolic elastance, WSmax = maximum wall stress, PAB= pulmonary artery banding, TAPSE= tricuspid annular plane systolic excursion, LVEDD= left ventricular enddiastolic volume, RVEDD= right ventricular enddiastolic volume, RA= right atrium, LA= left atrium, BW= bodyweight. Values are mean ± SEM. * p<0.05 vs CON; † p<0.05 vs ACS; $ p<0.05 vs PAB; # p<0.05 vs vehicle treated.

6SILDENAFILTREA TMENTINESTABLI SHEDRIGHTVENT RICULARDYSFUN CTIONIMPROVES DIASTOLICFUNCT IONANDAT TENU ATESINTERSTITIA LFIBROSISINDEPE NDENTFROMAFT ERLOADMAJ Borgdorff, B Bartelds, MG Dickinson, P Steendijk, M de Vroomen,

RMF Berger

An adapted version of this chapter is published as:

Am J Physiol Heart Circ Physiol. 2014 May 30. pii: ajpheart.00843.2013. [Epub

ahead of print]

Chapter 6136

ABSTRACTAims

Right ventricular (RV) function is an important determinant of prognosis

in congenital heart diseases, pulmonary hypertension, and heart failure.

Preventive Sildenafil treatment has been shown to enhance systolic RV function

and improve exercise capacity in a model of fixed RV pressure load. However, it

is unknown whether Sildenafil has beneficial effects when treatment is started

in established RV dysfunction, which is clinically more relevant. Our aim was to

assess the effects of Sildenafil treatment on RV function and fibrosis in a model

of established RV dysfunction due to fixed afterload.

Methods and Results

Rats were subjected to pulmonary artery banding (PAB) which induced

RV dysfunction after 4 weeks, characterized by reduced exercise capacity,

decreased TAPSE and RV dilatation. From week 4 onward, 50% of rats were

treated with Sildenafil (100mg/kg/d; PAB-SIL, n=9) or vehicle (PAB-VEH,

n=9). At 8 weeks, exercise capacity was assessed using cage wheels and RV

function using invasive RV pressure-volume measurements under anesthesia.

Sildenafil treatment, compared to vehicle, improved RV ejection fraction

(44±2 vs. 34±2%, p<0.05 PAB-SIL vs. PAB-VEH), reduced RV end diastolic

pressure (2.3±0.5 vs. 5.1±0.9mmHg, p<0.05), and RV dilatation (end systolic

volume 468±45 vs. 643±71 μl, p=0.05). Sildenafil treatment also attenuated RV

fibrosis (30±6 vs. 17±3‰, p<0.05), but did not affect end-systolic elastance or

submaximal exercise capacity.

Conclusion

Sildenafil improves RV diastolic function and attenuates interstitial fibrosis in

rats with established RV dysfunction, independent from afterload. These results

indicate that Sildenafil treatment has therapeutic potential for established RV

dysfunction.

Therapeutic Sildenafil treatment in the pressure loaded RV 137

INTRODUCTIONRight ventricular (RV) failure due to pressure overload is a major determinant

of outcome congenital heart diseases(24) and in pulmonary arterial

hypertension(14). RV function also determines outcome in congestive heart

failure(21, 34). Given the increasing incidence of heart failure as well as the

quickly expanding population of grown-ups with congenital heart disease,

there is a growing need for therapies that specifically support RV function.

Unfortunately, despite a growing interest in the mechanisms underlying RV

failure(2, 18), so far no RV specific therapy is available. Treatments successful

in left ventricular failure might be beneficial in RV failure, but application could

be limited due to the fact that the RV is morphologically, functionally and

embryologically different from the LV(18, 31, 33).

However, recent studies have shown that –like LV failure- experimental RV

failure is associated with ventricular dilatation, impaired systolic and diastolic

function and adverse myocardial remodeling, including hypertrophy and

interstitial fibrosis(2, 7, 31). In LV failure due to pressure load, inhibition of

phosphodiesterase type 5A (PDE5A), has been proven to successfully reduce

hypertrophy and interstitial fibrosis and improve diastolic function(29).

Therapeutic administration of PDE5A inhibitors (e.g. Sildenafil) is now being

tested in several clinical heart failure trials(13, 26).

PDE5A inhibitors have also been successfully used in patients with pulmonary

arterial hypertension(16). In these patients, the beneficial effects of PDE5A

inhibition on the RV might partly be explained by decreased RV afterload,

resulting from the Sildenafil effects on the diseased pulmonary vasculature.

However, there is emerging evidence that Sildenafil also exerts direct beneficial

effects on the pressure loaded RV. PDE5A is activated in the RV of patients

with a pressure loaded RV(23). We have previously shown that Sildenafil

administered from the onset of pressure load (preventive treatment) enhanced

systolic RV function, attenuated ventricular dilatation and limited the decline

in exercise tolerance in a rat model of fixed RV pressure overload, but also

modestly increased interstitial fibrosis(7). Since RV afterload was fixed in

these experiments (pulmonary artery banding is unaffected by the pulmonary

vasodilatory effects of Sildenafil), these findings indicated that Sildenafil directly

affected the RV myocardium.

Chapter 6138

MATERIALS AND

METHODS

In clinical practice however, most patients already present with RV dysfunction,

which disqualifies them for preventive treatment. Therefore, if Sildenafil also

has beneficial effects when started in established RV dysfunction, this would

be very relevant for the clinical setting. The aim of the present study was to

test whether Sildenafil could improve systolic and diastolic function (measured

with echocardiography and pressure volume analysis) and attenuate fibrosis in

a model of established, pressure load induced RV dysfunction. Secondary, we

assessed whether changes in RV function and remodeling were associated with

changes in exercise tolerance (measured as voluntarily run distance). To define

the applicability of Sildenafil treatment in different phases of RV dysfunction we

compared our results with those from the preventive strategy study.







To induce fixed RV pressure overload, pulmonary artery banding (PAB) was

performed on Wistar rats (n=20; male; 160-180g; Charles River, the Netherlands),

which leads to severe RV dysfunction in 4 weeks(7). The animals were randomly

assigned to two groups; PAB-VEH and PAB-SIL. The first four weeks after PAB,

both groups received regular drinking water. See figure 1A for experimental

set-up. At 4 weeks after PAB, RV dysfunction was confirmed by exercise testing

and echocardiography. From four weeks after PAB, the PAB-SIL group received

drinking water to which Sildenafil (Pfizer Inc, New York, NY, United States

of America) was added in a dose (100mg/kg/day) that specifically inhibits

PDE5A(7). In the same study we have shown effective plasma levels resulting

in activation of protein-kinase-G1(7). The PAB-VEH continued to receive regular

drinking water. At 8 weeks after PAB, all rats were evaluated by exercise testing,

echocardiography, and pressure-volume measurements.

Two animals (1 PAB-SIL, 1 PAB-VEH) died prematurely; one of severe bleeding

during surgery, the other died suddenly in the second week after surgery of

unknown cause, but without any sign of RV failure.


Exercise tolerance and clinical signs of failure

To measure voluntary exercise tolerance, running wheels were mounted in the

rat cages, as described previously(7). Five days before surgery, five days before

the 4 wks-mark (halfway the experiment) and 5 days before sacrifice (at 8 wks),

animals were allowed to run in the cage wheel. Running distance was thus

recorded daily using a digital magnetic counter (Commodoor Cycle Odometer,

Commodoor, the Netherlands)(2, 7). Exercise tolerance was measured as

percentage change in running distance at 4 or 8 wks compared to 0 or 4 wks

respectively, for each individual animal.

Throughout the experiment, rats were examined daily for clinical signs of right

ventricular failure according to a predefined ABCDE-checklist as described

previously(7, 8). This ‘ABCDE-criteria’ were defined as follows: A: appearance and

activity, B: bodyweight, C: cyanosis and circulation, D: dyspnea and tachypnea,

E: edema and effusion. The A-symptoms were considered present when the

animal had a ruffled fur, red discoloration of head and neck (due to decreased

cleaning-behaviour) or was less active than previously, despite stimulation.

Body weight symptom was scored if there was a change in bodyweight of more

than 15 grams in <48 hours. Cyanosis was checked at exposed skin on head,

paws and tail. Hampered circulation was considered present if both front paws

and hind legs/tail were pale and markedly colder than previously (regarded

as a consequence of decreased perfusion). Dyspnea and tachypnea were

qualitatively assessed and defined as markedly increased breathing-effort and,

-frequency, respectively. Edema and effusions were defined as fluid collection in

thorax and/or abdomen at euthanization.

Echocardiography

Echocardiography was performed at 4 wks to confirm RV dysfunction and

at 8 wks as described previously(7) using a Vivid Dimension 7 system and

10S-transducer (GE Healthcare, Waukesha, WI, USA). We used apical 3- and 4-

chamber views and parasternal short and long axis views to measure RV and

right atrial dimensions, tricuspid insufficiency, tricuspid annular plane systolic

excursion (TAPSE), and continuous wave Doppler for the gradient across the

PAB. Cardiac output was calculated as aorta diameter)2 × 3.14 × velocity time

integral (VTI) x heart rate, using systolic aorta diameter and pulsed wave

Doppler measurements of aorta flow. Measurements from 6-12 consecutive

beats were used to average out beat-to-beat variation.

Chapter 6140



studies, obtained by right ventricle catheterization using a combined pressure-

conductance catheter (SPR-869, Millar Instruments Inc., Houston, TX, USA) at 8

weeks after surgery according to a previous described protocol(7).

The volume signal of the conductance catheter was calibrated for parallel

conductance and slope factor in order to obtain absolute volumetric values. The

parallel conductance was estimated the hypertonic saline method by infusing

10μL of hypertonic (10%) saline via the jugular vein cannula(1, 19). The slope

factor was calculated as uncalibrated conductance catheter cardiac output

divided by LV cardiac output, measured by echocardiography.

Load independent parameters of contractility (end systolic elastance, preload

recruitable strokework) and diastolic function (end diastolic elastance) are

measured during transient vena cava occlusion and could be obtained in 15

out of 19 animals (n=6/9 for PAB-VEH/PAB-SIL). We calculated RV volume at a

normalized end systolic pressure of 70 mmHg (V70) as an additional measure of

systolic function.

Visual inspection of the EDPVRs revealed that relations were either highly linear

or clearly exponential. Subjective scoring indicated that in the untreated group

4 of 6 EDPVR were clearly exponential, whereas in the treated group only 2 of 9

EDPVRs. The presence of both linear and exponential EDPVR complicates direct

statistical comparison of both groups; slope factor (end diastolic elastance) can

be used for the linear EDPVRs; the exponential coefficient can be used for the

exponential EDPVRs, but neither of them can be used in both. Therfore, to enable

a single, objective analysis of all EDPVRs we used the following approach. Each

EDPVR data set was divided into an upper and a lower volume range, separated

by the median EDV. The upper and lower parts were each fitted with a linear

curve, respectively: Ped = Vo-up + Eed-up x Ved, and Ped = Vo-low + Eed-low x

Ved. For EDPVRs which are essentially linear over the full volume range Eed-up

and Eed-low should be approximately equal and Eed-low / Eed-up would be

expected to be close to 1. For exponential EDPVRs, Eed-up will be substantially

higher than Eed-low and Eed-low/Eed-up clearly less than 1. Thus, this ratio

can be used as a simple, objective linearity index, with 1 indicating linear

relationships and values below 1 (increasingly steeper) exponential curves.


RESULTS


After heart catheterization, the rats were euthanized by removing the heart


septum, LV and both atria were separated and weighed. Tissue sections were

fixated, trans-sectionally cut at 4μm-thick sections and stained with wheat germ

agglutinin (WGA) to assess cardiomyocyte size and with Masson Trichrome

to assess fibrosis. Cardiomyocyte size was measured as average surface area

of cross-sectionally cut cardiomyocytes with a visible nucleus (Image-Pro,

MediaCybernetics, Bethesda, MD, USA) photographed in a trans-section of the

entire RV. The extent of fibrosis was quantified as the blue-stained percentage

of the total tissue area, measured per whole ventricle (Image Scope 11, Aperio

Technologies, Inc. Vista, CA, USA) as described previously(7).

qRT-PCR

The expression of the fetal gene program (myosin heavy chain isoforms,

natriuretic pro peptides type A and B) and markers of hypertrophy (acta1, RCAN)

was measured to characterize the hypertrophy response and the effects of

Sildenafil. Total RNA was extracted using the RNeasy fibrous tissue kit (Qiagen),

following the manufacturer’s guidelines. Data were normalized to reference

gene 36B4.


All quantitative data were tested for normality and are expressed as

mean±standard error of the mean (SEM). PAB-VEH versus PAB-SIL differences

were evaluated using Students t-tests or Mann-Whitney U tests as appropriate.

Group size is 8-9, except when specified otherwise. P<0.05 was considered

significant (PASW Statistics 18 for Windows, SPSS, Chicago, Illinois).

Pulmonary artery banding induced RV dysfunction

At week 4, before treatment was started, PAB had induced fixed RV pressure

load (PAB gradient 53±4mmHg), which led to RV dysfunction. RV dysfunction

was characterized clinically by decreased exercise tolerance (running distance

vs. baseline: -57±8%). Echocardiography showed low TAPSE (2.1±0.1mm, normal

value ~3 mm), right ventricular dilatation (end diastolic diameter: 4.8±0.2mm,

Chapter 6142

normal value ~3.5 mm) and right atrial enlargement (maximal long-axis diameter:

4.3±0.2 mm, normal value ~3 mm(9). After this clinical and echocardiographic

evaluation, the rats in the PAB-SIL group started with Sildenafil treatment.

Before treatment, there were no differences in characteristics, including RV

function parameters between PAB-SIL and PAB-VEH at 4 weeks (Table 1).

The changes to 4 weeks of PAB were similar to those described in previous

studies(7, 9). In these studies, a similar magnitude of PAB led at 4 weeks to

increased contractility as measured by end systolic elastance (+164%), RV

dilatation measured by an increase in end diastolic volume (+48%), as well

as early phase deterioration of diastolic function measured by end diastolic

elastance.

PAB-VEH PAB-SIL p-value

Echocardiographic parametersPAB gradient (mmHg) 49±6 56±4 0.33HR (/min) 315±8 347±15 0.10CO (mL/min) 56±5 75±9 0.12TAPSE (mm) 2.1±0.2 2.1±0.3 0.94RVEDD (mm) 5.0±0.2 4.6±0.4 0.48RA diameter (mm) 4.6±0.2 4.1±0.4 0.34

Sildenafil beneficially affected RV dysfunction

After 8 wks, due to growth of the animals, PAB gradients had increased (74±3

vs. 77±5mmHg, PAB-VEH vs. PAB-SIL, ns). Sildenafil treatment improved ejection

fraction and reduced end systolic volume (Ves) compared to untreated rats

(Fig 1B,C) but did not significantly affect end diastolic volume (Table 2). In the

untreated rats, RV pressure-volume loops showed ventricular dilatation (Fig

2A). In the PAB-SIL group the pressure volume-loops showed a leftward shift on

the volume axis, reflecting the (non significant) reduced ventricular dilatation

(Fig 2A). Contractility, assessed by end systolic elastance (Ees) and preload

recruitable stroke work (PRSW), did not significantly differ between the two

groups (Table 2). The RV-PA coupling, expressed as Ees/Ea, tended to increase

with Sildenafil treatment although the changes failed to reach statistical

Table 1- Echocardiographic characteristics at start of Sildenafil therapy

PAB-VEH= untreated pulmonary artery banding, PAB-SIL= PAB treated with Sildenafil, PAB= pulmonary artery banding, HR= heart rate, CO= cardiac output, TAPSE= tricuspid annular plane systolic excursion, RVEDD= right ventricular end diastolic diameter, RA= right atrium.Values are mean ±SEM.


significance (0.68±0.15 vs. 0.82±0.15, PAB-VEH vs. PAB-SIL, p=0.054. However,

Sildenafil treatment improved diastolic function: the end diastolic pressure

was significantly lower in the Sildenafil treated group (Fig 1D) and the end

diastolic pressure-volume relationship (EDPVR) differed markedly between the

two groups (Fig 2B): in the majority of untreated animals (4/6; 66%) EDPVRs

were shifted upwards and displayed a clearly exponential behavior, indicating

increased ventricular stiffness. In contrast, in the Sildenafil treated animals

almost all RVs had relatively low end diastolic pressures and a normal linear

EDPVR (2/9; 22%) (Fig 2B). To enable comparison of linear and non-linear

EDPVRs (neither end diastolic elastance nor exponential coefficient can be used

in both) we divided the EDPVR data set of each animal into an upper and lower

volume range, separated by the median Ved, and determined the slope (Eed)

for each part with a linear fit (see Methods section). The ratio of Eed-low/Eed-

up was used as an index for linearity (with 1 indicating a linear EDPVR). In the

untreated rats, the linearity index was 0.35±0.12; in the Sildenafil treated rats

0.90±0.14 (p=0.01)(Fig 1D), indicating significantly improved diastolic function.

The improvement in RV function by Sildenafil could not be correlated with

improvements in exercise tolerance (Table 2) and symptoms of RV failure were

seen in both groups (all animals in both groups were tachypneic and showed

decreased cleansing behaviour); none of the rats needed to be terminated

prematurely because of severe RVF symptoms.

Sildenafil attenuated RV fibrosis, but not RV hypertrophy

Sildenafil attenuated RV myocardial fibrosis (29±5‰ vs. 17±3‰, PAB-VEH vs.

PAB-SIL, p<0.05). This difference was not due to severity of loading, as the

amount fibrosis per mmHg RV peak pressure was also decreased by Sildenafil

(Fig 3A-C). The degree of interstitial fibrosis correlated with end diastolic

pressure (R2=0.587, p=0.001) (Fig 3D), suggesting interaction between fibrosis

and diastolic function.

Pressure load led to hypertrophy as shown by RV weight (1.2±0.1mg/g

bodyweight) (Table 2) and RV cardiomyocyte cross sectional surface area

(0.13±0.01μm²)(7). The degree of RV hypertrophy in the Sildenafil treated

animals did not differ from that in the untreated animals (Table 2).

No difference in expression of RV remodeling associated genes (NPPA, NPPB,

acta 1, RCAN1, MYH7/MYH6) could be demonstrated between the treated and

untreated groups (data not shown).

Chapter 6144

Figure 1 Functional effects of 4wks of treatment with Sildenafil at 8 wks of pulmonary artery banding.

All indices measured by pressure-conductance catheter. A Study design, a schematic illustration. B Ejection fraction. C End systolic volume (Ves). D End diastolic pressure (Ped). E Linearity index of the end diastolic pressure volume relations (Eed_low/Eed_up). Mean±SEM * p<0.05.

PAB-VEH PAB-SIL p-value

Pressure-volume parametersHR (/min) 283±12 283±11 1.00CO (mL/min) 89±4 102±5 0.07Ees (mmHg/μL) •1000 96±19 124±25 0.38PRSW (mmHg) 39±12 41±7 0.89

Voluntary exerciseChange at 8 wks vs. 4 wks (%) -46±4 -45±5 0.94Change at 8wks vs. baseline (%) -79±10 -81±13 0.74

Organ weightsRV weight/BW (mg/g) 1.2±0.1 1.1±0.1 0.70LV+IVS weight/BW (mg/g) 2.1±0.03 2.1±0.05 0.60RA weight/BW (mg/g) 0.31±0.08 0.26±0.06 0.25BW (g) 423±15 442±21 0.48

Table 2 Pressure-volume parameters, exercise and organ weights at 8 weeks

PAB-VEH= untreated pulmonary artery banding, PAB-SIL= PAB treated with Sildenafil, HR= heart rate, CO= cardiac output, Ees= End systolic elastance, PRSW= preload recruitable stroke work, RV= right ventricle, BW= bodyweight, LV= left ventricle, IVS= interventricular septum, RA= right atrium. Values are mean ±SEM.


Figure 3 Effects of Sildenafil on fibrosis.

A Representative examples of Masson-Trichrome-stained right ventricles of untreated (PAB-VEH, n=7) and treated (PAB-SIL, n=9) rats with a PAB. Ruler is 125μm. B Permillage RV fibrosis per unit surface area. C Permillage RV fibrosis normalized for RV peak pressure (Ppeak). D Scatter plot showing correlation between fibrosis permillage and end diastolic pressure (Ped). Mean±SEM, * p<0.05

Figure 2 Pressure-volume analysis of Sildenafil or vehicle in pulmonary artery banding.

A Representative pressure-volume loops of untreated (PAB-VEH, upper panel) and treated (PAB-SIL, lower panel) rats with a PAB during vena cava occlusion. End systolic pressure-volume relations marked by solid black lines, end diastolic pressure- volume relations marked by dashed black lines. B Overview of end diastolic pressure-volume relations obtained during vena cava occlusion in PAB-VEH (black, n=6) and PAB-SIL (gray, n=9). The pressure-volume loops in the left upper corner are shown to indicate the area of the pv-loops that is enlarged.

Chapter 6146

DISCUSSIONIn this study in rats with fixed RV pressure overload, we show that Sildenafil

treatment, initiated in established RV dysfunction, leads to reduced end diastolic

pressure, altered end diastolic pressure volume relations, improved ejection

fraction and attenuated RV fibrosis. These beneficial effects on the RV were not

accompanied by a better voluntary exercise tolerance. These data demonstrate

that Sildenafil has a direct beneficial effect on the diastolic function of the

pressure-loaded RV independent from its afterload; the pulmonary vasodilatory

effects of Sildenafil are negated by the pulmonary artery banding. This study is

the first to show that Sildenafil may be effective in the treatment of established

RV dysfunction.

Sildenafil improves diastolic dysfunction in established RV dysfunction

Under normal physiological conditions the RV is coupled to the pulmonary

vascular bed, which is characterized by low resistance and high compliance.

When RV afterload increases, e.g. due to pulmonary hypertension or congenital

heart diseases, the RV initially responds with increased contractility and

RV hypertrophy. Eventually, RV adaptation progresses into RV dysfunction

and failure. Although the mechanisms of RV dysfunction are incompletely

understood, diastolic dysfunction is thought to be important in the progression

to RV failure(17, 25). Clinical studies show that increased right atrial pressure (as

an indirect measure of diastolic RV function) is associated with poor outcome

in pulmonary arterial hypertension(3). The importance of diastolic dysfunction

is confirmed in experimental studies showing diastolic dysfunction while

contractility (end systolic elastance, PRSW) is preserved(7, 15).

Using pressure-volume measurements, we here show that Sildenafil beneficially

affects the end diastolic pressure-volume relation (EDPVR) and lowers end

diastolic pressure. The EDPVR reflects intrinsic diastolic stiffness(11). In humans

the RV EDPVR cannot be recorded accurately but we, and others, have previously

shown that within a physiological range, the EDPVR in a normal rat RV is linear(7,

15, 19).

The observed linear-to-exponential transformation of the EDPVR reflects

increased stiffness of the diseased RV. In the low volume range, compliant

elastin fibers and titin-molecules are being stretched, resulting in shallow slope

of the EDPVR. In the higher volume range, slack length of titin and collagen

fibers is exceeded, resulting in much steeper slope of the EDPVR(11). Sildenafil


treatment prevents the linear-to-exponential transformation of the EDPVR.

This in turn, results in lower end diastolic pressures, highlighting the improved

diastolic function. Since diastolic dysfunction is a prominent feature of RV failure,

these beneficial effects of Sildenafil on diastolic function may have therapeutic

merit for patients with advanced RV dysfunction.

Figure 4 Summary of Sildenafil effects in the early (0-4 wks) and late (4-8 wks) stage of pressure load-induced RV dysfunction.

The data set of the effects of Sildenafil on rats with a PAB at week 4 are derived from our previous study(7), in which Sildenafil treatment was started at the day of surgery (preventive strategy). A End systolic elastance (Ees). B End diastolic elastance (Eed). To allow comparison of Eed between the studies, the Eed of the initial 2 mmHg end diastolic pressure drop during occlusion was used here (see Methods on characterization of diastolic function). C Permillage RV fibrosis per unit surface area. D Ejection fraction. Mean±SEM, * p<0.05

In addition to the observed improvement in diastolic performance, parameters

of systolic function and ventricular-arterial coupling tended to improve with

Sildenafil, although these changes failed to reach statistical significance. These

slight improvements in systolic performance might have contributed to the

Chapter 6148

increased cardiac output measured in this study. An improvement of ventriculo-

arterial coupling (Ees/Ea) in this PAB-model of fixed RV-afterload might be

interpreted as either improved efficiency of ventricular performance at its fixed

afterload or as a functional adaptation in the pulmonary trunk proximal to the

PAB.

Potential mechanisms of remodeling

The exponential behavior of the EDPVR observed in the untreated rats, is not

secondary to a shift towards a high volume range, but also occurs at relatively

normal volumes. These results suggest that Sildenafil targets ventricular

remodeling, rather than just preventing ventricular dilatation. Sildenafil

enhances PKG1 activity, as shown in our previous study(7), which may affect

relaxation via phosphorylation of titin(20). Another important component of

RV dysfunction in experimental models is fibrosis(5, 8). Fibrosis plays a central

role in the adaptation of the ventricle to stress in general and pressure load

in particular(4, 32). In congenital heart disease and pulmonary hypertension,

interstitial fibrosis has been shown to contribute to diastolic dysfunction(10,

12, 25). In the present study the reduction of fibrosis in the Sildenafil treated

rats was strongly related to the reduction in end diastolic pressures (Fig 3D),

suggesting an important component of the positive effects. Whether Sildenafil

has additional beneficial mechanisms to explain the improved hemodynamics

remains to be explored.

Clinical Implications

Sildenafil is increasingly being used in the treatment of various types of

cardiovascular disease(28). However, the place of PDE5A inhibitors in the

treatment of RV dysfunction to fixed pressure overload is yet to be determined.

Comparing with the results of preventive treatment strategy(7) indicates

differences and similarities in reponse (summarized in Fig 4). Whereas the

preventive strategy primarily further enhances parameters of contractility (Ees),

the reversal strategy affects diastolic function (end diastolic pressure volume

relations, end diastolic pressure), although Ees/Ea also appeared to subtly

improve. One explanation of the less pronounced effect on contractility could be

that contractility is almost maximally enhanced already. In contrast, a prominent

feature of (more progressed) RV dysfunction is diastolic dysfunction. This study


is the first to report a beneficial effect on diastolic dysfunction in a model of fixed

afterload, which circumvents the potential effects on the pulmonary vasculature.

Previous studies in rats have shown that the degree of RV dysfunction presented

in this study (reduced cardiac output at rest, RV dilatation, reduced exercise

tolerance) can be clinically tolerated for an extended period of time(6). Similarly,

in patients with congenital heart diseases increased RV afterload may be

clinically well tolerated (30). However, also similar to patients with a systemic

right ventricle, PAB rats exhibited further decline in RV parameters and clinical

function, i.e. RV dilatation and reduced exercise capacity(30).

These analyses also reveal that Sildenafil may be associated with increased

fibrosis in the early stage, whereas in the later stage of the disease, when

fibrosis is a more prominent feature of RV remodeling, Sildenafil attenuates

fibrosis in the RV (Fig 4). In both strategies, preventive and reversal, Sildenafil

limits ventricular dilatation(7) and has a positive effect on ejection fraction. The

positive effects of Sildenafil in our rats with a pressure load appear to be in

contrast with the recently reported negative results of the RELAX trial, studying

the long term effects of Sildenafil on patients with HF-PEF(26). However, in the

RELAX trial, pulmonary artery pressure was only mildly elevated (41 mmHg),

hence RV dysfunction was probably also mild. Redfield et al suggested that

RV dysfunction has to reach a certain limit for Sildenafil to become effective

in ventricular remodeling, a dose-effect relation that has been previously

suggested from studies in mice with a LV load(22). Therefore, further trials are

warranted to study the long term effects of PDE5inhibition in RV and LV failure

(SIL-Hf trial:(13) ; PITCH-HF trial (I.D: NCT01910389)).

It is unclear why these functional changes did not result in an improvement

of exercise tolerance. Exercise tolerance as measured with voluntary cage

wheel exercise can be compared with clinically often used 6-minute walking

distance test (6MWT) rather than a maximal exercise test. The 6MWT is a

valuable outcome parameter in many clinical trials in pulmonary hypertension

or heart failure(16, 27). In all clinical trials in pulmonary hypertension, Sildenafil

treatment was associated with an improvement in 6MWT, however, in all these

studies the pulmonary vascular resistance or pulmonary artery (PA) pressure

also decreased(16), suggesting that the effect might be due to reduced

afterload, hence improved RV-PA coupling. In the current study, the RV afterload

Chapter 6150

FUNDING

ACKNOWLEDGE-MENTS

was fixed due to the pulmonary artery banding. We suggest that the persistence

of afterload explains the lack of effect on exercise tolerance, despite preserved

contractility and improved diastolic function.

Limitations

Our model of rats with RV dysfunction due to a fixed afterload comes with some

limitations, which should be discussed. Firstly, we did not address the effects

of different dosages of Sildenafil, which should be performed in future studies.

Secondly, our relatively small animal study represents hypothesis generating

groundwork, based on which mechanistic studies and larger (clinical) trials

can be designed. Thirdly, no invasive pressure-volume measurements were

performed at 4 weeks, as the extent of this procedure precludes survival of the

animals. However, data from our previous studies in this model clearly show

RV dysfunction at 4 weeks(7), which is confirmed by the echocardiographic

measurements we performed in the present study. Lastly, survival analysis

would have provided supplemental information on the therapeutic potential

of Sildenafil in this disease model. Even so, the strong effects of Sildenafil on

hemodynamics and fibrosis observed in the current and previous study(7)

prompt further clinical study on Sildenafil in the fixed pressure loaded RV.

Conclusion

In this study in rats with fixed RV pressure overload, we show that Sildenafil

treatment, in established RV dysfunction, reduced end diastolic pressure, altered

end diastolic pressure volume relations, improved ejection fraction and limits

fibrosis. These data demonstrate that Sildenafil has a direct beneficial effect on

the pressure-loaded right ventricle independent from effects on RV afterload.

These results indicate that Sildenafil treatment has therapeutic potential for

treatment of established RV dysfunction.

This work was supported by the Sebald foundation and a grant from the Dutch

Heart Foundation (2007T068).

The authors are indebted to M. Weij for excellent surgeries and B. Boersma and

M. Dokter for expert technical assistance.


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7ACORNERSTONE OFHEARTFAILUR ETREATMENTISN OTEFFECTIVEINE XPERIMENTALRI GHTVENTRICULA RFAILURE

MAJ Borgdorff, B Bartelds, MG Dickinson, P Steendijk, RMF Berger

Int J Cardiol. 2013 Nov 5;169(3):183-9.

doi: 10.1016/j.ijcard.2013.08.102. Epub 2013 Sep 7.

Chapter 7156

ABSTRACTBackground

Right ventricular (RV) failure due to increased pressure load causes significant

morbidity and mortality in patients with congenital heart diseases and

pulmonary arterial hypertension. It is unknown whether renin-angiotensin-

aldosterone-system (RAAS) inhibition (the cornerstone of left ventricular failure-

treatment) is effective in RV failure. We investigated the effects of combination

treatment of aldosterone-blocker eplerenone + angiotensin II-receptor blocker

losartan (Ep/Lo) on RV remodeling and function in a model of RV failure due to

increased pressure load.

Methods and Results

Rats (n=48) were randomized for pulmonary artery banding (PAB) or sham

surgery and for losartan (20mg/kg/d)+eplerenone (100mg/kg/d) treatment

(Ep/Lo) or vehicle (VEH). RV function was assessed by echocardiography and

pressure-volume analysis at 5 and 11 weeks, or at the occurrence of clinical RV

failure symptoms necessitating termination.

PAB resulted in RV failure in all rats, as defined by reduced cardiac output, RV

stroke volume, increased RV end diastolic pressure and liver congestion as well

as RV fibrosis, hypertrophy and reduced capillary density. Clinical RV failure

necessitated termination in 5/12 PAB-VEH rats. Angiotensin II type 1-receptor

expression in the RV was reduced in PAB rats indicating local RAAS activation.

Treatment of PAB rats with Ep/Lo significantly lowered arterial pressures, but

had no significant effect on RV function, remodeling or survival compared to

PAB-VEH rats.

Conclusions

RAAS-inhibition does not beneficially affect experimental RV failure due to

chronic pressure load. This is of high clinical relevance, because it indicates that

the RV might respond fundamentally different to RAAS-inhibition than the LV.

A cornerstone of heart failure treatment is not effective in RV failure 157

INTRODUCTIONRight ventricular (RV) failure due to pressure load is a primary risk factor for

early mortality and morbidity in patients with congenital heart diseases and

the main cause of death in pulmonary arterial hypertension(1-4). Despite the

recognized clinical importance of preserving RV function, the mechanisms of

RV dysfunction and failure are yet unknown and as a consequence there are no

clinically established treatments for RV failure(5). This is in sharp contrast with

left ventricular (LV) failure(6,7) and it is tempting to extrapolate proven treatment

strategies for LV failure to the RV. This is, however, associated with a number

of potential hazards. The RV differs functionally and morphologically from the

LV(8,9) and the RV derives embryologically from a distinct set of precursor

cells(10), implicating that RV cardiomyocytes might respond fundamentally

different to stress(5,11,12). Furthermore, the RV is coupled to the pulmonary

circulation, physiologically a low pressure, high compliance circulation with

different properties than the systemic circulation, which might affect the RV

response to commonly used LV drugs(13). However, the implications of these

differences between RV and LV for the treatment of RV failure remain largely

speculative and the effects on RV failure of proven treatment strategies for LV

failure are insufficiently studied(14).

One of the cornerstones in the treatment of LV failure is inhibition of an over-

activated renin-angiotensin-aldosterone-system (RAAS) with an angiotensin II

converting enzyme-inhibitors (ACEi) or angiotensin II receptor-blocker (ARB)

combined with an aldosterone receptor-blocker(6,7). RAAS inhibition has been

shown to improve LV function and attenuate adverse remodeling (fibrosis,

hypertrophy, ventricular dilatation) in (pre)clinical studies of LV failure(15-19).

Clinical studies suggest that RAAS over-activation might also play a role in RV

adaptation to various forms of abnormal loading and RV failure(20). Such data

suggest patients with a systemic RV to be prime candidates for RAAS inhibiting

treatment, as their RV is chronically pressure loaded and prone to failure.

However, trials of ACEi (21,22) or ARBs (23-25) in this patient group, reported

negative results. Most of these studies were limited by insufficient power, short-

follow up or retrospective set-up(26).

Preclinical data could provide a proof-of-principle that RAAS inhibition is

beneficial for the chronically pressure loaded RV. Unfortunately, data regarding

the in vivo functional effects of RAAS-inhibition on the pressure loaded RV are

Chapter 7158

MATERIALS AND

METHODS

lacking. Therefore, we tested both functional and histological effects of long-

term combined pharmacological inhibition of the angiotensin II receptor (type

1) and the aldosterone receptor by losartan/eplerenone treatment in a model

of pressure load induced RV failure.

We hypothesized that this clinically applicable treatment would attenuate

remodeling and, as a consequence, sustain RV function and prevent RV failure.







Wistar rats (n=48; male; 160-180g; Charles River, the Netherlands) were

randomized to pulmonary artery banding (PAB) or sham surgery. PAB (n=33) was

performed to induce RV pressure overload, as described previously(27), except

that for this study we used a tighter PAB size (19G; 1.1.mm instead of 1.3mm).

Three rats died during PAB surgery (1 severe bleeding, 1 acute RVF, 1 trachea

perforation). The remaining 30 animals with PAB were randomly assigned to a

group of 15 rats to receive vehicle or a group of 15 rats to receive eplerenone

and losartan. Inadvertedly, the last three animals assigned to the vehicle-group

received eplo-treatment from the start, resulting in a vehicle-treated group

(PAB, n=12) and an eplerenone and losartan treated group (PAB-eplo, n=18).

After randomization, pairs were made between both groups, so that each PAB

rat had a paired PAB-eplo rat, which made it possible to terminate the rats pair-

wise in case of clinical deterioration. Sham operated animals (n=15) underwent

the PAB procedure without the actual banding of the pulmonary artery and

served as control (CON), and were randomized into vehicle-treated (CON, n=7)

and treated (CON-eplo, n=8) groups.

Termination

Rats were terminated when clinical RVF (see Definition of clinical RV failure

below) developed. Along with the failing rat, the paired rat was terminated at

the same time point. At 11 weeks after surgery, all remaining rats (all CON(-eplo)

rats and the remaining PAB(-eplo) rats) were terminated.


One rat (in the PAB-eplo group) developed an abdominal tumor within two

weeks after PAB and was terminated. This animal had severe unilateral

hydroureteronephrosis and was excluded from further analysis.

Definition of clinical RV failure

Rats were examined daily for clinical signs of RV failure according to a previously

described checklist examining for appearance, activity, bodyweight changes,

peripheral circulation, cyanosis, dyspnea/tachypnea and edema/effusions.

Clinical RV failure was defined as the presence of at least: inactivity, ruffled

fur, severe dyspnea and palpable ascites. The decision whether RV failure was

present or not was made by 2 experienced observers who were blinded to the

experimental group of the rats(27).

RAAS-inhibiting treatment

Treatment was given from the moment of surgery onward and consisted of

the combination of angiotensin II receptor (type 1) blocker losartan (20mg/

kg BW/d)(28,29) via the drinking water and mineralo-corticoid receptor

blocker eplerenone (100mg/kg BW/d)(15,30) mixed in conventional rat chow,

which have been previously shown to be effective dosages in models of LV

disease(15,28-30). The untreated groups received conventional rat chow and

regular drinking water throughout the experiment.

Echocardiography

Transthoracal echocardiography was performed under general anesthesia

(isoflurane/air mixture: 5% induction, 2-3% maintenance) in all animals at 5

weeks after surgery and at termination as described previously (27) using a Vivid

Dimension 7 system and 10S-transducer (GE Healthcare, Waukesha, WI, USA).

We used apical 3- and 4- chamber views and parasternal short and long axis

views to measure RV and right atrial dimensions, tricuspid insufficiency, tricuspid

annular plane systolic excursion (TAPSE), and continuous wave Doppler for the

gradient across the PAB. Cardiac output was calculated as (aorta diameter)2 ×

3.14 × velocity time integral x heart rate, using systolic aorta diameter and

pulsed wave Doppler measurements of aorta flow. Measurements from 6-12

consecutive beats were used to average out beat-to-beat variation.

Chapter 7160

Heart catheterization

Hemodynamic assessment of the RV was performed by pressure-volume

analysis, obtained at termination by RV catheterization according to a previously

described protocol(27).

Briefly, rats were anesthetized (isoflurane/air mixture, 5% induction; 2-3%

maintenance), intubated and ventilated. Following bilateral thoracotomy and

pericardiotomy a pressure-conductance catheter (SPR-869, Millar Instruments

Inc., Houston, TX, USA) was introduced into the RV apically and positioned in the

RV outflow tract. RV pressures and conductance were recorded using a MPVS

400 processor at a sample rate of 1.000 Hz with Chart 5 (Millar Instruments

Inc., Houston, TX, USA). Analyses were performed offline using custom-made

software (CircLab 2012, P. Steendijk). Stroke volume (in mL) measured by

echocardiography was used to calibrate stroke volume (in arbitrary units) derived

from the conductance signal. End systolic and end diastolic elastance were

determined using the single-beat method(31). Following the RV measurements,

the catheter was introduced in the aorta and the LV via the right carotid artery

to measure systemic pressures.


After heart catheterization, the rats were terminated by excising the heart-lung

block from the thorax. Heart, lungs and liver were dissected. RV, interventricular

septum, LV and both atria were separated and weighed. The liver lobe and lung

lobe were weighed, dried overnight at 65°C and weighed again to determine wet

weight/dry weight ratio. Midventricular RV sections were fixated (formalin) and

stained to assess cardiomyocyte cross-sectional area (wheat germ agglutinin),

fibrosis (Masson Tri-chrome) and capillary density (lectin) as described

previously(27,32).

Gene expression of RAAS and remodeling

To assess activation of the local RAAS, mRNA expression of the angiotensin II

receptors type 1 and 2 (AT1R and AT2R) were measured. To study the underlying

mechanisms of putative effects of Ep/Lo treatment the expression of key markers

of the fetal gene program (myosin heavy chain isoforms, natriuretic pro peptides

type A and B) were measured, as well as genes involved in myocardial remodeling:

hypertrophy (ACTA, RCAN1), fibrosis (TGFβ-1, OPN-1, Col1A2, Col3A1) and


RESULTS

oxidative stress (HO-1, NOX-4). RV (free wall) tissue was snap-frozen in liquid

nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen Corporation,

Carlsbad, CA, USA); high quality was confirmed (RQI 9.3) using Experion (Bio-

Rad, Veenendaal, the Netherlands), before conversion to cDNA by QuantiTect

Reverse Transcription (Qiagen, Venlo, the Netherlands). Gene expression was

measured with Absolute QPCR SYBR Green ROX mix (Abgene, Epsom, UK) in the

presence of 7.5ng cDNA and 200nM forward and reverse primers. qRT-PCR was

carried out on the Biorad CFX384 (Bio-Rad, Veenendaal, the Netherlands) using

a standard protocol of maximally 35 cycles. Primer sequences are available upon

request. mRNA levels are expressed in relative units based on a standard curve

obtained by a calibrator cDNA mixture. All mRNA levels were corrected for 36B4

reference gene expression.



CON versus PAB differences were evaluated using Students t-test or Mann-

Whitney U test as appropriate. Treatment effects were tested by ANOVA with

Bonferroni post-hoc testing for multiple comparisons or Fisher’s Exact Test as

appropriate. Group sizes were 7 (CON); 8 (CON-eplo); 12 (PAB); 17 (PAB-eplo),

unless specified otherwise. P<0.05 was considered significant (PASW Statistics

18 for Windows, SPSS, Chicago, Illinois).

Model characterization: Pulmonary artery banding induces RV failure in

vehicle-treated rats

In vehicle-treated rats, pulmonary artery banding resulted in severe pressure

overload which induced (sub)clinical RV failure, characterized by reduced

cardiac index (Fig 1A) and stroke volume (Fig 1B), reduced TAPSE (Fig 1C), RV

dilatation (Fig 1D), tricuspid insufficiency (Fig 1E), right atrial enlargement (Fig

1F), increased RV end diastolic pressure (Fig 1G) and liver congestion (Fig 1H).

Five of the 12 untreated rats developed overt clinical RVF within 11 weeks after

PAB surgery, which necessitated termination. Local RAAS was activated in the

RV, indicated by downregulation of AT1-receptor mRNA (Fig 2). AT2-receptor

mRNA expression was not detectable at the maximum of 35 PCR cycles (Fig 2).

Chapter 7162

Figure 1. Model characterization: Pulmonary artery banding induces RV failure in vehicle-treated rats

A Cardiac index B RV stroke volume C tricuspid annular plane systolic excursion (TAPSE) D RV end diastolic diameter (RVEDD) E percentage of rats with tricuspid insufficiency (TI) F right atrial diameter (RA) G RV end diastolic pressure (EDP) H liver wet weight: dry weight ratio. All parameters measured by echocardiography, except EDP, which was measured by catheterization. Mean±SEM. * indicates p<0.05 between groups. CON= control, PAB= pulmonary artery banding, both vehicle-treated

Figure 2. mRNA expression of angiotensin II receptor type 1 (AT1R) and 2 (AT2R)

AT1R (left panel) was downregulated in PAB. AT2R (right panel) was not detectable (nd) at the 35th PCR cycle. Mean±SEM. * indicates p<0.05 between groups. CON= control, PAB= pulmonary artery banding


This RV failure phenotype was accompanied by pathological remodeling,

including threefold increment of myocardial fibrosis (Fig 3A), RV hypertrophy

(expressed as RV weight/tibia length (Fig 3B) or cardiomyocyte cross-sectional

area (Fig 3C), reduced capillary density (Fig 3B-D) and upregulation of

hypertrophy related genes and the fetal gene program (Table 2).

Figure 3. Fibrosis, hypertrophy and capillary density

A RV fibrosis (representative images in two top rows of pictures: Masson-Trichrome stained RVs, ruler is 1mm, black box width 1.3mm) B RV free wall weight normalized for tibia length (measure of RV hypertrophy) C RV cardiomyocyte cross-sectional area (third row of pictures: representative images of RV sections stained with a membrane marker (wheat germ agglutinin, green), ruler is 125μm. D RV capillary density, expressed as number of capillaries per 100*100μm (bottom row: representative images of RV sections stained with capillary-marker lectin, ruler is 125μm). Mean±SEM. * indicates p<0.05 vs. CON. CON= control, PAB= pulmonary artery banding (untreated), PAB-eplo= PAB treated with eplerenone/losartan

Chapter 7164

Eplerenone/Losartan treatment effects

Ep/Lo significantly reduced left ventricular peak pressure and aortic systolic and

diastolic blood pressure in PAB (Fig 4A-C). However, Ep/Lo treatment did not

have any significant effect on RV hemodynamics (Table 1, S1), representative

pressure-volume loops in Fig 5A-C. Contractility (end systolic elastance), active

relaxation (tau) and passive diastolic properties (end diastolic elastance, end

diastolic pressure) were unaffected by Ep/Lo (Table 1). Neither did Ep/Lo prevent

dilation of the RV and RA, tricuspid insufficiency or liver congestion (Table 1).

In line with this lack of hemodynamic benefit, Ep/Lo treatment did not delay

(Fig 5D) or prevent development of RV failure (5/17 vs. 5/12, PAB vs. PAB-eplo,

p=0.494).

Myocardial fibrosis and RV hypertrophy were not prevented by Ep/Lo treatment

(Fig 3A-C). Ep/Lo also did not affect capillary density (Fig 3D). In line with this,

expression of genes of the fetal gene program and genes related to hypertrophy,

fibrosis and oxidative stress were unaffected by Ep/Lo treatment (Table 2).

All parameters in CON and CON-eplo were equal (p=ns, data not shown).

Figure 4. Effects of treatment on systemic pressures

A left ventricular (LV) peak pressure B aorta maximum pressure C aorta minimum pressure. Mean±SEM. * indicates p<0.05 vs. CON; † indicates p<0.05 vs. PAB. CON= control, PAB= pulmonary artery banding (untreated), PAB-eplo= PAB treated with eplerenone/losartan


Figure 5. Pressure-volume analysis

A-C Representative pressure-volume loops of CON, PAB and PAB-eplo. End systolic pressure volume relations during vena cava occlusion indicated by solid lines, end diastolic pressure volume relations indicated by dashed lines. D Survival analysis. RVF-mortality/total animals per group CON: 0/7, CON-eplo: 0/8, PAB: 5/12, PAB-eplo: 5/17. Both PAB groups had significantly reduced survival (p<0.05), but there was no significant difference between PAB and PAB-eplo (p=0.39). CON= control (untreated), CON-eplo= CON treated with eplerenone/losartan, PAB= pulmonary artery banding (untreated), PAB-eplo= PAB treated with eplerenone/losartan

In this study we assessed the effects of proven LV failure treatment, the

combination of eplerenone and losartan, in a rat model of RV failure due

to chronic pressure load. We found that Ep/Lo neither prevented adverse

remodeling, nor clinical RV failure nor affected RV function.

Our findings show that RAAS-inhibition does not beneficially affect experimental

RV failure due to chronic pressure load, which is in strong contrast to previous

DISCUSSION

Chapter 7166

findings in the left ventricle(15,33-37). These data indicate that response of the

pressure loaded RV to RAAS-inhibition might differ fundamentally from that of

the (pressure loaded) LV, which may have highly relevant clinical consequences.

Eplerenone and losartan: works in the LV, not in the RV?

Pharmacotherapeutic guidelines developed for treating LV failure serve as a

roadmap in the search for effective treatments for RV failure. Even though an

increasing catalog of clinical studies of ACEi (21,22) or ARBs (23-25) in patients

with a pressure loaded RV has reported negative results, the paradigm remains

that RAAS inhibition should work in RV dysfunction. The negative results of the

studies are assumed to be attributable to insufficient power, short-follow up

or retrospective set-up of these studies(26). Although the putative benefits of

RAAS inhibition in this population certainly should not dismissed at this point,

the results of our study suggest a more fundamental explanation for the lack of

clinical effect.

A distinctive characteristic of the current study is the severity of the PAB model

which, in contrast to previously described PAB models(38-40), induces a clear

phenotype of clinical and functional RV failure. Previous studies of ACEi and

ARB in PAB models showed no effect on RV hypertrophy(41-44) in compensated

RV pressure loading. Our study adds the clinically relevant notion that Ep/Lo

treatment does not affect RV remodeling nor function in severe pressure load

induced RV failure. PAB rats had reduced cardiac output, activation of the

systemic RAAS (confirmed by blood pressure effect of Ep/Lo), and activation

of local RAAS (confirmed by downregulation of the AT1-receptor(45)). The

dosages of Ep/Lo and the administration regimens that were used in the current

study have been shown to effectively target LV disease(15,16,28,29). The lack

of treatment effects then, suggests that the RV responds differently to RAAS

inhibition than the LV.

The contribution of the RAAS to LV remodeling and function in fixed LV pressure

load is well established(46,47). Multiple studies show that RAAS inhibition

with losartan or eplerenone can attenuate remodeling and improve function

in the aortic constriction model(15,17,33-37). In contrast, the contribution of

RAAS to RV remodeling and function is insufficiently studied. RV pressure load


activates local RAAS, even in the absence of systemic RAAS activation in mild

PAB(41). However, blocking local RAAS activation by losartan did not prevent

remodeling or improve papillary length-tension relationship(41). The current

study shows that it also does not affect RV function in vivo. One explanation

might be that the local RAAS system of the RV functions differently than that of

the LV. This is supported by our observation that the ‘beneficial’ AT2-receptor,

which is upregulated in the pressure loaded LV(48), was not upregulated in the

pressure loaded RV. Additionally, in RV pressure load, the AT1-receptor has been

shown to be functionally uncoupled from its downstream effectors(42) and

protein kinase C isozymes (44), which are important regulators of remodeling

and function in the LV. This could explain why RAAS inhibition, as employed in

the current study, does not work in the RV. We added an aldosterone-receptor

blocker, eplerenone, to the losartan treatment to circumvent the possibility that

compensatory activation of the aldosterone-pathway (partially) negates the

inhibitory effects on AT1-receptor(15,49). Indeed, in the LV, pharmacological

inhibition on both levels of the RAAS resulted in more pronounced improvement

of remodeling and function than monotherapy(16).

Taken together, these data indicate important differences between the RV

and LV with regard to RAAS activation due to increased pressure load and the

response to RAAS inhibiting therapy. From the currently available data it is not

clear whether these different responses are caused by physiological differences

between the RV and LV, of by fundamental differences between right and

left ventricular cardiomyocytes, which embryologically derive from distinct

precursor cells.

Either way, the differences in both RAAS activation and response to therapy

might explain why clinical studies of RAAS inhibition in systemic RVs have failed

to show positive results (22,23,25). These studies certainly do not close the

book on RAAS inhibition in RV failure. A recent preliminary study in a murine

PAB model, has suggested that stimulation of the alternative ACE2-Ang-(1-7)

pathway might be beneficial for RV function(50). To take the exploration of RAAS

inhibition as a treatment strategy for RV failure a step further, the local RV RAAS

activity and its differences with local LV RAAS should be further unraveled.

Importantly, RAAS-inhibition has been reported to have beneficial effects in

models of pressure-loaded RV associated with pulmonary hypertension. Studies

Chapter 7168

in experimental pulmonary hypertension have described beneficial effects of

losartan/telmisartan treatment on RV function and remodeling(51-53). It is

important to realize that increased local RAAS-activity has been demonstrated

in pulmonary arteries of patients with pulmonary arterial hypertension (52).

Therefore, the described beneficial effects of RAAS-inhibition in these models

are not necessarily direct myocardial effects in the pressure loaded RV, but may

also be secondary to AT1R-inhibiting effects on the pulmonary vasculature,

leading to decreased RV-afterload and thereby secondary to improved RV

performance and remodeling. Effects of RAAS inhibition on the pulmonary

vasculature have been reported: losartan prevented pulmonary vascular

remodeling and decreased BMPR-2 expression in a model of shunt-induced

pulmonary hypertension (52, 53). The current PAB model, with a fixed afterload,

excludes such ‘confounding’ pulmonary vascular effects and thus allows

assessing direct effects of RAAS-inhibition on the RV-myocardium (54). The lack

of losartan effect in our study therefore indicates that RV effects of losartan

in (experimental) PH are secondary to the pulmonary vascular effects. These

experimental data suggest that RAAS-inhibition may be beneficial in patients

with RV-failure associated with pulmonary vascular disease, but not in patients

with RV-failure in the setting of CHD, in which RV-afterload is not determined

by pulmonary vascular resistance, including systemic RV, pulmonary branch

stenosis or other RV outflow tract obstructions.

Limitations

The current experiments were designed to test the preventive effects of a

‘clinical’ Ep/Lo treatment strategy on RV remodeling and function. In the clinical

setting, therapeutic effects that reverse established remodeling/dysfunction are

of high importance. However, given the lack of preventive benefits, it is unlikely

Ep/Lo would have therapeutic effects in the pressure loaded RV. Secondly, we did

not include monotherapy groups treated with losartan or eplerenone. Although,

in light of the lack of effects of the combination treatment, it seems unlikely

that monotherapy would have beneficial effects in PAB, we cannot conclude this

firmly based on the present study. As in all preclinical studies, caution is required

when data from experimental models are extrapolated to the clinical setting.


FUNDING

ACKNOWLEDGE-MENTS

Conclusion

Combination treatment with eplerenone and losartan did not prevent adverse

remodeling, clinical RV failure or benefit RV function in a model of pressure load

induced RV failure.

Our findings indicate that local RAAS activation in the pressure loaded RV and

its response to effective RAAS inhibition differ from that in the LV. This is of high

clinical relevance when treating patients with RV dysfunction due to abnormal

loading conditions. To further explore a potential role for RAAS inhibition in the

treatment of RV failure, the local RV RAAS activity and its differences with local

LV RAAS should be unraveled.

This study was supported by the Sebald fund.

The authors are greatly indebted to Michel Weij who performed the PAB

surgeries. We thank Bibiche Boersma and Martin Dokter for technical assistance.

Chapter 7170

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Supplemental table 1

CON PAB PAB-eplo p-value treatment

Pressure-volume parameters RVdPdtmax (mmHg/s) 1,256±59 2,484±168* 2,074±141* 0.18dPdtmin (-mmHg/s) 1,035±31 2,008±148* 1,712±146* 0.52dPdtmax indexed (1/s) 50±2 33±1* 31±1* 1.00dPdtmin indexed (-1/s) 48±2 31±2* 32±2* 1.00tau (ms) 18±1 25±2* 23±1* 1.00Echocardiographic parameters 5wkHR (/min) 368±8 303±12* 307±9* 1.00PAB gradient (mmHg) N/A 67±4* 61±4* 1.00SV (μL) 295±13 190±14* 187±14* 1.00TAPSE (mm) 2.9±0.1 1.5±0.1* 1.8±0.1* 1.00RVEDD (mm) 3.0±0.2 5.7±0.2* 5.5±0.3* 1.00RA diameter (mm) 3.5±0.1 6.3±0.5* 6.0±0.4* 1.00Tricuspid insufficiency (fraction) 0/7 12/12* 16/17* 1.00Pericardial effusion (fraction) 0/7 3/12 1/17 0.28

Table S1. Additional pressure-volume parameters and echocardiographic parameters at 5 weeks

dPdt max indexed= dPdt max/ peak pressure, dPdt min indexed= dPdt min/ end systolic pressure, PAB= pulmonary artery banding, TAPSE= tricuspid annular plane systolic excursion, RVEDD= RV end diastolic diameter, RA= right atrium. Means±SEM. p-values for eplerenone/losartan effect in right-hand column.

PART III

8RIGHTVENTRICUL ARFAILUREDUET OCHRONICABNO RMALLOADINGC ONDITIONS;WHA THAVEWELEARNE DOFPRECLINICAL RESEARCH?

MAJ Borgdorff, B Bartelds, MG Dickinson, RMF Berger

Under review

Chapter 8180

ABSTRACTRight ventricular failure is a major cause of morbidity and mortality in a broad

spectrum of cardiovascular disease. Seven years ago, a working group of the

National Heart, Lung and Blood Institute concluded that the right ventricle was

understudied and that as a consequence specific treatment options were lacking.

They urgently called for research aimed at filling this gap. Top priorities were the

development of preclinical models of RV failure, study of the pathophysiology

and pathobiology of RV failure and exploration of new therapeutic strategies.

We here review the progress of preclinical research since then.

RV failure due to chronic abnormal loading conditions: a review 181

INTRODUCTION

RV PHYSIOLOGY AND FAILURE

Right ventricular (RV) failure is the main cause of death in patients with

pulmonary hypertension (PH)1. RV failure or dysfunction is also a major predictor

of outcome in corrected congenital heart defects 2 and in left ventricular (LV)

failure due to ischemic heart diseases3, 4. Improved therapies for these diseases

have led to a growing population of survivors at risk for mortality due to right

ventricular (RV) failure. Currently over 4 million adults in Europe and the United

States suffer from late effects after treatment for congenital heart diseases.

Hence, there is an increasing need to support the failing RV. However, despite the

pivotal involvement of the RV in both common and rare cardiovascular diseases,

the RV has been given little attention in basic research, especially compared

to the LV. The “paucity of basic knowledge at all levels about its normal and

pathological function’5lead to an initiative in 2006 of the Working Group on

Cellular and Molecular Mechanisms of Right Heart Failure of the National Heart,

Lung and Blood Institute. Top priorities were to stimulate the development of

preclinical models of RV failure, study of the pathophysiology and pathobiology

of RV failure and exploration of new therapeutic strategies5.

Since 2006, there have been numerous studies in animal models studying RV

adaptation to stress and testing putative therapeutic strategies. In this review

we will summarize 7 years of preclinical RV failure research with a specific focus

on the models of RV failure, the hemodynamic profile and pathobiology of RV

failure, as well as possible therapeutic interventions.

The RV is not an LV

The RV serves to perfuse the low-resistance pulmonary vascular system. Before

birth, the RV and LV work parallel to support the systemic- and pulmonary

circulations, but after birth the systemic- and pulmonary circulation are

separated. While systemic resistance increases, pulmonary vascular resistance

decreases, thereby progressively unloading the RV. The contrasting physiological

environments of LV and RV are accompanied by differences in morphology,

function and physiology. The normal RV is thin-walled and compliant. Three

anatomical compartments can be discerned, termed inlet (sinus), apex and

outlet (cone), which give the RV a complex, crescent shape, complicating 2D

functional imaging. The RV contraction occurs in three phases: 1) contraction

Chapter 8182

of the papillary muscles, then 2) movement of the RV free wall toward the

interventricular septum and finally 3) contraction of the LV, which causes a

‘wringing’ that further empties the RV. As longitudinal shortening contributes

more to the stroke volume than the circumferential shortening, the RV has a

‘peristaltic’ contraction pattern6. Consequently, under normal conditions the

ejection into the low-resistance pulmonary circulation is sustained and peak

pressure is prolonged. Related to this, compared to the LV, the RV has a lower

oxygen requirement at both rest and exercise, and lower coronary flow that

mostly occurs during systole7, 8.

In addition to these morphological and functional differences between RV and

LV, the RV is derived from a distinct set of precursor cells, the so-called secondary

heart field9. However, it is largely unknown to what extent RV cardiomyocytes

differ from LV cardiomyocytes and whether the embryological difference

translates in a distinct response to chronic abnormal loading conditions10. In light

of the functional, morphological, and embryological differences, it is imperative

to study the pathophysiology of RV adaptation to abnormal loading conditions

in RV-specific models.

Evaluation of RV failure

For a meaningful discussion of the (translational) significance of findings in these

preclinical models an unequivocal definition of RV failure is necessary. Heart

failure has historically been defined as the inability of the heart to meet the

metabolic requirements of the body. Clinically, heart failure is not an entity as

such but a continuum of disease severity, which is graded using the NYHA class

based upon daily activities. RV failure may be defined accordingly, although the

clinical signs and symptoms of RV failure may differ from those in left sided heart

disease. Specifically, declining RV function leads to congestion in the systemic

venous circulation and decreased output to the pulmonary circulation and left

heart. The cardinal clinical characteristics of RV failure then, are fluid retention

(evident in peripheral edema, effusion, ascites) and decreased systolic reserve

or low cardiac output (evident in exercise intolerance, fatigue, dyspnea and poor

peripheral circulation)11(Table 1).

In clinical practice, exercise capacity is an important measure for the severity

of heart failure and a prognostic indicator. Exercise capacity is traditionally

determined by maximal cardiopulmonary exercise testing, but low intensity


voluntary exercise performance, evaluated with a 6 minute walk distance

(6MWD), can be used similarly 12. The 6MWD reflects physiological reserve

capacity. Exercise capacity is now increasingly being used in models of RV

disease, either by voluntary exercise performance (e.g. by measuring daily

spontaneous activity in a running wheel10, 13, 14), or forced exercise capacity 15, 16.

Mortality is the ultimate clinical sign of RV failure and survival analysis is included

in some studies. However, in some models other factors, rather than RV failure,

might impede survival 17, 18.

Parameter ExamplesType of loading proximal pressure load (e.g. pulmonary artery banding)

peripheral pressure load (e.g. pulmonary hypertension)volume load (e.g. aorto-cavalshunt)combined pressure/volume load (e.g. pulmonary hypertension + aortocaval shunt)

Clinical symptoms Appearance (decreased grooming or inactivity)Bodyweight changes (cachexia or fluid retention)Cyanosis or decreased peripheral circulationDyspnea/tachypnea (labored breathing)Effusions (palpable ascites)

Exercise Voluntary/ spontaneous activityForced exercise testing

Effusion at autopsy Pleural effusionAscitesLiver wet/dry weight ratio

Survival MortalityHuman endpoints reached

Table 1. Evaluation of RV disease in animal models

Characterizing the severity of RV dysfunction/failure in experimental models

using clinical symptoms, exercise and/or mortality (Table 1) allows correlating

disease states to hemodynamic and cellular changes (Table 2, 3).

Chapter 8184

Spec

ies

Mod

elSi

gns

& s

ympt

oms

Surv

ival

Exer

cise

RVP

EDP

Ees

Eed

CI o

r CO

EDV

Ref

Rem

ark

Peri

pher

al p

ress

ure

load

rat

mct

30no

neno

mor

talit

yn/

a27

5621

-16

913

S40

rat

mct

80↓

BW, i

nacti

vity

no m

orta

lity

n/a

67 S

3815

-9-2

689

S40

rat

mct

80Ye

s (s

ee R

1)no

mor

talit

yV

↓96

S19

9 S

188

S4

-830

14R1

, R2

rat

mct

60↓

BW, r

esp

dist

ress

↑ m

orta

lity

n/a

166

S20

0 S

400

S70

0 S

-60

Sn/

a36

rat

mct

60n/

an/

an/

a32

5 S

n/a

766

S50

0 S

-64

Sn/

a11

9ra

tm

ct40

none

no m

orta

lity

F ↓

120S

400

Sn/

an/

a -4

5 S

n/a

144

rat

mct

60Ye

s (s

ee R

3)n/

aF

↓18

0 S

650

Sn/

an/

a-1

0n/

a14

4R3

rat

mct

60n/

an/

aF

↓14

0 S

333

Sn/

an/

a -3

0 S

1931

rat

mct

60n/

an/

an/

a16

0 S

200

Sn/

an/

a -2

5 S

n/a

26ra

tm

ct60

n/a

n/a

n/a

110

Sn/

an/

an/

a -

60 S

n/a

95ra

tm

ct40

n/a

n/a

n/a

110

Sn/

an/

an/

a -2

9 S

n/a

146

rat

mct

40n/

an/

an/

a12

1 S

n/a

n/a

n/a

-39

Sn/

a14

7R4

rat

SuH

xn/

an/

aF

↓20

0 S

n/a

n/a

n/a

-63

Sn/

a31

rat

SuH

xn/

a↑

mor

talit

yF

↓22

2 S

n/a

n/a

n/a

-42

Sn/

a82

rat

SuH

xn/

ano

mor

talit

yn/

a20

8 S

n/a

n/a

n/a

-42

Sn/

a27

R5ra

tSu

Hx

n/a

n/a

n/a

283

Sn/

an/

an/

an/

an/

a87

rat

FHR

n/a

n/a

F ↓

36n/

an/

an/

a -4

2 S

n/a

148

rat

mct

80↓

BW

no m

orta

lity

n/a

n/a

n/a

n/a

n/a

-50

S25

S37

rat

mct

60n/

an/

an/

an/

an/

an/

an/

a -8

3 S

n/a

111

rat

mct

60n/

an/

an/

a12

6 S

n/a

n/a

n/a

n/a

n/a

46ra

tm

ct60

n/a

n/a

n/a

130

Sn/

an/

an/

an/

an/

a47

rat

mct

60Ye

s (s

ee R

6)↑

mor

talit

yn/

a13

3 S

n/a

n/a

n/a

n/a

n/a

45R6

rat

mct

60Ye

s (s

ee R

7)↑

mor

talit

yn/

a13

3 S

n/a

n/a

n/a

n/a

n/a

149

R7ra

tm

ct60

n/a

↑ m

orta

lity

n/a

133

Sn/

an/

an/

an/

an/

a15

0ra

tm

ct80

Yes

(see

R8)

no m

orta

lity

n/a

n/a

n/a

n/a

n/a

n/a

n/a

50R8

rat

mct

30no

neno

mor

talit

yn/

an/

an/

an/

an/

an/

an/

a90

rat

mct

80Ye

s (s

ee R

9)no

mor

talit

yn/

an/

an/

an/

an/

an/

an/

a90

R9ra

tm

ct60

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

108

rat

mct

60n/

an/

an/

an/

an/

an/

an/

an/

an/

a13

8ra

tm

ct60

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

109

pigs

AVS

n/a

no m

orta

lity

n/a

29 S

n/a

-13

n/a

-44

Sn/

a35

R10

pigs

AVS

n/a

no m

orta

lity

n/a

84 S

n/a

74 S

n/a

3n/

a34

R11


Prox

imal

pre

ssur

e lo

ad

lam

bpa

b>8

none

no m

orta

lity

n/a

433

S75

281

S62

S -3

7 S

-13

58R1

2ra

bbit

pab5

none

no m

orta

lity

n/a

271

S30

S18

5 S

62n/

an/

a63

dog

pab1

3n/

ano

mor

talit

yn/

a10

5 S

n/a

243

S11

6 S

0n/

a55

rat

pab4

mild

sym

ptom

sno

mor

talit

yV

↓16

9 S

500

S16

2 S

125

S -1

5 S

60 S

13

rat

pab8

ABC

DE

(see

R13

)↑

mor

talit

yn/

a20

4 S

300

S33

8 S

1053

S -5

0 S

n/a

84R1

3ra

tpa

b6no

nen/

an/

a11

7 S

4010

0 S

n/a

-5-1

825

rat

pab1

2no

nen/

an/

a97

S50

9n/

a -2

5 S

n/a

151

R14

rat

pab2

0no

nen/

an/

a11

3 S

17-9

n/a

-12

n/a

151

R15

rat

pab3

n/a

n/a

n/a

166

S20

0 S

n/a

n/a

-26

Sn/

a26

mou

sepa

b4no

neno

mor

talit

yV

↓30

0 S

n/a

n/a

n/a

020

S10

rat

pab4

n/a

n/a

F ↓

220

Sn/

an/

an/

a -5

3 S

n/a

31ra

tpa

b7n/

an/

an/

a15

2 S

n/a

n/a

n/a

-37

Sn/

a95

rat

pab6

Yes

(see

R16

)no

mor

talit

yn/

a20

0 S

n/a

n/a

n/a

0n/

a83

R16

rat

pab6

n/a

n/a

n/a

217

Sn/

an/

an/

an/

an/

a87

rat

pab2

2n/

ano

mor

talit

yn/

an/

an/

an/

an/

a0

n/a

27m

ouse

pab3

n/a

n/a

n/a

n/a

n/a

n/a

n/a

075

S51

rat

pab4

n/a

n/a

F ↓

n/a

n/a

n/a

n/a

-42

Sn/

a16

rat

pab6

n/a

no m

orta

lity

n/a

n/a

n/a

n/a

n/a

0n/

a27

rat

pab8

n/a

n/a

F ↓

n/a

n/a

n/a

n/a

-45

Sn/

a16

rat

pab

low

cun/

an/

an/

an/

an/

an/

an/

an/

an/

a27

rat

pab3

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

44ra

tpa

b6n/

an/

an/

an/

an/

an/

an/

an/

an/

a44

mou

sepa

b6Ye

s (s

ee R

17)

↑ m

orta

lity

n/a

n/a

n/a

n/a

n/a

n/a

n/a

28R1

7ra

tpa

b9n/

an/

an/

an/

an/

an/

an/

an/

an/

a44

Tabl

e 2.

Ove

rvie

w o

f hem

odyn

amic

cha

nges

in m

odel

s of

RV

pres

sure

load

Num

bers

are

per

cent

ages

incr

ease

/ de

crea

se v

ersu

s co

ntro

ls. F

or s

ome

stud

ies

and

para

met

ers

thes

e ar

e ap

prox

imati

ons,

dep

endi

ng o

n ho

w p

reci

se d

ata

wer

e re

port

ed. S

indi

cate

s a

sign

ifica

nt

chan

ge v

ersu

s co

ntro

ls. N

umbe

r be

hind

‘mct

’ ind

icat

es t

he d

osag

e of

mon

ocro

talin

e in

mg/

kg. N

umbe

r be

hind

‘pab

’ ind

icat

es n

umbe

r of

wee

ks a

fter

whi

ch m

easu

rem

ents

wer

e pe

rfor

med

. MCT

= m

onoc

rota

line,

PA

B= p

ulm

onar

y ar

tery

ban

ding

, FH

R= fa

wn-

hood

ed r

at, S

uHx=

Sug

en-H

ypox

ia, A

VS=

arte

rio-

veno

us s

hunt

, BW

= bo

dyw

eigh

t, R

VP=

righ

t ve

ntri

cula

r pe

ak o

r sy

stol

ic p

ress

ure,

ED

P=

end

dias

tolic

pre

ssur

e, E

es=

end

syst

olic

ela

stan

ce, E

ed=

end

dias

tolic

ela

stan

ce, C

I= c

ardi

ac in

dex,

CO

= ca

rdia

c ou

tput

, ED

V= e

nd d

iast

olic

vol

ume.

*fo

r exe

rcis

e V

indi

cate

s vo

lunt

ary

exer

cise

testi

ng, F

in

dica

tes

forc

ed e

xerc

ise

testi

ng; ↓

= de

crea

sed;

↑=

incr

ease

d; =

= un

chan

ged.

Rem

arks

: R1

Som

e w

eigh

tloss

, ina

ctivi

ty a

nd d

yspn

ea. R

2 V↓

was

tren

d (p

=0.0

8). R

3 >5

% lo

ss o

f bod

y m

ass

a da

y, le

thar

gy, c

yano

sis,

resp

irato

ry d

istr

ess.

R4

CO e

stim

ated

on

vent

ricu

lar d

iam

eter

s. R

5 Pe

rica

rdia

l flui

d on

ech

o; m

orta

lity

stee

ply

incr

ease

d aft

er 6

wee

ks. R

6/R7

BW

loss

>10%

for 2

day

s an

d ar

teri

al o

xyge

n sa

tura

tion<

80%

. R8

‘sig

ns o

f hea

rt fa

ilure

incl

udin

g pl

eura

l eff

usio

n an

d as

cite

s’. R

9 W

eigh

t los

s, p

leur

al e

ffus

ion,

asc

ites.

R10

/R11

Hem

odyn

amic

mea

sure

men

ts a

fter

shu

nt c

lam

ping

. R12

Dur

ation

of P

AB

vari

ed, b

ut e

xcee

ded

8 w

eeks

. Eed

is s

tiffne

ss c

onta

nt. R

13 A

ll A

BCD

E ca

tego

ries

(s

ee T

able

1).

R14/

R15

CO is

ver

y lo

w in

the

se s

tudi

es. R

16 F

ailu

re s

ympt

oms

are

not

defin

ed. R

17 F

ailu

re s

ympt

oms

wer

e: e

dem

a, b

odyw

eigh

t cha

nges

(bot

h ↑

and

↓).

EDP

was

300

S a

fter

10

days

in

the

tight

est P

AB

grou

p.

Chapter 8186

Modeling RV failure in chronic abnormal loading conditions

When a human disease is studied in an experimental model, the model should

ideally be induced by a clinically relevant hit, which leads to a phenotype that

closely resembles the clinical disease19. There are three main groups of diseases

that can lead to RV failure: 1) pulmonary hypertension (PH), 2) (corrected)

congenital heart defects, and 3) LV failure. While widely varying in etiology,

the common factor in all these diseases is that they impose abnormal loading

conditions on the RV, i.e. increased pressure load (PH, pulmonary stenosis),

increased volume load (congenital shunts, pulmonary valve insufficiency after

Fallot repair, tricuspid valve insufficiency), or a combination of both (systemic

RV in congenital heart defects, residual lesions after Fallot repair, end-stage PH

with tricuspid valve insufficiency). Increased pressure load can be dynamic and

peripherally located, as in PH or fixed and proximally located as in pulmonary

stenosis. To represent these chronic abnormal loading conditions models of PH,

pulmonary artery banding (PAB), and/or left-right shunts have been developed.

Pressure load

Models of pressure load

Historically, the monocrotaline (MCT) model of PH has been the main model

to study RV adaptation to pressure load (Table 2). MCT is an alkaloid that is

converted in the liver under the influence of cytP450 into the active metabolite

that induces remodeling of the lung vasculature. Although the MCT model has

been invaluable for PH research, interpretation of data with regard to the RV is

hampered by putative toxic effects of MCT 17; it has been questioned whether

MCT rats die from progressive PH and RV failure or from other organ dysfunction,

since MCT can also induce hepatic veno-occlusive disease, myocarditis, and

acute lung injury17, 18. Besides MCT, other models of PH have been developed,

as excellently reviewed elsewhere 19-21. These models are characterized mostly

by double hits, e.g. MCT + increased pulmonary bloodflow 22, 23 or hypoxia +

an angiogenesis blocker 24. Although all these models may mimic pulmonary

vascular disease quite adequately, again, the direct effects of the agents used

to induce PH on the RV limits their use to study the pathobiology of RVF.

Additionally, in PH models, direct effects of drugs on the RV are difficult to

distinguish from indirect effects caused by reduction of pressure load. This

yields PH models unsuitable to study interventions for fixed lesions causing RV

pressure load.

MODELING RV FAILURE


Spec

ies

Mod

elSi

gns

& s

ympt

oms

Surv

ival

Exer

cise

RVP

EDP

Ees

Eed

CI o

r CO

EDV

Ref

Rem

ark

volu

me

load

rat

AVS

↓ B

W o

r ta

chyp

nea

no m

orta

lity

V ↓

4210

0-3

10

54 S

113

S13

mou

seAV

Sn/

ano

mor

talit

yV

=0

n/a

n/a

n/a

147

S15

0 S

10do

gsAV

Spl

eura

l eff

usio

ns; a

scite

sno

mor

talit

yn/

a36

S53

S4

n/a

-17

18 S

72R1

mou

sePI

none

no m

orta

lity

F ↓

↑n/

a20

0 S

n/a

n/a

n/a

100

S69

swin

ePI

ste

ntn/

ano

mor

talit

yn/

a0

-30

S-1

5 -1

2 S

-32

S58

S68

R2

pres

sure

+vol

ume

load

swin

ePI

/PS

sten

tno

neno

mor

talit

yn/

a79

S58

S50

S24

S -9

S42

S74

R3ra

tM

CT+A

VSye

s (s

ee R

4)↑

mor

talit

yn/

a72

Sn/

an/

an/

an/

an/

a76

R4ra

tM

CT+A

VSA

BCD

E (s

ee R

5)↑

mor

talit

yV

↓10

7 S

182

S81

4757

S71

S14

R5

Tabl

e 3.

Ove

rvie

w o

f hem

odyn

amic

cha

nges

in m

odel

s of

RV

volu

me

load

or c

ombi

ned

pres

sure

/vol

ume

Num

bers

are

per

cent

ages

incr

ease

/ de

crea

se v

ersu

s co

ntro

ls. F

or s

ome

stud

ies

and

para

met

ers

thes

e ar

e ap

prox

imati

ons,

dep

endi

ng o

n ho

w p

reci

sely

dat

a w

ere

repo

rted

. S

indi

cate

s a

sign

ifica

nt c

hang

e ve

rsus

con

trol

s. A

VS=

arte

rio-

veno

us s

hunt

, PI=

pul

mon

ary

insu

ffici

ency

, PS=

pul

mon

ary

sten

osis

, MCT

= m

onoc

rota

line,

BW

= bo

dyw

eigh

t, R

VP=

righ

t ve

ntri

cula

r pe

ak o

r sy

stol

ic p

ress

ure,

ED

P= e

nd d

iast

olic

pre

ssur

e, E

es=

end

syst

olic

ela

stan

ce, E

ed=

end

dias

tolic

ela

stan

ce, C

I= c

ardi

ac in

dex,

CO

= ca

rdia

c ou

tput

, ED

V=

end

dias

tolic

vol

ume.

*fo

r ex

erci

se V

indi

cate

s vo

lunt

ary

exer

cise

testi

ng, F

indi

cate

s fo

rced

exe

rcis

e te

sting

; ↓=

decr

ease

d; ↑

= in

crea

sed;

==

unch

ange

d.Re

mar

ks: R

1 M

easu

rem

ents

aft

er s

hunt

cla

mpi

ng. R

2/R3

CI i

s eff

ectiv

e (t

hus

min

us re

gurg

itant

frac

tion)

; Eed

is k

appa

(stiff

ness

con

tant

). R4

↓ B

W, e

ffus

ions

, let

harg

y, d

yspn

ea.

R5 A

ll A

BCD

E ca

tego

ries

(see

Tab

le 1

).

Chapter 8188

Besides the PH models, the PAB model is frequently used to study the pressure

loaded RV. PAB avoids both limitations of the PH models (Table 2). There are no

systemic and toxic effects and the fixed constriction of the pulmonary artery

ensures a constant pressure load, even when vasodilatory therapy is given. The

phenotype of this model depends on the size of constriction, age at surgery and

a number of other factors such as genetic strain. Mild constriction and surgery

at young age may lead to a chronically compensated state with no symptoms of

RVF13, 25, 26. Rats have been reported to survive up to 22 weeks in such condition,

despite (near)systemic RV pressures27. Alternatively, a tighter PAB may lead to

inactivity, decreased cleaning behavior (raised fur), poor peripheral circulation,

dys/tachypnea, ascites and pleural/pericardial effusions and, ultimately,

mortality in a high percentage of animals14, 28-30. High intensity exercise capacity31

and voluntary low intensity exercise10, 13, 14 are reduced in this model. Despite the

considerable variability in phenotype, which requires experience and technical

skill, a well-sized and timely PAB represents a valuable model of chronic pressure

load induced RVF.

Functional RV response to pressure load

The functional response of the RV to pressure load is characterized by increased

contractility, Frank-Startling mechanism, progressive diastolic dysfunction and

adverse interaction with the LV (Figure 1). However, it is important to note that

the RV response depends on the type of pressure load (e.g. fixed proximal vs.

dynamic peripheral)14.

The primary response of the RV to match increased pressure load is increased

contractility (Table 2). This concept of ventriculo-arterial coupling requires

contractility (defined as end systolic elastance, a load independent measure

of contractility) to increase proportionally with increased arterial elastance (a

measure of pressure load)32 to maintain stroke volume (Figure 1). When end

systolic elastance increases less than arterial elastance and thus the Ees/Ea

ratio decreases, this is considered to be a sign of RV failure 33. In a piglet model

of flow-induced PH contractility initially increases, but falls back to pseudo-

normal levels in a more advanced stage of RV dysfunction34, 35. Indeed, beneficial

pharmacological effects in the pressure loaded RV are frequently accompanied

by a restoration of Ees/Ea ratio 13, 33, 36.


Understanding the role of contractility in RV pressure load is hampered by

the fact that many studies do not include a load-independent measurement

of contractility, which can be obtained by pressure-volume analysis. Rather,

echocardiographic or CMR-derived parameters for systolic function are described,

which only indirectly relate to contractility. Compared to echocardiography,

CMR has the advantage of providing absolute volumes, allowing calculation of

stroke volume and cardiac output, two parameters influenced by contractility 10,

37, 38. CMR is therefore the gold standard in clinical practice. RV ejection fraction

can be measured by pressure-volume analysis or CMR and is highly preload

dependent, but has prognostic value in PH39. The ejection fraction is generally

reduced before overt RV failure ensues10, 40. Alternatively, systolic displacement

of the lateral tricuspid annulus along the base-apex axis (TAPSE), is readily

measured by echocardiography and is commonly used41. TAPSE decreases in

chronic pressure load, reflecting reduced longitudinal movement42 of the RV.

However, TAPSE has inherent limitations as independent measurement of RV

pumping function43. Lastly, fractional shortening of the RV outflow tract28 and

change in surface area44-47 are also reported, but the value of these parameters

is debated.

176

Figure 1. Pathophysiology of the pressure loaded RV

Conceptual representation of the progression of pathophysiological changes in the pressure loaded

RV. Example pressure-volume loops from compensation to failure. Volumetric changes were derived

from experimental studies and extrapolated using previously published normal values (Ref. 152).

Figure 1. Pathophysiology of the pressure loaded RV

Conceptual representation of the progression of pathophysiological changes in the pressure loaded RV. Example pressure-volume loops from compensation to failure. Volumetric changes were derived from experimental studies and extrapolated using previously published normal values (Ref. 152).

In acute pressure load, increased contractility is the only adaptive response48,

49. In chronic pressure load, ventricular dilatation is seen, even in mild to

moderately increased pressure10, 13, 14, 31, 40, 50, 51. This suggests that in addition to

Chapter 8190

increased contractility, also Frank-Starling mechanism contributes to adaptation

to chronic pressure load. However, ventricular dilatation leads to increased wall

stress unless it is compensated by sufficient hypertrophy10, 52. Increased wall

stress indeed is proposed to be a hallmark of the failing ventricle5.

Interestingly, contractility is enhanced even in late stages of RV dysfunction 13,

30, 36. What then is the role of diastolic dysfunction? Diastolic function has been

shown to be disturbed in patients with PH53. In these patients, higher right atrial

pressure (an indirect measure of diastolic function) is associated with worse

outcome54. From PAB models (Table 2) it is clear that diastolic dysfunction is an

inherent component of increased RV pressure load14. RV diastolic dysfunction

is partly compensated by right atrial adaptation. Right atrial contractility

increases and the right atrium serves more prominently as a reservoir, next to

functioning as a conduit55. Indeed, as RV stiffness increases, filling pressures

will have to increase, which in turn result in increased central venous pressure,

liver congestion, effusions, ascites, edema, fatigue: many of the clinical signs of

RVF11. In a recent study comparing PAB rats with and without clinical signs of

severe RVF, progressive RV failure was associated with a further deterioration of

diastolic function despite increased systolic function30. Deterioration of diastolic

function might thus play a central role in the transition from compensation to

failure in chronic afterload (Figure 1).

Diastolic function has a passive and an active component. The RV (compared

to the LV) has been suggested to be particularly vulnerable to disturbed active

relaxation, possibly due to insufficient expression of the sodium-calcium

exchanger (NCX)56. Indeed, in experimental PH, active relaxation is increasingly

disturbed with increasing MCT dose40, while passive stiffness has been reported

as normal14, 40 or increased57. Chronic PAB invariably leads to diastolic dysfunction,

with disturbances in both active relaxation and passive stiffness13, 14, 55, 58.

Interaction with LV function

Under normal conditions, the LV contributes significantly to RV function: up to

65% of the RV work can be attributed to LV contraction 59. However, adverse

interaction between the LV and the pressure loaded RV has been noted in

patients with PH60. The basis of this interference may relate to diastolic filling of

the LV60 or to the fact that the LV and RV share myocyte fibers 61.


In isolated hearts from MCT rats, pacing improved RV function by reducing

adverse interference during diastole62. Conversely, in a rabbit PAB model of

moderate RV dysfunction increasing LV afterload via aortic constriction improved

systolic RV function, while diastolic function was unaffected63.

In summary, the functional RV response to increased afterload is characterized

by increased contractility, RV dilatation and adverse interaction with the LV. The

increase in contractility may be insufficient, as ventriculo-arterial coupling ratio

decreases. However, progression to clinically overt RV failure is accompanied by

progressive diastolic dysfunction (Figure 1).

Volume load

Models of volume load

Volume load elicits a different RV response than pressure load in both mice10 and

rats13. The volume loaded RV represents therefore a distinct clinical problem, of

which the importance, most prominently in pulmonary valve insufficiency after

Fallot correction, is only recently recognized64. Consequently, preclinical data on

the volume loaded RV are sparse.

In patients, there is a considerable lag-time between the induction of the

volume overload of the RV and the development of symptoms64. A similar lag-

time in morbidity and mortality has been observed in the few studies comparing

pressure and volume load65. In those studies an aorto-caval shunt was used to

impose a volume load, which leads to a biventricular volume overload.

The creation of an aorto-caval shunt leads to inactivity, poor cleaning behaviour

(raised fur), loss of body mass, cyanosis, dyspnea and ascites, but not until after

about 4 months66. There are also a few reports of isolated RV volume load via an

atrial septum defect in cats67, or via a pulmonary valve insufficiency created by a

stent68 or surgical entrapment of the valve leaflets 69. So far, most studies in the

volume loaded RV are performed before symptoms of heart failure occur10, 13,

while voluntary low intensity exercise is either normal10, or reduced13.

Functional RV response to volume load

The aorta-caval shunt induces a high cardiac output state, with decreased

mean arterial blood pressure, hypertrophy of both ventricles with dilation of

the atria and ventricles70, 71. The RV tolerates chronic volume load for a long

time, although there are differences between models and species. Both systolic

Chapter 8192

and diastolic function is normal for a considerable period in rats13, 14. In dogs,

chronic RV volume load was shown to increase RV end diastolic pressure72. In

a mouse model of pulmonary insufficiency, RV systolic function was unaffected

but diastolic function deteriorated without overt clinical signs of heart failure 69. In summary, the preloaded RV is characterized by dilatation and long-term

preservation of function; ultimately diastolic dysfunction may cause progression

to RV failure (Figure 2A).

Combined pressure/volume load

Functional RV response to combined pressure/volume load

Interestingly, while chronic volume load per se does not impair RV contractile

function, it does affect the ability of the RV to adapt to additional pressure

load. The inotropic adaptation to increased pressure is limited, which is partly

compensated by the Frank-Starling mechanism68, 72, 73. Despite the apparent

attenuation of inotropic response, in models of corrected Fallot, the RV has been

shown to adapt to combined pressure/volume load for some time by increasing

systolic function74. Ultimately however, contractility parameters fall back to

pseudo-normal levels which could be regarded as a transition to RV failure75. In

a rat model of flow-induced PH (MCT+ aorto-caval shunt), which displays several

characteristics of RV failure14, 76, combined pressure/volume load induced

further RV dilatation but resulted in comprised contractility compared with PH.

In summary, combined pressure/volume load is poorly tolerated by the RV;

systolic function decreases over time, possibly along with diastolic dysfunction

(Figure 2B).


180

Figure 2. Pathophysiology of the volume loaded and combined pressure/volume loaded RV

Conceptual representation of the progression of pathophysiological changes in the volume loaded RV

(A) and combined pressure/volume loaded RV (B). Example pressure-volume loops from

compensation to failure. Volumetric changes were derived from experimental studies and

extrapolated using previously published normal values (Ref. 152).

Figure 2. Pathophysiology of the volume loaded and combined pressure/volume loaded RV

Conceptual representation of the progression of pathophysiological changes in the volume loaded RV (A) and combined pressure/volume loaded RV (B). Example pressure-volume loops from compensation to failure. Volumetric changes were derived from experimental studies and extrapolated using previously published normal values (Ref. 152).

Pathobiology of RV pressure load

In the pressure loaded RV a myriad of (extra)cellular changes take place that

may initially serve as adaptive remodeling, but are also present in the failing RV.

Rather than caused by a single ‘malignant’ pathway, RV failure is the resultant

of many biological changes, both adaptive and maladaptive (Figure 3). Findings

in studies about RV adaptation to abnormal loading conditions, should be

interpreted in relation to the state of RV dysfunction, assessed clinically and

hemodynamically. Additionally, the trigger inducing the increased load (e.g.

MCT or PAB) should be taken into account. For instance, it is possible that in

PH, other factors than increased pressure, perhaps products of the sick lung

circulation, contribute to RV dysfunction77.

PATHOBIOLOGY OF RV FAILURE

Chapter 8194

Hypertrophy and isoform switch

RV hypertrophy is an adaptive response of the pressure loaded RV (and LV)

to reduce wall stress and improve contractility. In left sided heart disease,

LV hypertrophy is a strong predictor for outcome78. The relation between

RV hypertrophy and outcome is less clear. In all models of pressure load RV

hypertrophy is present 13, 14, 25, 27. It has been suggested that RVH is more severe

in peripheral pressure load (PH) than in proximal pressure load (PAB)27, but this

could not be verified in other studies14, 15, 25. This is important to note, as the

degree of RV hypertrophy in different models is used by some authors to classify

the state of adaptation 16.

Pressure load induces a switch in myosin heavy chain composition favoring the

slower but energetically favorable beta-MHC isoform 10, 13, 14, 26, 79. This seems to

be part of a general adaptive response since no correlation can be made with

the state of adaptation. RV hypertrophy is induced by well-known pathways

described in the LV, including the MAPK-ERK1/2-, the PI3K-Akt-, and the

calcineurin-NFAT pathway10, which is a putative target for treatment80.

Fibrosis

RV fibrosis has been reported to in both PH- 27, 36, 81 and PAB models14, 27, 67.

Along with other extracellular matrix changes may lead to increased ventricular

stiffness. There is, however, a wide variation in the amount of collagen measured 14, 27, 40, 44, 82-85 and there is no relation with the state of RV adaptation14, suggesting

that fibrosis may be a by-product rather than a cause of RV dysfunction. RV

fibrosis can be reduced with several interventions, i.e. beta blockade36, ROS-

scavenger37, prostacyclin81, but strategies to target specifically fibrosis have not

been successful in improving RV function84.


182

Figure 3. Overview of the pathobiological changes in the abnormally loaded RV

Pathobiological hallmarks of the abnormally loaded RV. A myriad of genetic and epigenetic changes result in tissue-damage related processes (oxidative stress, fibrosis, apoptosis) and activation of (mal)adaptive processes on the tissue (capillary formation, inflammation) or cellular (hypertrophy, energy substrate use, mitochondrial function and calcium handling). These processes are regulated by a complex network of signaling pathways related to contactile function, cellular growth, energy metabolism and neurohumoral signaling. ATP= adenosine triphosphate, NFAT= nuclear factor of activated T-cells, MAPK= mitogen activated protein kinase, Mef2= myocyte enhancer factor-2, PKG-1= protein kinase G-1, PDE3/5= phosphodiesterase type 3/5, PKA= protein kinase A, SERCA2= sarcoplasmic reticulum Ca2+-ATPase, RyR= ryanodine receptor, PLB= phospholamban, NCX= sodium calcium exchanger, SP3= transcriptional repressor SP3, RAAS= renin angiotensin aldosterone system

RV oxygen supply/capillary formation

The RV requires less oxygen than the LV at both rest and exercise, and has a

lower coronary flow, which occurs during both systole and diastole7. However, in

patients with PH coronary perfusion is limited to diastole, while increased work

load increases oxygen consumption8, 86. Hence, a chronic oxygen demand-supply

mismatch could be an underlying mechanism of RV failure. RV coronary flow has

not been measured yet in experimental studies, but myocardial capillary density

has been assessed. Capillary density is reduced in several PH models that display

signs of RV failure27, 36, 81. Because it had previously been shown to be less reduced

in a model of mild PAB (without signs of failure) it was inferred that inadequate

Chapter 8196

capillary density may be responsible for the transition to failure87. Yet, these

studies compared to different models and diseases states. Recently, two studies

comparing the same trigger with different disease states, i.e. MCT15 or PAB 30,

found a slightly different pattern, i.e. increased capillary density in compensated

versus pseudo-normalized density in decompensated RVF. Inadequate oxygen

supply therefore, does seem to play a role in RV failure. Indeed, higher capillary

density resulting from prostacyclin treatment is accompanied by improved

survival in rats81. This then, raises the question which cellular processes are

most affected by reduced oxygen supply? Candidates are substrate metabolism,

mitochondrial function, and calcium handling.

Energy substrate metabolism

Under normal conditions, cardiac metabolism is mainly dependent upon fatty

acid oxidation (FAO). In overload conditions, the heart may switch back to its

fetal program, in which glucose and lactate (and not free fatty acids) are the

main energy substrates88. In addition, glucose metabolism then shifts from

complete oxidation via the Krebs cycle to glycolysis only, which yields much less

ATP than oxidative phosphorylation, but also uses less oxygen per molecule ATP.

Metabolic shifts are also observed in the pressure loaded RV. FAO is reduced

along with an increase in glycolysis. SPECT imaging in a small group of patients

with PH (WHO class I and III) suggested impaired FAO in severe, but not in

mild RV hypertrophy89. Micro-array data in rats with MCT-induced PH show a

downregulation of CPT-1b90. Also, in a severe PH model, but not in a compensated

PAB model, PGC-1a, a master regulator of fatty acid metabolism, and its nuclear

receptors PPAR-alpha and ERR-alpha are downregulated91. The reduction of FAO

as part of the metabolic switch appears to be an adaptive mechanism. Indeed,

preventive pharmacological inhibition of FAO with trimetazidine increased

cardiac output in mild PAB16.

Patients with PAH have an increased uptake of 18FDG in the RV92, 93, which

may indicate a switch towards glucose metabolism. In line with this, there is

increased expression of glycolysis-related genes94 and enzymatic glycolysis rates

are higher95 in experimental PH. This has been shown in MCT95, 96, hypoxia97, 98,

Fawn-Hooded rats 95, 99 and PAB16, 95, but importantly, many of these experimental

studies were done in compensated pressure load, without signs of RV failure.

Upregulation of pyruvate dehydrogenase kinase (PDK), which uncouples


glycolysis from the Krebs cycle and oxidative phosphorylation, has been shown

in both PH and PAB models of adaptive RV remodeling95, 99. Also, in rats with

compensated RV remodeling due to PAB, a shift in cytoplasmatic proteins was

observed from FAO towards glycolytic enzymes100.

The importance of these shifts in glucose metabolism has been questioned

in a study in PH patients86. In experimental PH, inhibition of PDK with the use

of dichloroacetate (DCA) has been shown to increase oxygen consumption

and improve RV function, although the improvement might be attributed to

a reduction in afterload95, 101. Inhibition of PDK also reverses mitochondrial

hyperpolarization, which is regarded as a sign of mitochondrial dysfunction102.

In mild PAB, DCA treatment has been reported to improve RV function, although

the results on cardiac function, evaluated by echocardiography are partly

contradictory95: DCA decreased RV systolic pressure in the setting of a fixed

pressure load while cardiac output mildly increased. Further evaluation of these

effects in more severe models of RV dysfunction would be of interest because

DCA therapy has also been shown to benefit experimental PH101, which could

indicate a strategy that targets both RV and pulmonary vasculature.

In summary, extensive metabolic remodeling occurs in the pressure loaded RV.

Whereas the substrate switch from FAO to glucose oxidation may be part of

the adaptive response, the simultaneous switch from glucose oxidation towards

glycolysis may ultimately deprive the RV of sufficient ATP and contribute to RV

failure103.

Mitochondrial function

Mitochondria are the ‘power-houses’ of the cardiomyocyte, but also regulate

many processes involved in stress-response: formation of oxygen radicals,

oxygen sensing, apoptosis and inflammation104. The pressure loaded RV may

be subjected to increased ‘oxidative stress’. MCT-rats show increased activity

of complex II and oxygen radicals, and also increased production of radical

scavengers50. Apoptosis and inflammation signaling is increased in a model

of flow-induced PH35. Treatment of MCT rats with radical scavenger EUK-134

improved RV systolic function37, although also here reduction of pressure

load may explain the effect105. In the Sugen-hypoxia-model of PH, treatment

with protandim, an anti-oxidant plant extract inducing nrf2 expression, mildly

increased cardiac output, suggesting that in experimental PH increasing defense

Chapter 8198

mechanisms against oxidative stress may be beneficial27. It is so far unclear

whether this is a direct RV effect, as data on this pathway in a PAB model are

lacking.

PKG-1-PDE5 pathway

The importance of the protein kinase-G-1-phosphodiesterase 5 (PKG-1-

PDE5)-pathway in cardiac remodeling was rediscovered by a study in mice

with transverse aortic constriction in 2005106. PKG-1 is activated by cGMP

and generally suppresses proliferative pathways107. In 2007, increased PDE5

expression, indicating (adverse) suppression of PKG-1 was demonstrated

in RV tissue from patients and rats with increased RV pressure load108. PDE5

inhibition with Sildenafil has beneficial effects on the RV, both in vitro108 and in

vivo13. However, whether these effects can be completely attributed to changes

in the PKG-1-PDE5 pathway is yet unclear13. Importantly, PKG-activity is not

uniformly suppressed in all models of experimental RV afterload 13. In addition,

Sildenafil has pleiotropic effects (e.g. stimulating the PDE3-PKA pathway,

opening mitochondrial (k)ATP channels, stabilizing mitochondria, suppression

of inflammation 107-111) that might account for the beneficial effects on the RV.

Involvement of RV-specific pathways

A major question remains what the consequences are of the different

embryologic origin of the RV as compared with the LV for the response to

overload conditions10, 112, 113. PAB induces upregulation of dHand, an RV-specific

precursor, as well as upregulation of RV-specific transcription factors GATA-4,

MEf2, and NKX2.579. On the other hand, well-known signaling pathways involved

in LV remodeling, such as the calcineurin pathway, have been shown to be

activated in a murine model of PAB10. However, overall major differences may

exist between RV and LV pathobiology. This is particular evident from studies

showing opposing response to therapies, which were beneficial in LV pressure

load, but lacked effect84 or even proved detrimental 83 in RV pressure load.

Micro-array studies have been performed in an attempt to unravel the pathways

underlying RV failure30, 51, 69, 90, 100, 114, 115. Kreymborg and coworkers describe

several pathways in the pressure loaded RV, that are not induced in the pressure

loaded LV, most prominently Wnt pathway, a component of calcium cycling

(annexin) and the apoptosis related gene clusterin51. Kreymborg also showed


that the degree of up/downregulation of common pathways differs between RV

and LV51. Other micro-array studies have yielded divergent results with regard to

new signaling pathways, due to the fact that they were performed in different

models (MCT, PAB), different species (rat, mouse, rabbit) and different states

of RV adaptation (compensated vs. decompensated). For instance, in rats with

MCT-induced PH, p38 MAPkinase was suggested to be an inductor of RV failure,

yet this was not confirmed in murine PAB models 28, 51. Interestingly, differences

in microRNA profiles in mice before and after RV loading were mainly found in

the non-myocyte fraction of RV tissue, underlining the importance of other cell

types for remodeling114. Again, the functional relevance of these findings remains

to be determined, especially since many of these studies were performed in

early stages of RV dysfunction.

Pathobiology of RV volume load

The pathobiology of volume load is different from that of pressure load: pressure

load induces concentric hypertrophy while volume load induces eccentric

hypertrophy. Toischer et al showed divergent activation of pathways in the

volume loaded- vs. the pressure loaded LV, providing evidence that there may

be different pathways involved65. Additionally, specific up- or downregulation

of ERK1/2 in pressure- and volume load respectively, may result in contrasting

phenotypes of concentric and eccentric hypertrophy116.

The few studies in the RV also indicate pathobiological differences in pressure

vs. volume load. Marino et al showed in cats that though the amount of RV

hypertrophy was similar, the collagen content was higher in cats with PAB than

in cats with an atrial septum defect 67. Likewise, in mouse models, despite similar

amounts of RV hypertrophy, the volume loaded RV had a less pronounced switch

in MHC isoforms and lower activation of calcineurin-NFAT pathway10. PDE5

inhibitor Sildenafil inhibits PKG-1 activity in the volume loaded RV, whereas it

activates PKG-1 in the pressure loaded RV 13, further indicating the differences

in signaling. In a study in newborn lambs, transcriptional repressor Sp3 was

specifically downregulated in the volume loaded RV, while it was upregulated in

the pressure loaded RV117. Sp3 inhibits Sp1-mediated activation of cTNT but also

regulates SERCA2, thereby providing a possible link with disturbances in calcium

cycling118. In line with this, in a murine model of pulmonary insufficiency, gradual

down regulation of calcium cycling proteins SERCA2 and CAMK2 is described,

Chapter 8200

which may underlie the diastolic dysfunction that ultimately ensues in volume

overload69. However, the exact functional consequences of these changes have

yet to be discovered.

Pathobiology of combined pressure/volume load

Experimental studies designed to study combined loading conditions are

scarce. However, severe pressure load is invariably complicated by additional

volume load (e.g. tricuspid insufficiency in PH) and some of the changes seen

in severe pressure load might actually be caused by the volume load. In a

study in which models of volume load (aorto-caval shunt) and pressure load

(MCT) were compared with a combination of both (PH+shunt), rats with the

combined load had significantly more RV hypertrophy than PH or shunt alone14.

Possibly, hypertrophy due to pressure load en volume load add up, either

through augmented activation of one pathway or simultaneous activation of

different pathways. Combined overload had a strong additive effect on MYH-

isoform switch and NPPA expression, whereas RCAN1 upregulation was similar

in PH, PH+shunt and shunt. However, most well-known signaling pathways (Akt,

ERK, PKG) were activated similarly at the end-stage. More studies in models of

combined loading conditions are needed to clarify the additional hypertrophy

and specific pathobiological changes in this clinically relevant state.

Currently no RV specific medical treatment strategies exist. Experimental

studies assessing medical treatment of the overloaded RV (Table 4) fall into

one of three categories. Firstly, treatment strategies for LV failure (e.g. beta

adrenergic blockade, RAAS inhibition) have been applied in the abnormally

loaded RV. Secondly, drugs that target the pulmonary vasculature in PH (e.g.

PDE5-inhibitors, endothelin-receptor antagonists) might have direct beneficial

effects on the RV. Thirdly, several proof-of-concept studies with experimental

treatments have been published.

TREATMENT OF RV FAILURE


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Chapter 8202

Beta adrenergic blockade

Beta blockers are a cornerstone in the treatment of LV failure. Although clinicians

have long been reluctant to give beta blockers to patients with failing RVs in the

setting of pulmonary hypertension, several pre-clinical studies show beneficial

effects on RV remodeling. In a rat model of PH, carvedilol, a non-selective beta

blocker with also alpha1 blocking effects, reduced development of hypertrophy,

fibrosis, capillary rarefaction and a reduction of cardiac output and TAPSE82.

Metoprolol, a selective beta blocker, has comparable effects, although part of

the effects might be mitigated through reduction of the pulmonary vascular

remodeling82. Bisoprolol, another selective beta blocker, in the MCT model does

not prevent hypertrophy or capillary rarefaction, but prevents fibrosis along

with delaying the decline in cardiac output and TAPSE36. Interestingly, bisoprolol

increased the phosphorylation of the ‘myocardial spring’ titin, suggesting

that beta blockade has direct RV effects. Complicating the rationale behind

beta blocking therapy are reports that downregulation and desensitization

of adrenoreceptors and dopamine receptors by G protein–coupled receptor

kinase-2 leads to decreased inotropic reserve in the pressure loaded RV31.

Unfortunately, no data are published on beta blockade in the failing RV due to

fixed, proximal-type pressure overload (e.g. PAB), which might further elucidate

the direct protective effects of beta blockade on the RV.

RAAS-inhibition

Blockade of the renin-angiotensin-aldosteron-system (RAAS) is the other

cornerstone in the treatment of LV failure. Clinical data in PH suggest that the

RAAS is activated in at least a subgroup of patients119. In animal models of PH,

RAAS-inhibition with losartan of telmisartan may have shown beneficial effects

on the RV, but these might be secondary to afterload-reduction due to effects

in the pulmonary vasculature119-121. There are however also suggestions that the

RAAS system is active in congenital heart disease122, and combinations of RAAS-

inhibiting drugs (such as an angiotensin receptor blocker+eplerenone) might

potentiate the effect and target oxidative stress, fibrosis and improve diastolic

dysfunction like in the LV123, 124. However, recently no effect of RAAS inhibition

was shown in a PAB model of severe RV dysfunction/failure84.


PDE5 inhibitors

If PDE5 inhibition had direct beneficial effects on the RV, it would be an

excellent therapeutic approach for RV failure in PH, as it also reduces pulmonary

vasculature resistance125, thereby killing two birds with one stone. However,

the first two studies that used mild PAB models to study direct effects of PDE5

inhibition on the RV, disappointingly showed no prevention or reduction of

hypertrophy and fibrosis or functional improvement 26, 44. The functional analysis

in these studies was limited to thermodilution-measured cardiac output and

echocardiography. Using pressure-volume analysis in a PAB model of moderate

RV dysfunction, we showed that preventive PDE5-inhibition with Sildenafil

increases contractility, reduces dilatation and attenuates decline of exercise,

but leaves diastolic function unchanged13. The differences between the studies

could be due to differences in loading condition as in the LV it was shown that

the effects of PDE5-inhibition were dependent upon the severity of the loading

condition126. Adding to this is the observation that Sildenafil treatment in

established RV dysfunction improves diastolic dysfunction85.

However, as mentioned before, the mechanisms by which Sildenafil exerts its

effects are pleiotropic and not limited to the PDE5-PKG-1 system. Sildenafil inhibits

PDE5, thereby increases compartimentalized cGMP which in turn activates

PKG-1, which has inhibitory effects on pathways of pathological remodeling127,

128. Sildenafil also phosphorylates titin, thereby reducing stiffness129, 130. In the

pressure loaded RV, Sildenafil treatment as a preventive strategy increased

fibrosis13, 26, 44. However, when given in a later stage of established pressure load

Sildenafil reduces fibrosis and reduces ventricular stiffness85, again supporting

the concept that timing and loading severity determines cardiac response.

Other pharmacological approaches to manipulate the PDE5-PKG-1 axis include

stimulation of soluble guanylate cyclase, but so far no direct RV effects have

been demonstrated131, 132.

Sildenafil had beneficial effects in a model of mitral insufficiency which imposes

a volume load on the LV110. Unfortunately, these beneficial effects were absent

in the volume loaded RV13.

Chapter 8204

CONCLUSION

Endothelin receptor antagonists

Endothelin receptor antagonists (ERAs) have an anti-hypertrophic and anti-

fibrotic effect on the RV in PH133-135. However, it is unclear whether this is a direct

effect on the RV or a consequence of decreased pressure load which results

from vasodilating/antiproliferative effects on the pulmonary vasculature133, 136.

Unfortunately, no studies address this issue. However, ET-1 has been shown to

affect contractility in mouse RV cardiomyocytes via an increase in intracellular

Ca2+ transients through activation of the ETR-A receptor and the Na+-

Ca2+ exchanger137. Upregulation of ET-1 in RV myocardium of patients and models

of compensated hypertrophy63, 138 suggests a rationale for endothelin receptor-

inhibiting treatment. However, isolated heart studies showed that ERAs depress

both contractility and relaxation in the hypertrophic RV. This adverse effect on

the RV might explain the negative outcomes of both preclinical139 and clinical140,

141 studies on ERAs in RV pressure load without pulmonary vascular remodeling.

Intriguingly, the negative inotropic effect of ERAs is absent in overtly failing left

ventricles, likely due to differences in ET-1 activation. It is therefore possible that

ERAs have contrasting therapeutic effects in compensated and failing RVs, but so

far this has not been studied.

Exercise training

Apart from medical intervention, exercise training may be beneficial for the

pressure loaded RV. Regular exercise can induce several effects both in the

myocardium and in the vascular system142. In patients with pressure loaded RVs,

exercise training has been show to increase exercise capacity mildy143. However,

Handoko et al showed in the MCT model, that exercise was beneficial in rats

with mild PH, but detrimental in rats with severe PH144. Exercise training has not

been tested yet in other models of RV load. The RV may also be damaged by

high intensity endurance sports145, hence caution is warranted with regard to

exercise in patients with an abnormally loaded RV.

In recent years, substantial progress has been made in understanding the

pathophysiological and pathobiological mechanisms of RV failure due to chronic

abnormal loading conditions, especially pressure load. Study of different models

has underlined the value of the ‘tight’ PAB model. In addition, the progression of


pathophysiological changes in abnormal loading conditions has been described

in detail, revealing the importance of diastolic dysfunction in RV failure. The

pathobiology of RV failure resembles that of LV failure in some aspects, but

RV-specific changes have been marked out with regard to for instance energy

metabolism, signaling pathways and calcium handling. A number of ‘new’

targets has yet to prove their value, but hopefully will add to the very limited

treatment options for RV failure. Lastly, RV failure in the setting of volume load

or combined pressure/volume load are distinct clinical entities of increasing

importance but their study is largely unexplored territory.

Chapter 8206

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107 Kass DA. Cardiac role of cyclic-GMP hydrolyzing phosphodiesterase type 5: from experimental models to clinical trials. Curr Heart Fail Rep. 2012; 9: 192-199.

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109 Nagendran J, Gurtu V, Fu DZ, Dyck JR, Haromy A, Ross DB, Rebeyka IM, Michelakis ED. A dynamic and chamber-specific mitochondrial remodeling in right ventricular hypertrophy can be therapeutically targeted. J Thorac Cardiovasc Surg. 2008; 136: 168-78, 178 e1-3.

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111 Xie YP, Chen B, Sanders P, Guo A, Li Y, Zimmerman K, Wang LC, Weiss RM, Grumbach IM, Anderson ME, Song LS. Sildenafil prevents and reverses transverse-tubule remodeling and Ca(2+) handling dysfunction in right ventricle failure induced by pulmonary artery hypertension. Hypertension. 2012; 59: 355-362.

112 Olson EN. Gene regulatory networks in the evolution and development of the heart. Science. 2006; 313: 1922-7.

113 Srivastava D. Making or breaking the heart: from lineage determination to morphogenesis. Cell. 2006; 126: 1037-48.

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Nagayama T, Hsu S, Zhang M, Koitabashi N, Bedja D, Gabrielson KL, Takimoto E, Kass DA. Pressure-overload magnitude-dependence of the anti-hypertrophic efficacy of PDE5A inhibition. J Mol Cell Cardiol. 2009; 46: 560-567.

Takimoto E, Koitabashi N, Hsu S, Ketner EA, Zhang M, Nagayama T, Bedja D, Gabrielson KL, Blanton R, Siderovski DP, Mendelsohn ME, Kass DA. Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice. J Clin Invest. 2009; .

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130 Kruger M, Kotter S, Grutzner A, Lang P, Andresen C, Redfield MM, Butt E, dos Remedios CG, Linke WA. Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. Circ Res. 2009; 104: 87-94.

131 Andersen A, Nielsen JM, Holmboe S, Vildbrad MD, Nielsen-Kudsk JE. The Effects of Cyclic Guanylate Cyclase Stimulation on Right Ventricular Hypertrophy and Failure Alone and in Combination with Phosphodiesterase-5 Inhibition. J Cardiovasc Pharmacol. 2013; .

132 Lang M, Kojonazarov B, Tian X, Kalymbetov A, Weissmann N, Grimminger F, Kretschmer A, Stasch JP, Seeger W, Ghofrani HA, Schermuly RT. The soluble guanylate cyclase stimulator riociguat ameliorates pulmonary hypertension induced by hypoxia and SU5416 in rats. PLoS One. 2012; 7: e43433.

133 Hill NS, Warburton RR, Pietras L, Klinger JR. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats. J Appl Physiol (1985). 1997; 83: 1209-1215.

134 Chen SJ, Chen YF, Meng QC, Durand J, Dicarlo VS, Oparil S. Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats. J Appl Physiol (1985). 1995; 79: 2122-2131.

135 Choudhary G, Troncales F, Martin D, Harrington EO, Klinger JR. Bosentan attenuates right ventricular hypertrophy and fibrosis in normobaric hypoxia model of pulmonary hypertension. J Heart Lung Transplant. 2011; 30: 827-833.

136 Prie S, Stewart DJ, Dupuis J. EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension. Circulation. 1998; 97: 2169-2174.

137 Nagasaka T, Izumi M, Hori M, Ozaki H, Karaki H. Positive inotropic effect of endothelin-1 in the neonatal mouse right ventricle. Eur J Pharmacol. 2003; 472: 197-204.

138 Nagendran J, Sutendra G, Paterson I, Champion HC, Webster L, Chiu B, Haromy A, Rebeyka IM, Ross DB, Michelakis ED. Endothelin axis is upregulated in human and rat right ventricular hypertrophy. Circ Res. 2013; 112: 347-354.

139 Jiang BH, Tardif JC, Shi Y, Dupuis J. Bosentan does not improve pulmonary hypertension and lung remodeling in heart failure. Eur Respir J. 2011; 37: 578-586.

140 Packer M, McMurray J, Massie BM, Caspi A, Charlon V, Cohen-Solal A, Kiowski W, Kostuk W, Krum H, Levine B, Rizzon P, Soler J, Swedberg K, Anderson S, Demets DL. Clinical effects of endothelin receptor antagonism with bosentan in patients with severe chronic heart failure: results of a pilot study. J Card Fail. 2005; 11: 12-20.

141 Kalra PR, Moon JC, Coats AJ. Do results of the ENABLE (Endothelin Antagonist Bosentan for Lowering Cardiac Events in Heart Failure) study spell the end for non-selective endothelin antagonism in heart failure? Int J Cardiol. 2002; 85: 195-197.

142 Gielen S, Schuler G, Adams V. Cardiovascular effects of exercise training: molecular mechanisms. Circulation. 2010; 122: 1221-1238.


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Handoko ML, de Man FS, Happe CM, Schalij I, Musters RJ, Westerhof N, Postmus PE, Paulus WJ, van der Laarse WJ, Vonk-Noordegraaf A. Opposite effects of training in rats with stable and progressive pulmonary hypertension. Circulation. 2009; 120: 42-9.

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Mouchaers KT, Schalij I, de Boer MA, Postmus PE, van Hinsbergh VW, van Nieuw Amerongen GP, Vonk Noordegraaf A, van der Laarse WJ. Fasudil reduces monocrotaline-induced pulmonary arterial hypertension: comparison with bosentan and sildenafil. Eur Respir J. 2010; 36: 800-807.

Mouchaers KT, Schalij I, Versteilen AM, Hadi AM, van Nieuw Amerongen GP, van Hinsbergh VW, Postmus PE, van der Laarse WJ, Vonk-Noordegraaf A. Endothelin receptor blockade combined with phosphodiesterase-5 inhibition increases right ventricular mitochondrial capacity in pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol. 2009; 297: H200-7.

Piao L, Sidhu VK, Fang YH, Ryan JJ, Parikh KS, Hong Z, Toth PT, Morrow E, Kutty S, Lopaschuk GD, Archer SL. FOXO1-mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) decreases glucose oxidation and impairs right ventricular function in pulmonary hypertension: therapeutic benefits of dichloroacetate. J Mol Med (Berl). 2013; 91: 333-346.

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Alfakih K, Plein S, Bloomer T, Jones T, Ridgway J, Sivananthan M. Comparison of right ventricular volume measurements between axial and short axis orientation using steady-state free precession magnetic resonance imaging. J Magn Reson Imaging. 2003; 18: 25-32.

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9GENERALDISCUSS IONANDFUTUREP ROSPECTS

MAJ Borgdorff

Chapter 9220

General discussion and future prospects 221

INTRODUCTION

PATHO-PHYSIOLOGY

In recent years, an increasing body of literature has underscored the importance

of the right ventricle (RV) in a wide spectrum of cardiovascular disease, including

congenital heart diseases and pulmonary arterial hypertension. At the same

time, the RV could be regarded as the elusive heart as a paucity of knowledge

exists at all levels about its normal and pathological function1. This becomes

most evident in the fact that currently no clinically established treatments exist

that specifically target the failing RV. In the introduction we suggested three

routes towards such treatments: exploration of new therapeutic targets by

studying the pathophysiology and pathobiology of RV failure, assessing the

direct effects of pulmonary hypertension drugs on the RV and evaluating the

efficacy of treatment strategies for left ventricular (LV) failure in RV failure.

Consequently, the main aim of this thesis was to expand the current knowledge

of RV failure due to chronic abnormal loading conditions using preclinical models,

by describing in detail the physiological and biological consequences of different

types of abnormal loading and by studying the effects of therapeutic agents with

a putative beneficial effect on the RV. With these data, RV failure can be better

understood and recommendations for possible therapeutic strategies can be

given. We here discuss the major findings of this thesis in an integrated way, by

asking a number of fundamental questions with regard to the pathophysiology

and treatment of RV failure.

The pathophysiology of right ventricular failure

What is RV failure?

Virtually every preclinical study published on the abnormally loaded RV is

introduced by statements about the prevalence and importance of RV failure

in different forms of cardiovascular disease. Few studies however, specifically

address the question whether the animal model used in that study, designed

to study RV failure, actually is an accurate and representative model of RV

failure. Answering that question requires a clear definition of RV failure and

(consequently) the recognition that chronic abnormal loading conditions do not

necessarily lead to RV failure. In the absence of a clear definition, confusion

easily arises whether the observed changes belong to a compensated (adaptive)

phenotype or a failing (maladaptive) phenotype. The establishment of a clear

definition of RV failure, however, is not easy. In the clinical setting, RV failure is

defined as clinical symptoms and signs (fatigue, dyspnea, exercise intolerance,

Chapter 9222

ascites etc.) in the context of abnormal parameters of RV function and

morphology as measured by echocardiography or cardiac magnetic resonance

imaging (MRI)2. This ‘subjective’ definition seems hardly applicable in animal

studies, and thus most researchers define RV failure using only the abnormal

echocardiography and/or cardiac MRI parameters. However, it is important

to recognize that many patients with an abnormally loaded RV will show

abnormalities in echo and MRI studies, while being asymptomatic and thus by

definition not in RV failure.

Interpreting a compensated phenotype as RV failure is hazardous: it may lead to

incorrect labeling of adaptive processes as maladaptive and, more importantly,

lead to overestimation of the therapeutic potential of (pharmacological)

interventions. In this thesis we have systematically assessed ‘clinical’ symptoms

and signs of RV failure in experimental models. We conclude that the occurrence

of clinical symptoms is closely related to the type of abnormal loading and the

progression of RV dysfunction (e.g. chapters 2-5). In chapter 4 we show that

RV function parameters (whether measured by echocardiography or pressure-

volume analysis) change markedly in pressure load per se, but are significantly

more affected in rats with clinical symptoms of RV failure. We therefore propose

that systematic assessment of ‘clinical’ parameters in animal studies (observation

of symptoms and signs, exercise measurements) is very useful in positioning

the model in the compensation- failure spectrum. However, a disadvantage of

the ‘clinical’ assessment is that it requires experience to assess the animals in

this way and that the system is partly subjective. Future studies may explore

techniques to objectify the symptoms in the different categories, by for instance

telemetric measurement of breathing rate/effort or skin temperature. Also the

spontaneous exercise measurements may be refined, for instance by techniques

using continuous monitoring of activity.

Is RV failure ’systolic failure’?

Regardless of the exact definition of RV failure, the relative contributions of

systolic and diastolic function over time in the development of RV adaptation

and RV failure are incompletely understood. The classic paradigm of failing

contractility as hallmark of pressure load induced RV failure is challenged

by recent findings in multiple models (PAB3, MCT4, and chapters 2, 4 and

8) showing enhanced contractility, even in advanced stages of RV failure. On


the other hand, awareness of the importance of preserving diastolic function

is increasing. In chapter 4 we have shown that in experimental pressure load,

clinical deterioration is associated with a decline of diastolic function. A recent

study in patients with pulmonary hypertension also suggests that diastolic

function contributes to RV failure5. We conclude that RV failure is not ‘systolic

failure’, but that diastolic dysfunction may play a central role in the transition

from compensation to failure, at least in some forms of abnormal RV loading.

To gain further insight in the relative contributions of systolic and

diastolic dysfunction in the different subtypes of RV failure, currently

invasive measurements that allow pressure-volume analysis are needed;

echocardiography and cardiac MRI do not provide load independent parameters

of ventricular function. Especially diastolic function is difficult to assess by these

techniques. On one hand, this implies that in preclinical studies pressure-volume

measurements should be performed to fully appreciate the functional changes

in RV failure and the impact of interventions on them.

On the other hand, parameters of RV function with prognostic significance that

can be non-invasively measured could be valuable in both preclinical research

and clinical practice. We suggest that as echocardiography and cardiac MRI

techniques rapidly advance, specific attention be given to the establishment of

new parameters of RV systolic and diastolic function and anatomy that reliably

assess the presence and extent of RV failure6.

Pressure load, volume load, mixed loads: different conditions, one response?

Another fundamental question in RV research is whether all abnormal loading

conditions elicit a common adaptive RV response, or that the RV responds in

different ways depending on the type of loading. Chapters 2 and 3 of this thesis

specifically address this question and show that indeed important differences

exist in the RV response to pressure load, volume load and combined pressure-

volume overload respectively.

These differences may imply that different ‘subtypes’ of RV failure require

distinct therapeutic approaches, which should be subject of future research.

For instance, the therapeutic goals may be different: the pressure loaded RV

may benefit primarily from interventions that preserve diastolic function, while

the volume loaded RV, which displays less diastolic dysfunction may be rather

treated with inotropics or new treatments that may limit ventricular dilatation.

Chapter 9224

Even when the therapeutic goals would be similar, the different RV failure

‘subtypes’ might be more or less responsive to the same therapy. We indeed

seem to encounter this phenomenon in chapter 5, where Sildenafil has multiple

beneficial effects in the pressure loaded, but not the volume loaded RV.

Apart from therapeutic implications, the comparisons between the different

loading conditions, also provide clues for a deeper understanding of RV

physiology. A particular clue is that the RV copes more easily with volume

load than with pressure load: the extent to which compensatory mechanisms

are activated is less pronounced in volume load, and volume load elicits less

symptoms of RV failure even as the work performed is equal to that in pressure

load (chapters 2 and 5). Previous studies in the aorto-caval model suggest

that with (biventricular) volume load eventually heart failure will ensue, but

particularly because of LV-, not RV, dysfunction7-9. So then, is it actually true

(as often stated) that the RV is the LVs weaker brother? We would argue that

the answer depends on the circumstances. The RV indeed seems much more

vulnerable to pressure load than the LV, evidenced by the striking differences in

prognosis for pulmonary- and systemic hypertension10. However, the LV might be

much more vulnerable to volume load that the RV. Conceptually, this highlights

the distinct physiological role of both ventricles. The LV is a high pressure pump,

receiving a tightly controlled preload, while working against a high resistance

vascular bed. The RV is a low pressure pump, able to cope with considerable

fluctuations in venous return, but working against a low resistance vascular

bed. This could lead one to the conclusion that the RV is inherently unable to

withstand pressure load. It seems however, that the RV was not born that way.

Is the RV inherently unable to withstand pressure load?

During fetal life, because of the unique physiology of the feto-placental

circulation, pressure load on the RV and LV is roughly equal11. Right and left

ventricular wall thickness and force development are equal throughout fetal

development12, 13. Shortly after birth, pulmonary vascular resistance drops

while systemic vascular resistance rises. This results in the four-fold difference

between left and right sided peak pressure that normally will persist throughout

life, and as a consequence ‘unloads’ the RV and ‘pressure loads’ the LV, marking

the distinct physiological roles of the LV and RV.


In some congenital heart defects, however, this ‘unloading’ does not take place.

Examples include defects in which the RV serves as systemic ventricle (e.g. levo-

transposition of the great arteries, dextro-transposition of the great arteries

palliated with a Mustard-, or Senning-procedure) or defects in which the

pulmonary vascular resistance fails to drop (e.g. large ventricular septal defect

with Eisenmenger physiology). In these cases, the RV is not unloaded and has

to sustain a high pressure circulation. Remarkably, the RV appears to be able to

do so for decades in a substantial proportion of cases14-17. At least, the prognosis

of these patients appears to be better than patients in whom the RV is pressure

loaded after initial unloading18, 19. The RV thus does not seem inherently unable

to withstand pressure load.

Has the RV ‘forgotten’ how to withstand pressure load?

If the RV is not inherently unable to withstand pressure load, then why does

it fail quickly if it is pressure loaded? One may hypothesize that while being

unloaded after birth, the RV ‘unlearns’ the ability to withstand pressure load.

The physiological and morphological changes in the RV in this period, are

accompanied by major changes cellular signaling process activation. Possibly,

through permanent transcriptional inhibition of one or more vital adaptive

genes/ transcription factors, some signaling pathways or cellular processes that

are vital parts of fetal RV physiology, fail to be re-activated once the RV is pressure

loaded. For example, our studies in chapter 4 suggest that in pressure load, the

switch back towards fetal myocardial energy metabolism20, 21 may be incomplete.

Mitochondrial dysfunction and/or alterations in energy substrate use appear

to play a central role in the pathophysiology of RV failure22-24. Modification of

the balance between glucose- and free fatty acid (FFA) metabolism has yielded

promising results in preclinical models of abnormal RV loading25-27. The current

paradigm is that the switch from FFA metabolism to glucose metabolism is

incomplete and needs to be enhanced to sustain RV function. Our data suggest

that the myocardium of the failing pressure loaded RV might be energy deprived

due to combined downregulation of both FFA and carbohydrate metabolism

(chapter 4). One may hypothesize that therefore stimulation of FFA-metabolism

(which yields more energy than glucose metabolism) is advantageous for RV

function, especially in the long term. However, some authors have suggested

the opposite approach: stimulation of carbohydrate metabolism by inhibition of

Chapter 9226

TREATMENT

FFA-metabolism (the so-called Randle cycle)25. Testing these different strategies

in our PAB model of RV failure would be a pragmatic option to establish which

one is most effective. However, a more basic (and perhaps insightful) approach

would be to unravel the exact nature of the differences between myocardial

metabolism before and after unloading. Is an RV that never was unloaded more

energy efficient that one that is pressure loaded after initial unloading? If so,

does that efficiency relate to usage of a specific substrate (e.g. glucose vs. FFA)?

If so, why does the RV that has been unloaded, fail to reach that same efficiency?

Animal experiments comparing myocardial energy metabolism in the normal

RV of near term fetal rats and the pressure loaded RV of juvenile rats may point

out specific enzymes or substrate routes that are incompletely re-activated/

inhibited. Ideally, the experiment would compare RV’s that were physiologically

unloaded and then loaded (as in PAB in juvenile animals) to RV’s that were never

unloaded (e.g. PAB in fetal animals), but this will require a larger animal model

for practical reasons.

Treating right ventricular failure

Is inhibition of the PDE5-PKG1 axis the cornerstone of RV failure treatment?

The answer depends on the context in which RV failure is placed. Sildenafil, an

inhibitor of phosphodiesterase type 5A (PDE5A), is effective in different forms of

PAH28-30. Sildenafil reduces RV hypertrophy, attenuates remodeling and improves

RV function31, 32. However, these effects are largely owing to the pulmonary

vascular effects of Sildenafil; the reduction in hypertrophy and improvement

in function are proportional to the reduction in afterload. PDE5A is highly

expressed in pulmonary vascular smooth muscle cells, and its inhibition causes

smooth muscle cell relaxation, which reduces the resistance of the pulmonary

vascular bed and thereby reduces RV pressure load. However, apart from these

indirect ‘unloading’ effects, Sildenafil might also directly target the RV33.

Studies in the LV suggested that PDE5A plays a role in regulating ventricular

function34, 35. Even more importantly, PDE5A activation, possibly via inhibition

of protein kinase G-1, was hypothesized to be a main cause of heart failure36-38.

In 2005, Takimoto and coworkers showed that Sildenafil completely prevented

and even reversed adverse remodeling and dysfunction in experimental

pressure load induced LV failure39. The PDE5A-PKG-1 axis became a focus of


basic heart failure research and a growing body reports suggest it has a central

role in clinical heart failure 40-46. However, it is important to note that despite the

profound effects of Sildenafil in preclinical research39, 41, clinical trials in patients

with LV failure show predominantly disappointing results, ranging from no effect

to mildly positive 47-51.

This naturally raised the question about the importance of the PDE5A-PKG-1 axis

in RV failure. While virtually absent in the normal RV, PDE5A has been suggested

to be highly expressed in the pressure loaded RV52. Interestingly, these results

have not been reproduced so far. We measured PDE5A (mRNA and protein)

expression in different stages of pressure-, volume-, and combined pressure/

volume loading and found consistently expression levels that were comparable

to controls. It may be that PDE5A activity (rather than expression) correlates

more closely to RV function, but it could also indicate that the role of PDE5A in

adaptation/maladaptation is less important than previously thought.

Are the effects of Sildenafil on target or off target?

How does Sildenafil exactly exerts its effects? Are the effects of Sildenafil

indeed mediated through the PDE5A-PKG1 axis? Inhibition of PDE5A would

increase cyclic GMP and consequently activate PKG-1, targeting downstream

effector calcineurin-NFAT and RGS2/Akt/MAPK-kinase pathways39, 43, 45. Although

Sildenafil activated PKG-1 in our PAB model, we did not observe alterations

in the downstream pathways in the Sildenafil treated group vs. the vehicle

treated groups (chapter 5). An exciting possible explanation is that the balance

of activation and interaction of these pathways differs between the LV and the

RV. The marked changes in downstream pathways could also be temporary,

or might only be observed in profound targeting of the pathway by genetic

modification 38, 39, 53, 54 in severe pressure overload42. However, the attenuating

effects on ventricular hypertrophy, as reported in LV loading 39, 41, that would be

expected by inhibition of hypertrophy pathways were not seen in the RV neither

by us, nor by others31, 55, 55, 56. Taken together, these findings should prompt one

to reconsider the mechanism by which Sildenafil works.

There is ample evidence that Sildenafil has pleiotropic effects beyond the

PDE5A-PKG1 axis57-61, which may provide a more logical explanation of the

functional effects of Sildenafil, at least with regard to diastolic function (chapter

6). Very recently, Sildenafil has been shown to phosphorylate the giant structural

Chapter 9228

protein titin62. Titin plays an important role in myocardial stiffness, and evidence

shows that rather than a passive fiber, titin is a highly regulated and modulated

protein63-66. Sildenafil phosphorylates titin in the N2-B unique sequence via PKG-

1 activation, thereby reducing myocardial stiffness in the LV of old, hypertensive

dogs67, 68. Given the importance of diastolic dysfunction in RV failure, it would be

interesting to see if the N2-B unique sequence is relatively dephosphorylated

in failing RVs, whether the phosphorylation status correlates to parameters of

diastolic stiffness, and whether pharmacological modification of this sequence

sustains diastolic RV function.

Should we start Sildenafil treatment in patients with RV failure?

A direct beneficial effect on the pressure loaded RV could make Sildenafil the first

drug to treat other forms of pressure load induced RV failure than pulmonary

hypertension, such as in systemic RVs after atrial switch surgeries for dextro-

transposition of the great arteries or in levo-transposition of the great arteries

, or in RV outflow tract obstruction in patients with repaired Fallot’s Tetralogy.

In this thesis we report two studies exploring the direct effects of Sildenafil

on RV remodeling and function in the PAB model in two treatment strategies:

preventive (chapter 6) and therapeutic (chapter 7). Taken together, the results

of these studies imply the intriguing concept that treatment effects depend on

the stage of the disease42. Early Sildenafil treatment during 4 weeks enhanced

contractility, reduced dilatation, but did not affect the already compromised

diastolic function that occurred after 4 weeks in the model. Late Sildenafil

treatment for 4 weeks, had no effect on contractility and mildly reduced

dilatation but improved the more severely compromised diastolic function that

occurred after 8 weeks in the model. At first sight, this might seem paradoxical,

but when the progression of RV dysfunction is taken into account, these findings

make sense. As reviewed in chapter 8, and shown in chapters 2 and 4, the early

RV response to pressure load is enhancing contractility and dilatation, while

diastolic dysfunction appears in later stages of the disease, when contractility

may reach a plateau-phase. A drug capable of enhancing contractility, reducing

dilation and improving diastolic dysfunction will therefore in early phases of RV

pressure load mainly enhance contractility and reduce dilatation, while effects

on diastolic function will be weak until pronounced diastolic dysfunction occurs.

This concept is supported by the observation that Sildenafil had no effect in the

normally loaded (and thus not-activated) RV.


Beyond the setting of RV failure due to pressure load our data provide no support

for Sildenafil treatment. While an experimental study in the volume loaded

LV showed beneficial effects of Sildenafil69, our experiments in the volume

loaded RV (chapter 6) failed to show beneficial effects. It might be that in the

LV, which is inherently more vulnerable to volume load, diastolic dysfunction

(which can be targeted by Sildenafil) plays a more prominent role than in the

volume loaded RV. However, our data suggest a more fundamental explanation:

Sildenafil failed to activate PKG-1 in the volume loaded RV. This may again reflect

distinct adaptive responses to different loading conditions.

We come back to our original question: Should we start Sildenafil treatment in

patients with RV failure? The studies in this thesis suggest that Sildenafil might

have a place in the treatment of patients with pressure load induced RV failure

outside the context of pulmonary hypertension. There is, however, a number

of concerns. Firstly, as evident from the previous discussion, the mechanisms

by which Sildenafil exerts its effect are poorly understood, which warrants

caution for adverse side-effects. Important in this respect is that we observed

accelerated fibrosis in the preventively treated PAB group (chapter 5). Secondly,

the categories of patients that could benefit from Sildenafil should be more

precisely delineated. Sildenafil’s effects appear to be highly dependent on both

the severity and type of abnormal loading condition, and the progression of

RV disease. Thirdly, the effects of Sildenafil on diastolic dysfunction were not

accompanied by improvement of exercise tolerance or RV failure symptoms.

Lastly, it is currently very challenging to measure diastolic RV function in

patients, and invasive (pressure-volume) measurements cannot systematically

be performed in patients.

It is therefore too early to start treating patients in the above mentioned

categories with Sildenafil. If the effects on diastolic dysfunction can be

reproduced in a larger animal model, preferably with concomitant improvement

of exercise capacity, in a set-up specifically designed to track adverse effects and

to evaluate effects on diastolic function by echocardiography or cardiac MRI, a

small clinical trial is indicated.

Chapter 9230

Right and left or right and wrong? Can we treat RV failure with drugs for LV

failure?

Treatment strategies for heart failure in congenital heart disease are mostly

based on the therapeutic guidelines for heart failure in adults with acquired

heart disease70. This is not completely irrational, because some patients

with congenital heart disease have similar levels of functional incapacity and

a neurohormonal profile resembling that seen in acquired heart failure71.

However, when the substantial differences between the two categories of heart

failure are appreciated (etiology, physiology, temporal development etc.), it

soon becomes apparent that the application of the treatment algorithms for

acquired heart failure in congenital heart disease is complicated. Moreover, the

functional, morphological and embryological differences between the right and

left ventricle may render chronic RV failure a unique clinical entity in itself that

does not necessarily respond to conventional therapy.

Nonetheless, attempting to treat the RV with conventional heart failure therapy

is a worthwhile approach, especially because currently no RV-specific treatments

are available. Inhibition of the renin-angiotensin-aldosteron system (RAAS) is

one of the cornerstones of heart failure therapy72, 73. In chapter 7 we studied

the effects of RAAS inhibition by combined losartan + eplerenone treatment in

a model of pressure load induced RV failure. We showed that, in contrast to in

the LV74-78, losartan + eplerenone treatment does not blunt adverse remodeling,

sustain ventricular function or prevent failure in pressure load induced RV

failure.

The significance of these findings lies primarily in contributing proof for the

principle what works in the LV, does not necessarily work in the RV. Such preclinical

data are especially important because conducting well-designed trials in patients

with congenital heart disease to address these questions is notoriously hard79,

for a variety of reasons (small, heterogeneous target population, short follow-

up attainable etc.)80. The few trials of RAAS inhibition (angiotensin converting

enzyme inhibitor or angiotensin II receptor blocker) performed in patients with

a chronically abnormally loaded RV (systemic RV) did not provide convincing

evidence for their effectiveness 81-85. Beyond RAAS inhibition, also results of

studies with β-adrenergic receptor blockers are inconclusive 86-89. The safety and

benefit of diuretics and lifestyle modification appears so evident that further

study in the RV might not be needed90. Resynchronization therapy could be

beneficial for certain subgroups of patients91-94.


CONCLUSION

In summary, there is a lack of both preclinical and clinical studies assessing

the efficacy of conventional heart failure treatment strategies for RV failure,

which should be a focus of future research. The risk of disqualifying effective

treatments strategies based on clinical studies that due to insufficient power fail

to show a significant treatment effect should be a strong incentive to perform

well-designed preclinical studies. Animal models of pressure load (pulmonary

artery banding), volume load (aorto-caval shunt; pulmonary valve insufficiency)

and combined pressure+ volume load as described in this thesis, provide an

excellent platform for this.

Lastly, the negative results of (pre)clinical studies testing LV therapies for the RV

convey an important message. RV failure is a unique clinical entity that needs

tailor-made treatment. This calls for the identification of new and more directly

relevant targets for intervention.

In the last decade, more knowledge about the abnormally loaded RV has been

accumulated than ever before. This knowledge may have rendered the RV less

elusive, but no less remarkable14. At the same time, concrete pharmacological

candidates for treatment of patients are still very few. In this thesis we show

that there are at least three routes towards new treatment strategies for RV

failure that merit exploration. With the suffering of patients and their families in

mind, it is to be hoped that these explorations will soon turn RV failure from a

detrimental into a manageable condition.

Chapter 9232

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APPENDICES

SUMMARYINDUT CH

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Falen van de rechter hartkamer (RV falen) door chronische abnormale

belasting is een belangrijke veroorzaker van ziekte en sterfte van patiënten

met pulmonale hypertensie, een aangeboren hartafwijking of hartfalen. Het

belang van de rechter kamerfunctie is pas recent onderkend en daarom is er

betrekkelijk weinig wetenschappelijk onderzoek gedaan naar de rechterkamer

(RV), zeker vergeleken met de linkerkamer (LV). De kennis opgedaan ten aanzien

van de LV kan niet zonder meer worden toegepast op de RV omdat beide kamers

aanmerkelijk verschillen qua functie, anatomie en embryologische oorsprong.

Ook de etiologie van LV falen (ischaemische hartziekten, cardiomyopathieen

en hypertensie) verschilt van RV falen. RV falen is meestal het gevolg is van

chronisch abnormale belasting. Het gebrek aan kennis vertaalt zich in het feit

dat er momenteel geen specifieke behandelingen beschikbaar zijn voor RV falen.

Onderzoek naar de RV respons (het geheel van genetische, post-translationele,

morfologische en functionele veranderingen) op verschillende vormen van

abnormale belasting is daarom van groot belang. Het nauwgezet ontrafelen van

de RV respons maakt het mogelijk om de gunstige en ongunstige veranderingen

in de RV te onderscheiden. Herkenning van gunstige en ongunstige processen is

de basis voor de ontwikkeling van behandelingen voor RV falen.

Diermodellen van abnormale RV belasting zijn om verschillende redenen een

belangrijk middel in dit onderzoeksveld. Ten eerste bieden ze het conceptuele

raamwerk waarop begrip van de klinische situatie geënt kan worden. Ten tweede

maken ze het mogelijk pathofysiologische en pathobiologische veranderingen

nauwgezet te meten onder strikt gecontroleerde omstandigheden, hetgeen

onmogelijk is in patiënten. Tot slot kunnen in diermodellen experimentele

behandelingen getest worden, zonder patienten in gevaar te brengen.

De studies in dit proefschrift zijn voor bovenstaande doeleinden ontworpen.

Het kader waarin de onderzoeksvragen werden gesteld wordt weergegeven

in hoofdstuk 1. In de daarop volgende hoofdstukken beschrijven we studies in

ratten en muizen met verschillende vormen van abnormale RV belasting om

1) de pathofysiologie en pathobiologie van RV falen beter te begrijpen en 2)

mogelijke behandelingen voor RV falen te testen.


Deel I: pathofysiologie en pathobiologie

Een fundamentele vraag in dit onderzoeksveld is of alle soorten abnormale

belasting worden beantwoord met dezelfde RV respons, of dat deze varieert

met de soort belasting. Hoewel de beschikbare literatuur suggereert dat

er verschillende soort-specifieke RV respons patronen bestaan, directe

vergelijkingen veelal ontbreken. In hoofdstuk 2 maken we twee van dergelijke

directe vergelijkingen in ratten modellen. We vergeleken de RV respons op twee

soorten druk belasting (pulmonale hypertensie (PH) versus banding van de

truncus pulmonalis (PAB)) en we vergeleken de RV respons tussen pulmonale

hypertensie met en zonder additionele volume belasting. In hoofdstuk 3

vergeleken we de RV respons op drukbelasting en volume belasting in muizen.

Beide studies tonen belangrijke verschillen in functionele RV respons op

verschillende soorten abnormale belasting. In hoofdstuk 2 beschrijven we dat

drukbelasting door PAB resulteerde in diastolische disfunctie na 4 weken, terwijl

PH dat niet deed, onafhankelijk van de mate van RV hypertrofie. Daarnaast gaf

PAB meer RV dilatatie and klinische tekenen van falen dan PH. De combinatie

van PH en volume belasting resulteerde in toegenomen hypertrofie en een

patroon van pseudonormalisatie van functionele parameters van met name

contractiliteit.

In hoofdstuk 3 reduceerde drukbelasting de spontane inspanning van muizen,

terwijl het hartminuutvolume normaal bleef. Bij volume belasting daarentegen,

bleef de spontane inspanning intact en was het hartminuutvolume toegenomen.

Ook de moleculaire response verschilde tussen de soorten belasting:

drukbelasting leidde tot een activatie van de calcineurine-NFAT pathway, volume

belasting niet.

In hoofdstuk 4 beschrijven we de pathofysiologie en –biologie van het PAB

model. PAB induceerde (sub)klinisch RV falen binnen 11 weken in alle ratten.

In pathofysiologisch opzicht werd RV falen gekenmerkt door toegenomen

contractiliteit en verslechterende diastolische functie. De rechterkamers

van alle ratten hadden pathologische veranderingen zoals hypertrofie van de

cardiomyocyten, fibrose, verminderde dichtheid van capillairen, expressie van

het foetale gen-programma (natriuretische peptiden, isoform switch van de

myosin heavy chain) en activatie van de calcineurine-NFAT pathway. Echter, de

DEEL I

Appendices246

capillair-myocyt ratio was significant lager in ratten met klinische tekenen van

RV falen, dan in ratten zonder klinische tekenen van RV falen. De ratten met

klinische tekenen van RV falen hadden ook toegenomen mRNA expressie van

hemoxygenase-1, een uiting van oxidatieve stress, en significant afgenomen

expressie van de genen die coderen voor de actine filamenten.

Deel II: behandelopties

In dit proefschrift rapporteren we twee studies naar de directe effecten

van Sildenafil op RV remodellering en –functie in het PAB model. We testten

twee verschillende behandelingsstrategieën: preventief (hoofdstuk 5) en

therapeutisch (hoofdstuk 6). Preventieve behandeling werd gegeven gedurende

4 weken vanaf de PAB operatie. Therapeutische behandeling werd ook gegeven

gedurende 4 weken, maar startte 4 weken na de PAB operatie, wanneer er al

sprake was van RV disfunctie. Samengenomen impliceren de resultaten van

deze twee studies het intrigerende concept dat de effecten van Sildenafil

behandelingen afhankelijk zijn van de fase waarin de ziekte zich bevindt.

Preventieve Sildenafil behandeling verhoogde RV contractiliteit, verminderde

dilatatie, maar liet diastolische functie ongewijzigd. Therapeutische Sildenafil

behandeling had een zwak (niet significant) effect op contractiliteit, een matig

reducerend effect op dilatatie maar verbeterde de diastolische functie van de

rechterkamer. Dit lijkt paradoxaal, maar is in het licht van de progressie van RV

disfunctie begrijpelijk. Zoals aangetoond in hoofdstukken 2 en 4 is de vroege

RV respons op drukbelasting verhoging van de contractiliteit en dilatatie, terwijl

diastolische disfunctie pas in de latere fase van de ziekte een rol gaat spelen,

wanneer de contractiliteit een plateau-fase bereikt. Een farmacon dat in staat

is om contractiliteit te verhogen, dilatatie te remmen en diastolische functie

te verbeteren zal daardoor in de vroege fase van drukbelasting voornamelijk

contractiliteit verhogen en dilatatie remmen, terwijl de effecten op diastolische

functie pas duidelijk worden in de fase waarin diastolische disfunctie toeneemt.

Dit concept wordt ondersteund door de observatie dat Sildenafil geen enkel

effect heeft op een normaal belaste rechterkamer (hoofdstuk 5).

DEEL II


Tot slot beschrijven we in hoofdstuk 7 de effecten van remming van het renine-

angiotensine-aldosteron systeem (RAAS) op RV falen ten gevolge van chronische

drukbelasting. Hiervoor werden ratten met een PAB gedurende maximaal 11

weken behandeld met losartan (angiotensine II type 1 –receptor blokker) en

eplerenone (aldosteron-receptor blokker). Losartan+eplerenone behandeling

remde RV remodellering niet, had geen ondersteunend effect op RV functie en

voorkwam het ontstaan van RV falen niet. Dit is in schril contrast met eerdere

bevindingen in de drukbelaste linkerkamer. Het belang van deze observaties

ligt voornamelijk in het feit dat ze het paradigma onderschrijven dat wat ‘links’

werkt, niet per se ‘rechts’ ook werkt.

Deel III: bespreking en toekomstperpectieven

In hoofdstuk 8 en 9 worden de belangrijkste bevindingen van dit proefschrift

besproken en in een breder perspectief geplaatst van de huidige literatuur

over de pathofysiologie en pathobiologie van RV falen en behandelopties,

veelal verkregen in diermodellen. Tevens doen we suggesties voor toekomstig

onderzoek.

Concluderend geven de studies in dit proefschrift het beeld van rechterkamer-

falen als een uniek en heterogeen klinisch syndroom dat niet vanzelfsprekend

gevoelig is voor conventionele hartfalen therapie. Diermodellen van chronisch

abnormale RV belasting moeten ook in de toekomst gebruikt worden om

meer inzicht te verkrijgen in de soort-specifieke pathofysiologische en

pathobiologische veranderingen in verschillende soorten van belasting. Op

basis van deze studies kunnen experimentele behandelingen (zoals Sildenafil)

hun weg vinden naar klinische toepassingen.

DEEL III

ACKNOWLEDGEM ENTS

Appendices250

I am well aware of the fact, dear reader, that most likely only now I finally have

your attention. I started a PhD project because my curiosity was tickled by an

elusive heart and also because I wanted to mature and broaden my perspective

before dedicating my life to clinical pediatrics. (and because it allowed me to

listen to the prelude of BWV 872 over and over and over again throughout

the week). From my current vantage point, I’d say that my curiosity was met

by greater mysteries than I suspected, my freshmanship by a tough and steep

learning curve and with regard to perspective, I’d say the horizon is still stretching

further. Most importantly though, I look back on 4 years in which I thoroughly

enjoyed meeting and working with a host of people, who contributed in big

and small ways to the book that you are now holding. I am honored to have

coordinated their collective effort. As far as I am concerned, it is appropriate

that their names would get the best of your attention.

Prof. dr. R.M.F. Berger, beste Rolf. Ik heb een diep respect voor je gedrevenheid

en enthousiasme om topkwaliteit klinische zorg, onderwijs en onderzoek te

leveren. Je hebt een uitzonderlijk vermogen om met weinig middelen grote

doelen te bereiken. Onze samenwerking heeft een waardevolle rol gespeeld in

mijn vormingsproces tot klinicus en wetenschapper; ik hoop dat ik permanent

besmet ben geraakt met jouw onverzettelijkheid en professionaliteit. Bovendien

gaf je op de hoogte- en dieptepunten aan het thuisfront altijd volop ruimte om

mijn aandacht en tijd thuis te besteden. Dank je wel voor de gelegenheid om dit

project bij jou te doen. Een voorrecht!

Dr. B. Bartelds, beste Beatrijs. Ik kan me geen co-promotor voorstellen die

beter dan jij in staat is om mij tot de uitersten van mijn kunnen te pushen. Je

uitdagende en inventieve vragen, kritische commentaar op presentaties en

manuscripten en je messcherpe analyses van data en literatuur waren goud

waard voor mijn promotietijd. Al hebben schaarste in tijd, energie en soms wat

tegenspoed af en toe een stempel gedrukt op de voortgang van de projecten, je

deed altijd je best om onze onderzoekstijd ook prettig en verrijkend te laten zijn

(‘this is holiday’). Dank!


I would like to thank the members of the reading committee, prof. dr. W.H. van

Gilst, prof. dr. R. Naeije and prof. dr. T. Ebels for their critical evaluation of my

thesis.

Dr. M. de Vroomen, beste Maartje. Dankjewel voor jouw onmisbare bijdrage aan

de verschillende onderzoeken. En natuurlijk voor een geweldige herintroductie

in de klinische kindergeneeskunde tijdens mijn dagje MCL.

Dr. P. Steendijk, beste Paul. Je werd mij ooit geïntroduceerd als ‘de koning van

de PV-analyse’. Mij is al vlot gebleken dat die term jou werkelijk te kort doet.

Dankjewel voor de vele uren waarin we samen naar CircLab staarden. Je hebt

me in korte tijd cruciale kennis en technieken geleerd die een centrale rol spelen

in mijn onderzoeksprojecten. Ik denk met plezier terug aan de vele bezoeken aan

Leiden. Op afstand heb je per mail en/of telefoon dikwijls oplossingen geboden

bij technische problemen of gewoon met je natuurkundige nuchterheid orde

gebracht in het wazige brein van de klinicus. Veel dank!

We found a welcoming and stimulating environment at the Experimental

Cardiology. I would like to thank the entire staff (including Danielle en Carla)

for facilitating that and the many useful comments and questions. Especially, dr.

H.H.W. Silljé, beste Herman. Dankjewel voor je hulp, inzichten en de altijd open

deur op het lab.

Dear collegues; it was a blast working with you over the years: Irma, Hisko, Jardi,

Willem-Peter, Liza, Meimei (zu zhou buh pah, kai shie tan!), Bo, Lili, Hongjuan,

Leonie, Mariusz (thanks!), Anne-Margreet, Atze (still need to continue our God-

talk), Frank, Irene (pleasure working with you), Jasper, Lennaert, Niek (you

recovered tons of data that I deleted (fail!); for that and many other reasons you

are rather brilliant), Vincent (wish you the best), Renee, Hasan (we should catch

up), Megan, Wardit, Maxi, Wouter Meijers, Wouter te Rijdt, Nicolas, Rogier,

Harmen.

Bibiche, Janny. Ik heb het jullie niet altijd makkelijk gemaakt. Jullie hebben het

mij wel vaak makkelijk gemaakt. Jullie leverden altijd topkwaliteit, of het nou

PCR of western of inbedden was. Wat fijn om met jullie samen te werken!

Appendices252

Inge, Martin (dokter Dokter), Linda en Silke (sorry for the smelly livers), bedankt

voor tips, trucks en hulp!

A number of students crunched data, pipetted, stained, did the Western. Re-

did it. Re-did it again. And then again. And … well you get a sense of their

awesomeness. Marnix, Germen, Anne-Marie, Maarten, thank you!

Vincent Bloks, array-genius, thank you for the tremendous effort on the array-

studies and your patience whenever Annemarie and me –once again- really

didn’t know what we were talking about.

Staff at the animal facility. Thanks for all the hard supportive work! Ramon,

prettig je elke dag te spreken en je met zoveel overgave te zien werken. Ik wens

je het beste!

Annemieke, Andre, koffie? Thanks for many, many hours of work on my

experiments. And for your cooperation and flexibility whenever I came up with

a new idea (project/ experiment/model …) at the last minute. And of course for

10,000 jokes about my ‘love’ for rodents. Het ga jullie goed!

Michel, als dit boek een held had, dan was jij het zeker weten! Dankjewel voor

je enorme toewijding aan dit werk en de bergen talent en energie die je in mijn

projecten hebt gestoken. Erg prettig om met je samen te werken. Jij bent de

eerste die ik bel voor mijn volgende project ... en ook als mijn blindedarm eruit

moet.

Collega onderzoekers in het Centrum voor Aangeboren Hartafwijkingen; Laura,

Menno, Djoeke, Mark-Jan, Willemijn, Floris, Jan-Renier, Diederik, Lysanne. Zet

‘m op!

Mentors of the project management course; prof. dr. K.N. Faber, prof. dr. M.G.

Rots and prof. dr. H. Snieder: dear Klaas Nico, Marianne and Harold, thanks for

your advice and support on pivotal moments during my PhD years.


De kinderartsen en collega assistenten van het Martini Ziekenhuis, wat een

voorrecht om voor en met jullie te werken! Jullie hebben me moeiteloos weten

om te vormen van eigenwijze kindercardiologisch onderzoeker tot kinderarts-in-

wording. Dank ook voor de gelegenheid om mijn proefschrift af te ronden naast

het klinische werk.

My paranimfen, Michael and Joel:

Dear Dr. Dickinson, brother-in-arms, what a privilege to work alongside of you!

I am still in awe of your many talents, endurance and dedication. Your positive

and good-natured spirit has been very helpful for me, especially when the

animals seemed to be against us. I couldn’t have done this project without you.

Good luck to you and Janine. I hope we will soon work together again in both the

clinical setting as well as in research.

Dear Joel, you and Mirjam have contributed to this period in many ways, both

directly and indirectly. It’s awesome that you get to be here for the defense,

eh? We have noticed the lesson of the heart; that life is best lived only when

you find a rhythm in which effort and relaxation alternate. I hope we can

support eachother in learning to live accordingly with our families. Thank you

for everything.

Christiaan, dear dr. Wesselink, I am very thankful that after all these years our

lives are still closely intertwined. It’s been quite a ride last couple of years; more

life-events than we can count. I think it’s time for a party!

Dank ook aan mijn schoonfamilie voor geduld met een drukbezette schoonzoon/

zwager.

Maarten, David, my big brothers, I guess we were very, very, fortunate to grow

up in a family that nurtured, stimulated and delighted in our young minds.

I ordered extra bitterballs to celebrate that.

Appendices254

Mama, Papa, hoe ouder ik word, hoe beter ik begrijp wat een fantastische

ouders jullie zijn. Ik hoop dat mijn leven getekend wordt door dankbaarheid

voor alles wat door jullie heen ons gegeven is.

Josephine en Pieter, sorry dat er zo weinig plaatjes in dit saaie boek staan.

Gelukkig staat er een prachtige dolfijn (Orcinus orca linnaei) op de rug van

de kaft! Wat was het heerlijk om na lange dagen op het lab altijd thuis weer

onthaald te worden door het vrolijke geluid van rennende blote voetjes op de

kamervloer. Gaan jullie vandaag gewoon op tijd naar bed? Promotiefeest is echt

erg saai voor jullie. Morgen heeft papa weer gewone kleren aan en dan gaan we

iets veel leukers doen.

Mijn lieve, onovertroffen en fantastische vrouw, Nienke. Ik draag dit proefschrift

aan jou op, al is het eigenlijk net zo goed het resultaat van jouw inzet, flexibiliteit

en incasseringsvermogen als van het mijne. Je uiterlijke schoonheid verhult nog

zoveel meer moois van binnen. Telkens weer een onwerkelijke gewaarwording

dat ik met jou het leven mag delen. Mijn beker stroomt over.

B I B L I O G R A P H Y

Appendices256

Right ventricular adaptation in congenital heart diseases

Bartelds B, Borgdorff MA, Berger RM. J. Cardiovasc. Dev. Dis. 2014, 1(1), 83-97

Right ventricular failure due to chronic abnormal loading conditions. What

have we learned from preclinical research?

Borgdorff MA, Bartelds B, Dickinson MG, Berger RM. Submitted.

A cornerstone of heart failure treatment is not effective in experimental right

ventricular failure

Borgdorff MA, Bartelds B, Dickinson MG, Steendijk P, Berger RM. Int J Cardiol.

2013 Nov 5;169(3):183-9

Right ventricular failure in chronic pressure load is associated with progressive

diastolic dysfunction

Borgdorff MA, Koop AM, Dickinson MG, van Wiechen MP, Steendijk P, Silljé HH,

Berger RM, Bartelds B. Under revision.

Distinct loading conditions reveal various patterns of right ventricular

adaptation

Borgdorff MA, Bartelds B, Dickinson MG, Steendijk P, de Vroomen M, Berger RM.

Am J Physiol Heart Circ Physiol. 2013 Aug;305(3):H354-64

The role of disturbed blood flow in the development of pulmonary arterial

hypertension: lessons from preclinical animal models

Dickinson MG, Bartelds B, Borgdorff MA, Berger RM. Am J Physiol Lung Cell Mol

Physiol. 2013 Jul 1;305(1):L1-14

Sildenafil treatment in established right ventricular dysfunction improves

diastolic function and attenuates interstitial fibrosis independent from

afterload

Borgdorff MA, Bartelds B, Dickinson MG, van Wiechen MP, Steendijk P, de

Vroomen M, Berger RM. Am J Physiol Heart Circ Physiol. 2014 May 30. pii:

ajpheart.00843.2013. [Epub ahead of print]

Bibliography 257

A critical role for Egr-1 during vascular remodelling in pulmonary arterial

hypertension

Dickinson MG, Kowalksi P, Bartelds B, Borgdorff MA, Kamps JA, van der Feen DE,

Sietsma J, Molema G, Berger RM. Cardiovascular Research 2014, in press

Inhibition of Calcineurin activation prevents ventricular dilatation in the

pressure loaded RV

Bartelds B, Borgdorff MA, Smit-van Oosten S, Weij M, Takens J, Boersma B,

Nederhoff MG, van Gilst WH, de Windt L, Berger RM. Under revision

Sildenafil enhances systolic adaptation, but does not prevent diastolic

dysfunction, in the pressure-loaded right ventricle

Borgdorff MA, Bartelds B, Dickinson MG, Boersma B, Weij M, Zandvoort A, Silljé

HH, Steendijk P, de Vroomen M, Berger RM. Eur J Heart Fail. 2012 Sep;14(9):1067-

74

Differential responses of the right ventricle to abnormal loading conditions in

mice: pressure vs. volume load

Bartelds B, Borgdorff MA, Smit-van Oosten A, Takens J, Boersma B, Nederhoff

MG, Elzenga NJ, van Gilst WH, De Windt LJ, Berger RM. Eur J Heart Fail. 2011

Dec;13(12):1275-82

Egr-1 expression during neointimal development in flow-associated pulmonary

hypertension

Dickinson MG, Bartelds B, Molema G, Borgdorff MA, Boersma B, Takens J, Weij

M, Wichers P, Sietsma H, Berger RM. Am J Pathol. 2011 Nov;179(5):2199-209.

Epub 2011 Sep 13.

Multiple voxel 1H MR spectroscopy of phosphorylase-b kinase deficient

patients (GSD IXa) showing an accumulation of fat in the liver that resolves

with aging

Sijens PE, Smit GP, Borgdorff MA, Kappert P, Oudkerk M. J Hepatology

2006;45:851-5

Appendices258

The right auricle tunnel in total cavopulmonary connection does not prevent

atrial arrhythmias in the long term

Borgdorff MA, van den Heuvel F, Bartelds B, Waterbolk TW, Berger RM. In

preparation

Experimental right ventricular failure is associated with dysregulation of genes

involved in dilated

cardiomyopathy

Borgdorff MA*, Koop AMC*, Bloks VW, Silljé HHW, Berger RMF, Bartelds B. Eur

Heart J 2013;34(suppl):622 (ESC Congress 2013, Amsterdam, the Netherlands)

Right ventricular failure is diastolic heart failure and is not caused by

hypertrophy or fibrosis

Borgdorff MAJ, Bartelds B, Dickinson MG, Steendijk P, de Vroomen M, Berger

RMF. J Am Coll Cardiol. 2013;61(10_S) E453 (ACC Congress 2013; San Francisco,

CA, USA).

Sildenafil improves established right ventricular dysfunction via enhancement

of diastolic function Borgdorff MAJ, Bartelds B, Dickinson MG, Steendijk P, de

Vroomen M, Berger RMF. J Am Coll Cardiol. 2013;61(10_S) E459 (ACC Congress

2013; San Francisco, CA, USA).

Distinct patterns of functional right ventricular adaptation to experimental

right ventricular pressure vs volume overload

Borgdorff MAJ, Bartelds B, Smit-van Oosten A, Steendijk P, de Vroomen M,

Berger RMF. Cardiol Young 2011;21(suppl):S16 (AEPC Congress 2011; Granada,

Spain)

Sildenafil improves the pressure-loaded right ventricle independent from the

pulmonary vascular resistance

Borgdorff MAJ, Bartelds B, Dickinson MG, Takens J, Boersma B, Smit-van Oosten

A, Weij M, Zandvoort A, Steendijk P, de Vroomen M, Berger RMF. Am J Respir Crit

Care Med 183;2011:A4981

(ATS Congress 2011, Denver, CO, United States of America)

Bibliography 259

PDE5A-inhibition benefits the pressure loaded, but not the volume loaded

right ventricle

Borgdorff MAJ, Bartelds B, Dickinson MG, Steendijk P, De Vroomen M, Berger

RMF.

Eur Heart J 2011;32(suppl):435 (ESC Congress 2011, Paris, France)

The right ventricle from adaptation to maladaptation. Correlations in a rat

model

Borgdorff MAJ, de Vroomen M, Bartelds B, Dickinson MG, Takens J, Boersma B,

Steendijk P, Berger RMF. Eur Heart J 2010;31(suppl):751 (ESC Congress 2010,

Stockholm, Sweden)

ABOUTTHEAUTH OR

Appendices262

Reinout Borgdorff (1982) was born in Zeist. He earned his MD at the University

Medical Center Groningen, University of Groningen, where he subsequently

worked as a PhD-fellow at the Center for Congenital Heart Disease, department

of Pediatric Cardiology (Beatrix Children’s Hospital) under the supervision of

prof. dr. R.M.F. Berger. The studies performed then are presented in this thesis.

He is currently working as a resident in the pediatrics department of the Martini

Ziekenhuis in Groningen. In 2015 he will start his pediatric residency at the

Beatrix Children’s Hospital. He lives in Groningen with his wife and children.

T H E E L U S I V E H E A R T the right ventricle in chronic abnormal loading conditions

M.A.J. Borgdorff

THE EL

USIVE H

EART

M.A.J. BorgdorFF

‘ t h e r i g h t v e n t r i c l e i s a n e l u s i v e h e a r t ; i t s f u n c t i o n

a n d f o r m a r e o n l y r e c e n t l y b e g i n n i n g t o b e u n c o v e r e d .

Y e t i t s i m p o r t a n c e i n t h e p h y s i o l o g y o f c o n g e n i t a l

h e a r t d i s e a s e s a n d p u l m o n a r y a r t e r i a l h y p e r t e n s i o n c a n

h a r d l y b e u n d e r e s t i m a t e d . ’

adjective \ē-ˈlü-siv, -ˈlü-ziv\

: hard to find or capture, understand, define, isolate or identify

: tending to evade grasp or pursuit

Example: the blue whale (Balaenoptera musculus) is one of the ocean's most elusive inhabitants

elu·sive

ISBN 978-90-367-7172-6 Also available

as an ebook www.theelusiveheart.wordpress.com

T H E E L U S I V E H E A R T

the right ventricle in chronic abnormal loading conditions

door Marinus A.J. Borgdorff, op maandag 27 oktober 2014 om 12.45 uur precies in het Academiegebouw van de Rijksuniversiteit Groningen, Broerstraat 5, Groningen

Direct aansluitend aan de promotie in het Academiegebouw

Vanaf 21.00 uur bent van harte welkom in Land van Kokanje, Oude Boteringestraat 9, Groningen

Uitnodiging voor het bijwonen van de openbare verdediging van het proefschrift

RECEPTIE

FEEST

PARANimfen

Reinout Borgdorff Leisteenstraat 13 9743 VA Groningen [email protected]

Michael G. Dickinson Joel M. Harding [email protected]

T H E E L U S I V E T H E E L U S I V E T H E E L U S I V E T H E E L U S I V E

H E A R TH E A R TH E A R TH E A R T

the right ventricle in chronic abnormal loading

conditions

door Marinus A.J. Borgdorff, op maandag 27oktober 2014 om 12.45 uur precies in hetAcademiegebouw van de RijksuniversiteitGroningen, Broerstraat 5, Groningen

Direct aansluitend aan de promotie in hetAcademiegebouw

Vanaf 21.00 uur bent u van harte welkom inLand van Kokanje, Oude Boteringestraat 9,Groningen

Uitnodiging voor het bijwonen van de openbareverdediging van het proefschrift

RECEPTIE

FEEST

PARANimfen

Reinout Borgdorff Leisteenstraat 139743 VA [email protected]

Michael G. DickinsonJoel M. Harding

[email protected]

THE EL

USIVE H

EART

M.A.J. BorgdorFF

Documents

Proefschrift Borgdorff