The Effect of Adenosine or Diazoxide on Apoptosis

AHMED SHALABY

The Effect of Adenosine or Diazoxide on Apoptosis, Aquaporin-7 and Glycocalyx integrity

during Myocardial Protection in Coronary Bypass Surgery

ACADEMIC DISSERTATIONTo be presented, with the permission of

the Board of the School of Medicine of the University of Tampere,for public discussion in the Small Auditorium of Building M,

Pirkanmaa Hospital District, Teiskontie 35, Tampere, on August 30th, 2013, at 12 o’clock.

UNIVERSITY OF TAMPERE

ACADEMIC DISSERTATIONUniversity of Tampere, School of MedicineTampere University Hospital, Heart Center Finland

Reviewed by Docent Vesa Anttila University of OuluFinland Docent Peter RaivioUniversity of HelsinkiFinland

Supervised by Professor Matti TarkkaUniversity of TampereFinlandDocent Ari MennanderUniversity of TampereFinland

Copyright ©2013 Tampere University Press and the author

Cover design byMikko Reinikka

Acta Universitatis Tamperensis 1841 Acta Electronica Universitatis Tamperensis 1321ISBN 978-951-44-9182-5 (print) ISBN 978-951-44-9183-2 (pdf )ISSN-L 1455-1616 ISSN 1456-954XISSN 1455-1616 http://tampub.uta.fi

Suomen Yliopistopaino Oy – Juvenes PrintTampere 2013

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To my professor, Matti Tarkka and my Family.

4

LIST OF ORIGINAL COMMUNICATIONS

This dissertation is based on the following four original publications, referred to in

the text by their Roman numbers I-IV

I Shalaby A, Rinne T, Järvinen O, Saraste A, Laurikka J, Porkkala H, Saukko P,

Tarkka T. Initial results of a clinical study: adenosine enhanced cardioprotection

and its effect on cardiomyocytes apoptosis during coronary artery bypass grafting.

Eur J Cardiothorac Surg (2008) 33(4):639-644.

II Shalaby A, Rinne T, Järvinen O, Latva-Hirvelä J, Nuutila K, Saraste A, Laurikka

J, Porkkala H, Saukko P, Tarkka M. The Impact of Adenosine Fast Induction of

Myocardial Arrest during CABG on Myocardial Expression of Apoptosis-Regulating

Genes Bax and Bcl-2. Cardiology Research and Practice (2009) 1-6.

III Shalaby A, Mennander A, Rinne T, Oksala N, Aanismaa R, Narkilahti S,

Paavonen T, Laurikka J, Tarkka M. Aquaporin-7 expression during coronary artery

bypass grafting with diazoxide. Scandinavian Cardiovascular Journal (2011)

45(6):354-359.

IV Mennander A, Shalaby A, Oksala N, Leppanen T, Hamalainen M, Huovinen S,

Zhao F, Moilanen E, Tarkka T. Diazoxide may protect endothelial glycocalyx

integrity during coronary artery bypass grafting. Scandinavian Cardiovascular

Journal (2012) 46(6):339-344.

The original publications are reprinted with the permission of the copyright holders.

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ABBREVIATIONS

AIF Apoptosis inducing factor

Apaf-1 Apoptotic protease activating factor 1

ATP Adenosine triphosphate

Bax Bcl-2 Associated X protien

Bcl B cell Lymphoma 2

Ca2+

Calcium

CABG Coronary artery bypass grafting

CI Cardiac index

CK-MB Creatine Kinase myocardial band

CO Cardiac output

COPD Chronic obstructive pulmonary disease

CPB Cardiopulmonary bypass

DNA Deoxynucleotideamine

ECG Electrocardiomyogram

EF Ejection fraction

FADD Fas-associated death domain protein

FLICE FADD-like interleukin-1β-converting enzyme

GSH Glutathione (reduced form)

GSSG Glutathione (oxidized form)

HR Heart rate

IRI Ischemia-reperfusion injury

ICU Intensive care unit

IHC Immunohistochemistry

IL Interleukin

ISF Interstitial fluid

K+ Potassium

mitoKATP Mitochondrial ATP sensitive potassium channel

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MAP Mean arterial pressure

Mg + Magnesium

MI Myocardial Infarction

MPAP Mean pulmonary artery pressure

Na+ Sodium

NADP Nicotinamideadeninedinucleotidephosphate

NO Nitric oxide

PCWP pulmonary capillary wedge pressure

PKC Protein kinase C

PVRI Pulmonary vascular resistance index

ROS Reactive oxygen species

SVRI Systemic vascular resistance index

TNF Tumor necrosis factor.

TnT Troponin T

TUNEL Terminal deoxynucleotidyl- transferase mediated dUTP

nick end- labeling

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Table of Contents

ABBREVIATIONS ................................................................................................................ 5

Table of Contents ................................................................................................................... 7

3. ABSTRACT ....................................................................................................................... 9

4. INTRODUCTION ........................................................................................................... 10

5. REVIEW OF THE LITERATURE ................................................................................ 11

5.1. Background .............................................................................................................. 11

5.2. Mechanisms involved during ischemia-reperfusion injury ................................. 12 5.2.1. Systemic Inflammatory Response ...................................................................... 14 5.2.2. Myocardial Stunning .......................................................................................... 14 5.2.3. Arrhythmias ........................................................................................................ 15

5.3. Myocardial protection techniques ......................................................................... 16 5.3.1. Non cardioplegic techniques .............................................................................. 16 5.3.2. Cardioplegic techniques ..................................................................................... 17

5.4. Myocardial Necrosis and Apoptosis ...................................................................... 21

5.5. Bax and bcl2 gene expression ................................................................................. 21 5.5.1. Pathophysiology of cardiomyocyte apoptosis .................................................... 22 5.5.2. Activation and regulation of apoptosis ............................................................... 22

5.5.3. Diagnostic methods and techniques of apoptosis ............................................... 24

5.6. Myocardial protection and apoptosis .................................................................... 24

5.7. Myocardial Aquaporins .......................................................................................... 25

5.8. Endothelial glycocalyx integrity ............................................................................. 25

5.9. Adenosine and myocardial protection ................................................................... 26 5.9.1. Adenosine preconditioning and pretreatment .................................................... 26

5.9.2. Mechanism of action of Adenosine .................................................................... 26

5.10. Diazoxide and myocardial protection .................................................................. 27

6. AIM OF THE WORK...................................................................................................... 29

7. MATERIAL AND METHODS ....................................................................................... 30

7.1. Patient selection ....................................................................................................... 30 7.1.1. Patients receiving Adenosine (I, II) .................................................................... 30 7.1.2. Patients receiving Diazoxide (III, IV) ................................................................ 30

7.2. Anesthesia ................................................................................................................ 31

7.3. Operative and perfusion techniques ...................................................................... 31 7.3.1. Cardioplegia and Adenosine administration (I, II) ............................................. 31

7.3.2. Cardioplegia and Diazoxide administration (III, IV) ......................................... 32

7.4. Tissue harvesting ..................................................................................................... 32 7.4.1. Patients receiving Adenosine (I, II) .................................................................... 32 7.4.2. Patients receiving Diazoxide (III, IV) ................................................................ 32

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7.5. Sample analysis ........................................................................................................ 33 7.5.1. Patients receiving Adenosine (I, II) .................................................................... 33 7.5.2. Patients receiving Diazoxide (III, IV) ................................................................ 34 7.5.3. Statistical analyses .............................................................................................. 36

8. Results .............................................................................................................................. 37

8.1. Adenosine and TUNEL-positive cells (I) ............................................................... 37

8.2. Expression of bax and bcl2 genes and Adenosine (II) .......................................... 37

8.3. Histopathological changes, Aquaporin-7 expression and Diazoxide (III) .......... 39

8.4. Endothelial glycocalyx integrity and Diazoxide (IV) ........................................... 39

9. DISCUSSION .................................................................................................................. 41

10. SUMMARY................................................................................................................... 45

11. ACKNOWLEDGEMENTS ......................................................................................... 46

12. References .................................................................................................................. 48

13. ORIGINAL COMMUNICATIONS .............................................................................. 63

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3. ABSTRACT

Background:

Myocardial dysfunction after coronary bypass surgery (CABG) is associated with

ischemia-reperfusion injury (IRI) and tissue damage. Adenosine and Diazoxide are

ATP sensitive mitochondrial potassium channel (KATP channel) openers that may

have cardioprotective value after myocardial dysfunction and CABG.

Objective:

We evaluated whether Adenosine and Diazoxide added to blood cardioplegia

during CABG have a myocardial cardioprotective impact.

Materials & methods:

This thesis consists, in a double blinded randomized fashion, two sets of

hemodynamically stable patients:

1. Patients with stable coronary artery disease (CAD) without recent myocardial

infarction treated with Adenosine. During CABG, Adenosine 250 μg/kg was

injected into the aortic root immediately after cross clamping. For myocardial

apoptosis, a dUTP nick-end labeling (TUNEL) detection kit was used and

expressions of bax and bcl-2 genes were evaluated.

2. Patients had a stable coronary artery disease eligible for elective CABG, but

with a history of a recent less than a month old myocardial infarction. Diazoxide 50

µg/l was mixed with blood cardioplegia and injected into the aortic root. Expression

of Aquaporin-7 and peripheral plasma levels of syndecan-1 and hyaluronan were

investigated.

Results:

Adenosine decreased apoptosis without hemodynamic compromise. Diazoxide

decreased relative Aquaporin-7 expression associated with postoperative tissue

damage, while decreasing syndecan-1 and hyaluronan early postoperative levels.

Conclusions:

Adenosine and Diazoxide added to blood cardioplegia during CABG may have a

myocardial cardioprotective impact in decreasing myocardial apoptosis and

preserving endothelial glycocalyx integrity, respectively.

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4. INTRODUCTION

Current cardioprotective strategies during cardiopulmonary bypass (CPB) allow

patients to undergo coronary artery bypass surgery (CABG) with an operative

mortality rate ranging from less than 2% to 4%. However, some patients may

experience myocardial infarction, severe ventricular dysfunction, heart failure, and

death, to name but a few postoperative complications. (Colapinto ND et al. 1971,

Iyengar SR et al. 1972, Park JL et al. 1999, Piper HM et al. 1999)

Few studies investigate novel cardioprotection, emphasizing on the outcome of

high risk patients (Lemasters JJ et al. 1996, Wan S et al. 1997, Walport M 2001,

Eigel BN et al. 2001), in whom electrocardiographic (ECG) abnormalities,

elevations of plasma creatine kinase and troponin I and T (TnT), and

echocardiographic wall motion abnormalities (Bigelow WG et al. 1950, Melrose DG

et al. 1955, Tyers GF et al. 1974) may, however, influence the interpretation of the

results. Again, many studies claim that cardioprotective measures are to be

initiated after cessation of CPB (Downey JM et al. 1994).

For cardioprotection, it would be ideal to interfere at an early stage during

intraoperation, while irreversible ischemia-reperfusion injury (IRI) may be avoided

and only reversible changes have occurred (Annecke T et al. 2101). Experimental

evidence suggests that early administration of ATP-sensitive K+ channel (KATP

channel) openers, such as Adenosine and Diazoxide, may ameliorate the recovery

from cardiac IRI (Armstrong S et al. 1994, Beresewicz A 2004).

This thesis summarizes the impact of intraoperatively administrated Adenosine and

Diazoxide on modifying cardiac IRI in hemodynamically stable patients undergoing

CABG. The efficacity of treatment was investigated with early markers

representing reversible cardiac IRI such as myocardial apoptosis, cell membrane

Aquaporin-7 and endothelial glycocalyx integrity.

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5. REVIEW OF THE LITERATURE

5.1. Background

In 1950, Bigelow et al described hypothermia "as a form of anesthesia" that would

expand the scope of surgery; it was proposed that hypothermia would permit

surgeons to operate safely on the bloodless heart (Bigelow WG et al. 1950). Since

then, many cardioprotective techniques have been developed (Tyers GF et al.

1974, Abd-Elfattah AS et al. 1994). Though many techniques have reported

positive enhancement of myocardial protection, the ideal cardioprotective solution

(cardioplegia) has yet to be found.

Consequences of inadequate myocardial protection during surgery are usually

apparent in the immediate postoperative period, but also affect long-term outcome

(Wan S et al. 1997, Sawa Y et al. 1998, Walport M et al. 2001). While the

consequences of inadequate myocardial protection are usually apparent in the

immediate postoperative period, the full impact may not be fully appreciated for

months; postoperative elevation of cardiac biomarkers after CABG has been

shown to associate with both early and late mortality (Klatte K et al. JACC 2001,

Costa MA et al. Circulation 2001, Kathiresan S et al. Am J Cardiol 2004). Boli et al

reported that patients with increased level of plasma creatine kinase-myocardial

band enzyme (CK-MB) after CABG exhibited greater mortality after six months

(Boli et al. 1999). Specifically, the 6-month mortality rates for patients with peak

CK-MB ratios of <5, 5 to <10, 10 to <20, and 20 upper limits of normal were

3.4%, 5.8%, 7.8%, and 20.2%, respectively. Conversely, the cumulative 6-month

survival was inversely related to CK-MB. These observations support the concept

that myocardial injury, occurring as a result of inadequate intraoperative

myocardial protection, is associated with increased risk of mortality. The aim of the

ideal cardioplegia would enhance reversibility of IRI, while avoiding irreversible

changes.

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5.2. Mechanisms involved during ischemia-reperfusion injury

Several myocardial stresses occurring during cardiac surgery, including ischemia

reperfusion, inflammatory response, operative trauma and oxidative stress have

been reported to trigger the myocardial injury. Significant evidence now exists that

the primary mediators of reversible and irreversible myocardial IRI (Wan S et

al.1997, Sawa Y et al. 1998) include intracellular systolic Calcium (Ca2+) overload

and oxidative stress induced by reactive oxygen species (ROS) generated at the

onset of reperfusion (Bolli R et al. 1999, Park JL et al. 1998, Piper HM et al.1999).

Nitric oxide (NO) may interact with ROS to generate various reactive nitrogen

species that appear capable of both contributing to and reducing injury (Beckman

JS et al. 1996, Droge W 2002, Mallet RT et al. 1994). In addition, metabolic

alterations occurring during ischemia can contribute directly and indirectly to Ca2+

overload and ROS formation. Restoration of intracellular pH at the onset of

reperfusion via Na+ -H+ exchange contributes to intracellular Ca2+ overload via

reversed Na+ -Ca2+ exchange (Lemasters JJ et al. 1996, Eigel BN et al. 2003).

The metabolic changes that occur during ischemia also reduce the endogenous

antioxidant defense systems of cardiac myocytes. The first line of defense against

mitochondrial ROS formation and its deleterious effects is the reduced glutathione/

oxidized glutathione (GSH /GSSG) system. The depletion of glutathione levels

increases ROS formation, oxidative stress, and Ca2+ (Verbunt RJ et al. 1997,

Vaage J et al. 1997, Palace V et al. 1999, Sharikabad MN et al. 2003).

Although Ca2+ may return to normal levels early in reperfused stunned

myocardium, the transient increases in intracellular Ca2+ can activate Ca2+-

dependent protein kinase (PKC); proteases, such as calpain; and endonucleases

(Vasquez-Vivar J et al. 1998, Matsumura Y et al. 2001). Calpain activation and

subsequent action on contractile proteins has been implicated in the reduction in

myofilament Ca2+ sensitivity observed in stunned myocardium (Urthaler F et al.

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1997, Tsuji T et al. 2001). Similarly, there is significant evidence that ROS are

involved in mediating myocardial stunning. Various spin trap agents and chemical

probes have demonstrated the rapid release of ROS into the vascular space during

reperfusion after brief ischemia in vivo (Sekili S et al. 1993, Obata T et al. 1994,

Tang XL et al. 1995, Gorodetskaya EA et al. 2001).

Mitochondria are a primary source of intracellular ROS in cardiac myocytes

(Vanden Hoek TL et al. 1997, Narayan P et al. 2001). Scavengers of ROS and

antioxidants attenuate myocardial stunning in vitro and in vivo, and these

interventions are effective when administered prior to or at the onset of reperfusion

(Li Q et al. 1998, Sun JZ et al. 1996). This may explain why myofibrils isolated from

in vivo reperfused stunned, but not ischemic, myocardium exhibit reduced Ca2+

sensitivity (Miller WP et al. 1996).

More prolonged ischemia, which produces irreversible injury, is associated with

more severe intracellular Ca2+ overload and further depletion of endogenous

antioxidants, conditions which both contribute to and are exacerbated during

reperfusion by the production of ROS. The production of ROS during reperfusion

appears to contribute to Ca2+ overload, as exposure of normal myocytes to

exogenous ROS is associated with increased L-type Ca2+ channel current and

increased Ca2+ (Josephson RA et al. 1991, Thomas GP et al. 1998).

Conversely, increases in Ca2+ during IRI may adversely affect mitochondrial

function, leading to further ROS production (Halestrap AP et al. 1998, Delcamp TJ

et al. 1998). Mitochondria can buffer small increases in intracellular Ca2+ via the

Calsium-uniporter, a process that is energetically favorable due to the Ca2+

gradient and the mitochondrial membrane potential. During reperfusion, the

increase in cytosolic Ca2+ enhances mitochondrial Ca2+ uptake. Since excess

cytosolic Ca2+ has been associated with the loss of myocyte viability, mitochondrial

Ca2+ buffering is initially cardioprotective (Miyata H et al. 1998). However,

14

continued mitochondrial Ca2+ buffering in the face of decreased antioxidant

reserves and excess ROS formation sets up a cycle which may ultimately lead to

the total collapse of mitochondrial membrane potential and cell death (Josephson

RA et al. 1991).

The synergistic interactions between Ca2+ overload and ROS formation during

conditions of decreased antioxidant reserves may also provide an explanation of

why ROS scavengers are not very effective at reducing irreversible injury when

administered at reperfusion (Tanaka M et al. 1993, Watanabe BI et al. 1993).

5.2.1. Systemic Inflammatory Response

A growing body of evidence suggests that cardiopulmonary bypass (CPB) is

believed to trigger whole body inflammatory response, which may be responsible

for some of the major postoperative complications following open heart surgery

(Wan S et al. 1997). The inflammatory response includes complex humoral and

cellular interaction with numerous pathways contributing to inflammation including

activation, generation, or expression of thrombin, complement, cytokines,

neutrophils, adhesion molecules, and other multiple inflammatory mediators

(Walport M 2001, Brady AJ et al. 1993, Kloner R et al. 2000,Wan S et al. 1997).

Nitrous oxide desensitizes the myofilament to Ca2+, resulting in decreased

contractility. There are several ways that proinflammatory cytokines may induce

myocardial functional depression. (Meldrum D 2000. Sawa Y et al. 2001,Menasche

P et al. 1998, Teoh KH et al. 1995).

5.2.2. Myocardial Stunning

Stunning refers to a loss of contractility that follows immediately sublethal ischemic

insult salvaged by reperfusion. Although no cell death results from the ischemia,

the myocytes are reversibly injured (stunned) and exhibit no signs of ultrastructural

damage. Myocardial stunning is an injury that may last for only a few hours or

15

persist for several days despite the restoration of normal blood flow. The length of

time for function to return is dependent on a number of parameters, including the

duration of the original ischemic insult, the severity of ischemia during the original

insult, and the adequacy of the return of the arterial flow. Stunning includes flow-

function mismatch. When coronary blood flow is restored to normal or near normal,

and ischemia is resolved, the myocardium does still not contract (Al-Dadah AS et

al. 2007, Deja MA et al. 2006, McCully JD et al. 2006).

Most of the studies refer to the oxyradical hypothesis as a cause of stunning.

Actually, 50%-70% of the stunning effect is due to a burst of O2-derived free

radicals liberated during the first few minutes of reperfusion with arterial blood

(Kloner RA et al. 2001). Alterations in the availability of Ca2+ and the sensitivity of

the contractile apparatus to Ca2+ may be one of the possibilities. The exact

changes leading to the failure of contraction during stunning are unknown (Murry

CE et al. 1991).

Stunned myocardium is able to contract when exposed to inotropic stimuli, though

is independent on the inotropic stimuli. Patients often require inotropes for the first

hours to days after surgery until stunning resolves. (Arnold JM et al. 1985, Patel H

et al. 2001)

5.2.3. Arrhythmias

Arrhythmia is another manifestation of the injured myocardium. Ischemic heart

disease carries an increased risk of malignant arrhythmia, myocardial infarction,

and sudden cardiac death (Stienberg J et al. 1999). Postoperative arrhythmias

have been considered to be a manifestation of IRI and have been used as a

variable to compare strategies for myocardial protection during cardiac operations.

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5.3. Myocardial protection techniques

5.3.1. Non cardioplegic techniques

5.3.1.1. Intermittent cross-clamping with fibrillation

One of the earliest forms of cardioprotection, still used at some centers today, is

known as intermittent aortic cross-clamping with fibrillation and moderate

hypothermic perfusion (30°C to 32°C) (Rousou JA et al. 1988). Using this

approach, CABG can be performed on the unarrested heart with ascending aorta

cannulation and generally a two-stage single venous cannula. This technique

allows the surgeon to operate in a relatively stable field during ventricular

fibrillation, and to avoid the consequences of profound metabolic changes that

occur with more prolonged periods of ischemia. The duration of fibrillation is

determined by the time to complete the distal anastomoses. After completion of the

last distal graft, the heart can be defibrillated and the proximal aortic-based graft

anastomoses performed on the beating heart, using an aortic partial occlusion

clamp (Bretschneider HJ et al 1975).

5.3.1.2. Systemic hypothermia and elective fibrillation

Although infrequently used, this technique appears to be a safe approach to the

heart during CABG. In 1984, Atkins et al reported a low incidence of perioperative

infarction and a low hospital mortality rate in 500 consecutive patients using this

technique (Akins CW 1984). With this method, systemic hypothermia (28°C),

elective fibrillation, and maintenance of systemic perfusion pressure between 80

mmHg and 100 mmHg are key elements.

Upon fibrillation, the local vessel can be isolated and myocardial revascularization

performed. The limitations of this technique include: 1. The surgical field may be

obscured by blood during revascularization; 2. Ventricular fibrillation is associated

with increased muscular tone, which can limit the surgeon's ability to position the

17

heart for optimal exposure; and 3. The technique is generally not applicable for

intracardiac procedures.

5.3.2. Cardioplegic techniques

Cardioplegic solutions contain a variety of chemical agents that are designed to

arrest the heart rapidly in diastole, create a quiescent operating field, and provide

reliable protection against IRI. In general, there are two types of cardioplegic

solutions: crystalloid cardioplegia and blood cardioplegia. These solutions are

administered most frequently under hypothermic conditions (Rosenkranz ER et al.

1982).

5.3.2.1. Cold crystalloid cardioplegia

There are basically two types of crystalloid cardioplegic solutions: the intracellular

and the extracellular types. The intracellular types are characterized by absent or

low concentrations of sodium and calcium (Teoh KH et al. 1986). The extracellular

types contain relatively higher concentrations of Na+, Ca2+ and Mg +. Both groups

avoid concentrations of K+ greater than 40 mmol/L, contain bicarbonate for

buffering, and are osmotically balanced. In both types the concentration of

potassium used ranges between 10 mmol/L and 40 mmol/L (for potassium 1

mmol/L = 1 mEq/L) (Teoh KH et al. 1986).

Numerous studies have been performed to determine the efficacy of using cold

crystalloid cardioplegic solutions to protect the heart during cardiac surgery

(Rousou JA et al. 1988). While there is considerable controversy regarding the

"ideal" solution and its components, there is evidence that in those centers in

which crystalloid cardioplegia is used almost exclusively, excellent myocardial

protection can be achieved. In many reports the perioperative myocardial infarction

rate is less than 4%, and the operative mortality rate is less than 2% (Martin TD et

al. 1998).

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5.3.2.2. Cold blood cardioplegia

Cold blood cardioplegia is widely employed throughout the world (Hayashida N et

al. 1994). Although there are a variety of formulations, it is usually prepared by

combining autologous blood obtained from the extracorporeal circuit while the

patient is on cardiopulmonary bypass with a crystalloid solution consisting of

citrate-phosphate-dextrose (CPD), tris-hydroxymethyl-aminomethane (tham) or

bicarbonate buffers, and potassium chloride. CPD is used to lower the ionic

calcium, the buffer is used to maintain an alkaline pH of approximately 7.8, and the

final concentration of K+ is used to arrest the heart (approximately 30 mmol/L)

(Martin TD et al 1994).

Prior to administering blood cardioplegia, the temperature of the solution is usually

lowered with a heat exchanging coil to between 12°C and 4°C. The ratio of blood

to crystalloid varies among centers, with the most common ratios being 8:1, 4:1,

and 2:1. This in turn affects the final hematocrit of the blood cardioplegia infused.

For example, if the hematocrit of the autologous blood obtained from the

extracorporeal circuit is 30, these ratios would result in a blood cardioplegia with a

hematocrit of approximately 27, 24, and 20, respectively (Rousou JA et al. 1988).

The use of undiluted blood cardioplegia or "miniplegia" (using a minimum amount

of crystalloid additives) has also been reported to be effective. In an acute IRI

canine experiment, Velez and colleagues tested the hypothesis that an all-blood

cardioplegia (66:1 blood to crystalloid ratio) would provide superior protection

compared to a 4:1 blood cardioplegia delivered in a continuous retrograde fashion

(Velez DA et al. 2001). They found very little difference between the animal groups

with respect to infarct size or postischemic recovery of function. This is consistent

with the findings that the level of hypothermia is important in blood cardioplegia,

and not necessarily the hematocrit (Rousou JA et al. 1988).

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5.3.2.3. Warm Blood cardioplegia

The concept of using warm (normothermic) blood cardioplegia as a

cardioprotective strategy in humans dates back to the 1980s. In 1982, Rosenkranz

et al reported that warm induction with normothermic blood cardioplegia, with a

multidose cold blood cardioplegia maintenance of arrest, resulted in better

recovery of function in canines than a similar protocol using cold blood induction

(Rosenkranz ER et al. 1982).

An experimental study demonstrating that a terminal infusion of warm blood

cardioplegia before removing the cross-clamp (hot shot) accelerated myocardial

metabolic recovery (Teoh KH et al. 1986). Normothermic blood cardioplegia in

humans is an effective cardioprotective approach (Lichtenstein SV et al. 1991).

The operative mortality in a warm cardioplegic group consisting of 121 consecutive

patients was 0.9% compared to 2.2% for 133 patients that received antegrade

hypothermic blood cardioplegia (Lichtenstein SV et al. 1991). However, Martin et al

showed that the use of warm cardioplegia may be associated with increased

incidence of neurological deficits (Martin TD et al. 1994).

5.3.2.4. Tepid blood cardioplegia

In a study by Hayashida et al., 72 patients undergoing coronary artery bypass

grafting were randomized to receive cold (8oC) antegrade or retrograde, tepid

(29oC) antegrade or retrograde, or warm (37oC) antegrade or retrograde blood

cardioplegia. While protection was adequate for all three, tepid antegrade

cardioplegia was the most effective in reducing anaerobic lactate acid release

during the arrest period. These authors reported similar findings when the tepid

solution was delivered continuously retrograde and intermittently antegrade

(Hayashida et al. 1994).

20

5.3.2.5. Different ways of administering cardioplegic solutions

Cardioplegic solutions may be administered using different techniques; these

include: intermittent antegrade, continuous antegrade, continuous retrograde,

intermittent retrograde, antegrade followed by retrograde, and simultaneous

antegrade and retrograde infusions. While all methods are generally efficient,

comparisons are difficult due to numerous confounding factors such as: 1.

composition of the solution, 2. temperature of the solution, 3. duration of the

infusion, 4. infusion pressure, 5. type and complexity of the operation, 6. need for

surgical exposure, and 7. expected versus actual cross-clamp time

(Ruengsakulrach P et al. 2001).

In 1956, Lillehei et al used retrograde coronary sinus perfusion to protect the heart

during aortic valve surgery (Ruengsakulrach P et al. 2001). The theoretical

advantage includes ensuring a more homogeneous distribution of the cardioplegic

solution to regions of the heart poorly collateralized. It is also effective during: 1.

the setting of aortic valve surgery, 2. reducing the risk of embolization; and 3.

delivering cardioplegia in a continuous manner.

Delivering cardioplegia both antegrade and retrograde is also feasible and safe

(Ihnken et al. 1984). Sonicated albumin and transesophageal echocardiography

were used intraoperatively to assess the effects of delivering a cardioplegic

solution antegrade and retrograde simultaneously (Cohen et al 1999).

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5.4. Myocardial Necrosis and Apoptosis

Necrosis represents irreversible IRI associated with loss of transmembrane ion

gradient and membrane disruption secondary to depletion of intracellular

adenosine triphosphate (ATP) causing an inflammatory reaction in surrounding

tissue. Apoptosis, on the other hand, represents reversible IRI associated with

specific DNA fragmentation (Gottlieb RA et al. 1994).

Apoptosis is activated through the caspase (cystein aspartic acid-specific

protease) cascade; there are at least 10 different members of intracellular

proteases that cleave substrate proteins (Baufreton C et al. 1999). Several hours

before the morphologic appearance of apoptosis, downstream caspases (caspase-

3, -6, -7) are activated (Bolli R et al 1999). Myocardial apoptosis may reflect

immediate cardioprotection (Martin SJ et al 1995, Kirshenbaum LA et al. 1997,

Kluck RM et al. 1997, Yang J et al. 1997, A et al. 1997, Halestrap AP et al. 1998,

Maulik N et al. 1998, Van Engeland M et al. 1998, Rucker-Martin C et al. 1998,

Freude B et al. 2000).

5.5. Bax and bcl2 gene expression

Cardiomyocytes apoptosis is influenced by the proapoptotic genes bax and bcl2

(Hamacher-Brady A et al. 2006, Kovacević M et al. 2007). In experimental studies,

IRI correlates with bax and bcl-2 changes (Condorelli G et al. 1999). Inhibition of

these apoptotic gene expressions during IRI is associated with reduction of

infarction size, improvement of regional contractility, vascular endothelial function

and myocardial blood flow (Misao J et al. 1996, Condorelli G et al. 1999, Yeh C-H

et al. 2006, Nagy N et al. 2007, Guo J et al. 2008, Lv X et al. 2008, Yan L, et al.

2008).

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5.5.1. Pathophysiology of cardiomyocyte apoptosis

Pure apoptotic cell death is significantly different from necrosis. Apoptosis is an

energy-requiring and precisely regulated process of cell death which is directed by

a genetic program (Paparella D. et al. 2002). Apoptotic cell initiates its own death

process by activating endogenous proteases. The early stages of microscopic

picture of the apoptosis refer to the following features: Cell shrinkage and

aggregation of chromosomal DNA into small masses and preparation for

exocytosis. These apoptotic bodies are membrane bounded and are subsequently

phagocytosed by macrophages and neutrophils. Necrosis, on the other hand, is

associated with loss of transmembrane ion gradient and membrane disruption

secondary to depletion of intracellular ATP causing an inflammatory reaction in

surrounding tissue. The specific DNA fragmentation is a hallmark of apoptosis, and

it is utilized as a detection method in the "DNA laddering" (Butterworth J et

al.1999).

5.5.2. Activation and regulation of apoptosis

Apoptotic cell death is activated through the caspase (cystein aspartic acid-specific

protease) cascade. At least 10 different members of these intracellular proteases

that cleave substrate proteins behind aspartate residues are known (Baufreton C

et al. 1999). Once the so-called downstream caspases (caspase-3, -6, -7) are

activated, cell death appears to be inevitable; this occurs several hours before the

morphologic appearance of apoptosis (Bolli R yet al. 1999).

Two major molecular pathways of caspase activation are recognized in myocardial

cells. The main pathway is initiated by the release of a mitochondrial respiratory

chain protein—the cytochrome c—from mitochondrial intermembranous space

(Appleyard R.F wt al. 1993). The cytochrome-c–mediated activation of apoptosis

has been demonstrated in animal and human models of heart failure (Luss H et al.

2002, Saraste A et al. 1999). Cytochrome c in conjunction of apoptosis protease-

activating factor (Apaf-1) and dextro-adenosine triphosphate (dATP) allow the

autocatalytic activation of the most upstream caspase on the mitochondrial

23

pathway (procaspase-9) (Abbate A et al. 2002). This cytosolic complex is mediated

through certain stimuli such as apoptosis-inducing factor (AIF), aberrant oncogene

expression, p-53, cytotoxic agents, and DNA damage (Abbate A et al. 2002).

The mitochondrial pathway is regulated through at least two classes of proteins.

The first class is antiapoptotic intracellular proteins (such as the proto-oncogene

Bcl-2 family proteins) and proapoptotic proteins (such as Bax) (Abbate A et al.

2002). The ratio between Bcl-2 and Bax in cardiac myocytes has been shown in

human and animal studies to be related to an increased apoptosis rate (Wan S et

al. 1997). Regulation of Bcl-2 is studied in various apoptogenic cardiac conditions

in humans and rats. Increased levels of Bcl-2 are detected soon after acute

coronary occlusion in human and rat heart (Velthius H et al. 1995, Krown K.A et al.

1996). However, in chronic heart failure secondary to pressure overload, the levels

of Bcl-2 are underexpressed in a rat model (Meldrum D.R 1998).

The second group of regulatory proteins is the inhibitor of apoptosis proteins (IAP)

family (Westhuyzen J 1997). The antiapoptotic characteristics of inhibitor of

apoptosis proteins are due to direct inhibition of different steps of signal

transduction in cytochrome-c–dependent or death-receptor–mediated apoptosis.

The alternative pathway for activation of apoptosis is called the death receptor

pathway. It is thought to play only a secondary role in initiation of cardiomyocyte

apoptosis (Yau TM et al 1994). The activation of the most upstream caspase

(procaspase-8) involves the binding of extracellular death signal proteins (egg,

Fas-ligand) to their cognate death receptor on the cell surface, such as Fas-

associated death domain protein (FADD) (Vento AE et al.1999). The cytoplasmatic

domain has a characteristic amino acid residue that is essential for the

proapoptotic activity. This domain is specific for each death receptor.

At least two different regulatory proteins for inhibition of death receptor pathway

are described. The first group is the inhibitors of receptor-mediated caspase

activation such as FADD-like interleukin-1β-converting enzyme (FLICE)-inhibitory

protein (FLIP), inhibitor of FADD-like interleukin-1β-converting enzyme (I-FLICE),

caspase homologue (CASH), and FADD-like antiapoptotic molecule-1 (FLAME-19)

24

(Pesonen EJ et al. 1999). This class of proteins inhibits competitively the receptor-

induced activation of upstream caspase. The second group is the inhibitor of the

apoptosis protein family that was discussed earlier.

5.5.3. Diagnostic methods and techniques of apoptosis

A few methods are available for identifying the prevalence of apoptotic cells in a

tissue sample (Hammill AK et al. 1999, Van Heerde WL et al. 2000, Narayan P et

al 2001, Maiese K et al. 2000). The most convincing proof of apoptosis is the

visualization of nuclear-stained cells. This method is limited owing to the difficulty

of finding a few cells that remain only 6 to 24 hours (Paparella D et al. 2002). Other

techniques that utilize electrophoresis of nuclear fragments (DNA-

laddering)(Butterworth J et al. 1999) or the histochemical staining of the

fragmented DNA—the so-called TUNEL (terminal deoxynucleotidyl-transferase

mediated dUTP nick end-labeling) — and its modifications such as comet assay

(Wan S et al. 2002). In-situ end-labeling are limited by the inability to specify cell

types and the fact that DNA fragmentation can occur during damage or repair.

5.6. Myocardial protection and apoptosis

Understanding mechanisms of apoptosis during cardiac surgery (Gottlieb R et al.

1994, Freude B et al. 2002) may help to identify new therapeutic targets. Open-

heart surgery and the use of CPB have been associated with increased apoptotic

index. Thus, prevention and attenuation of proinflammatory and apoptogenic (Yang

J et al. 1997) state of CPB remain important therapeutic goals.

Blood contact with foreign surface results in increased serum level of Fas and Fas-

ligand in humans undergoing cardiopulmonary bypass (Kawahito K et al. 2000).

This increases apoptotic cell death of the myocardium through the death receptor

pathway. The evidence of postbypass syndrome including cerebral dysfunction

and renal insufficiency may be secondary to apoptotic injury (Walinsky P et al.

25

1999). Potential therapeutic implications are reduction of the foreign surface,

shortening of bypass time, and addition of antiapoptotic medication to the priming

solution in the CPB circuit (Meldrum D et al. 1999).

5.7. Myocardial Aquaporins

Myocardial Aquaporin proteins form cell transmembrane channels that participate

in water trafficking during normal and pathological conditions. Increased

expression of Aquaporin-4 is associated with the formation of myocardial edema

(Braimbridge MV et al. 1976, Warth A et al. 2007). Its differential expression

pattern is dependent on tissue destruction and during cancer (Greenburg JJ et al.

1960, Xu H et al. 2009). Aquaporin-7 is a cell-membrane receptor facilitating both

glycerol and water trafficking from the cell to the interstitium thus controlling both

edema and cardiac energy supply (Webb WR et al. 1966, Hibuse T et al. 2009).

Excessive Aquaporin-7 expression may be considered as an early marker of

histological tissue damage after IRI and edema (Greenburg JJ et al. 1960, Liu Y et

al. 1998, Liu et al. 2010).

5.8. Endothelial glycocalyx integrity

Endothelial glycocalyx shedding is a novel concept reflecting tissue injury after

ischemia. The healthy vascular endothelium is covered by a glycocalyx layer

mainly consisting of hyaluronan and syndecan glycoproteins, the shedding of

which indicates vascular permeability and tissue edema (Prasad SM et al. 2006,

Van den Berg BM et al. 2003). In contrast, preserving the endothelial glycocalyx

and sustaining the vascular barrier reduces interstitial edema. Peripheral plasma

hyaluronan and syndecan-1 values may reflect endothelial glycocalyx integrity and

myocardial protection during CABG (Liu Y et al 1998, Broadhead MW et al 2004).

The evaluation of these parameters may prove clinically practicable to interpret the

state of endothelial glycocalyx after CABG (Rehm M et al 2007, Svennevig K et al.

2008).

26

5.9. Adenosine and myocardial protection

Administration of the nucleoside Adenosine retards the rate of ischemia-induced

ATP depletion and onset of ischemic contracture, attenuates myocardial stunning,

enhances myocardial function after IRI, and may reduce infarct size (Lasley RD

1998). Adenosine may play a role in mediating the infarct size–limiting effects after

IRI (Downey JM et al. 1994, Cohen MV et al. 2000). A transient Adenosine infusion

may suffice prior to ischemia to limit the infarct size (Randhawa MPS et al. 1995,

Sekili S et al. 1995). However, controversial data on the impact of Adenosine

receptor antagonists on IRI has been presented (Armstrong S et al. 1994).

5.9.1. Adenosine preconditioning and pretreatment

It is important to differentiate between Adenosine preconditioning and Adenosine

pretreatment. The former involves a brief infusion of Adenosine that is terminated

prior to the onset of ischemia, whereas the latter involves the continuous infusion

of Adenosine until the onset of ischemia. The significance of this difference lies

with the observation that Adenosine pretreatment, not Adenosine preconditioning,

attenuates myocardial stunning (Sekili S et al. 1995, Randhawa MPS et al. 1995).

The beneficial effects of Adenosine infusion prior to ischemia appears to be due to

the direct effects of Adenosine on the cardiomyocyte since: 1. Adenosine must be

infused at a dose that reaches the interstitial fluid space that surrounds the

cardiomyocyte; and 2. Adenosine reduction of ischemic and hypoxic injury can be

demonstrated in isolated myocyte preparations (Lasley RD et al. 1998, Rice PJ et

al. 1996).

5.9.2. Mechanism of action of Adenosine

Although the cardioprotective effects of Adenosine have been recognized for some

time, there are still many questions regarding its mechanism of action. Genetic,

biochemical, and pharmacologic studies indicate that there are at least four distinct

sarcolemmal Adenosine receptor subtypes, designated A1, A2a, A2b, and A3, that

27

couple to a variety of guanine nucleotide binding (G) proteins depending upon the

receptor subtype and tissue studied. Currently, there is direct evidence that two,

possibly three, of these receptors are expressed in the adult heart. Radioligand

binding studies have documented the presence of A1 and A2a Adenosine

receptors in mammalian myocardium, and numerous studies since have reported

the physiological roles of these receptors (Olanrewaju HA et al. 2000). The results

of recent studies suggest that Adenosine A2b receptors may be expressed in the

coronary vasculature (Morrison RR et al.2002, Zhou QY 1992). Although there are

some reports of A3 receptor mRNA expression in cardiac tissue, presently there is

no definitive evidence for the expression of this receptor in the normal mammalian

heart (Wang JL et al. 1997).

Activation of A1 receptors on normal atrial myocytes exerts negative chronotropic,

dromotropic, and inotropic effects via modulation of K+ and Ca2+ channel

conductances. Activation of the same receptor exerts few, if any, direct effects in

ventricular myocardium. However, A1 receptor activation significantly blunts the

metabolic and contractile effects of beta-adrenergic receptor stimulation (Wang JL

et al. 1997).

5.10. Diazoxide and myocardial protection

Diazoxide is a pharmacological activator of ATP-sensitive K+ channels (KATP

channels), which has been shown to decrease IRI (McCully JD et al. 2002). This

protective effect mimics ischemic preconditioning (Beresewicz A et al. 2004), and

may be blocked by KATP channel blockers such as Glibenclamide or 5-

hydroxydecanoate (Gross F et al.1999, Cohen et al. 2000). Mechanisms may also

include activation of KATP channels in the sarcolemma of myocardium

(sarcoKATP), leading to an energy-sparing effect through shortening of the action

potential and reduced Ca2+ entry (Deja MA et al. 2009).

However, several studies have shown poor correlation between action potential

shortening and protection, and further that protection can occur in the absence of

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1572983/#bib8


28

action potentials (Henley PJ et al. 2002). In addition to their expression in the

plasma membrane of cells, KATP channels have been described in the inner

membrane of mitochondria (mitoKATP) (Al-Dadah AS et al. 2007, Garlid et al.

1997).

Much of the evidence for the involvement of mitoKATP channels is pharmacological,

based especially on the selectivity of the channel opener diazoxide for mitoKATP

over sarcoKATP (Garlid et al. 1997). The details of the mechanism by which mitoKATP

channels might cause protection remain obscure, but in rabbit myocytes in short-

term culture, Diazoxide has been reported to increase flavoprotein

autofluorescence (Deja MA et al. 2006). This effect was potentiated by either

activation of protein kinase C or by Adenosine, both maneuvers known to trigger

cardioprotection (Deja MA et al. 2006) suggested that opening of the mitoKATP

channels might dissipate the inner mitochondrial membrane potential established

by the protein pump, and that this dissipation accelerates electron transfer by the

respiratory chain which if uncompensated by increased production of electron

donors, leads to net oxidation of the mitochondria (McCully JD et al 2006).









29

6. AIM OF THE WORK

The aim of this study is to investigate the effect of Adenosine and Diazoxide with

blood cardioplegia on myocardial protection during CABG. Evaluation of reversible

parameters of IRI, such as apoptosis, Aquaporin-7 expression and plasma levels

of syndecan-1 and hyaluronan are sought for.

More specifically the aim was to investigate:

The impact of Adenosine on the modification of cardiomyocyte apoptosis

and expression of apoptosis-regulating genes bax and bcl-2.

The impact of Diazoxide on Aquaporin-7 expression and endothelial

glycocalyx integrity through measuring plasma levels of syndecan-1 and

hyaluronan.

30

7. MATERIAL AND METHODS

7.1. Patient selection

After institutional approval by Tampere University Hospital Ethics Committee, the

protocol for this prospective randomized, double blind, placebo-controlled study

was reviewed by National Agency for Medicines, Finland. All 40 patients gave their

informed consent for the Adenosine group (ETL R05037M). Another 16 patients

offered their consent for the Diazoxide study (ETL R07027M). The

hemodynamically stable patients were scheduled for elective CABG using on

pump cardiopulmonary bypass technique.

7.1.1. Patients receiving Adenosine (I, II)

This part of the thesis included patients with a diagnosis of a non-recent CAD. The

exclusion criteria were diabetic patients with sulfonylurea medication, recent

myocardial infarction within the last month, redo cardiac operation, preoperative

diagnosis of asthma, chronic obstructive pulmonary disease (COPD), kidney

function impairment, liver dysfunction, patients with poor left ventricular function

(ejection fraction EF ≤ 30%), cardiac valve disease, and patients receiving

corticosteroids.

7.1.2. Patients receiving Diazoxide (III, IV)

This part of the thesis included patients with a diagnosis of CAD. The inclusion

criteria included stable myocardial coronary artery disease eligible for elective

CABG, but with a history of a recent less than 1 month old myocardial infarction

and detection of elevated TnT (TnT > 0.01 g/ l) or Creatinin kinase (CK-MB > 1U/ l)

release. The exclusion criteria were the same as for the patients receiving

Adenosine.

31

7.2. Anesthesia

A radial artery line and a pulmonary artery catheter were inserted for hemodynamic

monitoring. Anesthesia was induced with propofol (0.5-1.0 mg/kg), sufentanil (0.6-

0.8 μg/kg) and cis-atracurium. Sufentanil infusion was continued with a rate of

0.03-0.05 μg/kg/min. Sevoflurane was used as the main anaesthetic agent

throughout the operation, and also provided during the cardiopulmonary perfusion

with a vaporiser attached to the fresh gas inlet.

7.3. Operative and perfusion techniques

The surgical techniques were standardized in all cases. A median sternotomy was

performed, and one internal thoracic artery and from one to four peripheral vein

grafts from the lower extremities were taken in each case. A radial artery graft was

harvested whenever indicated. Cardiopulmonary bypass was established with

regular cannulation technique using mild hypothermia (35oC) with nonpulsative

flow with a membrane oxygenator. The circuit was primed with 1500 mL of

Ringer’s acetate. The proximal anastomoses were constructed during a single

cross-clamping period.

7.3.1. Cardioplegia and Adenosine administration (I, II)

Patients were allocated into two groups. In the Adenosine group, 20 patients

received Adenosine 250 µg/kg into the aortic root just after cross clamping. Twenty

patients in the Control group received normal saline as placebo. All patients

received routine blood cardioplegia delivered through antegrade route. As the

effect of Adenosine is dependent on temperature, the first cardioplegia infusion

was given at normothermia. After reaching asystole, cardioplegia temperature was

lowered to 10-12oC. Subsequent one-minute antegrade cardioplegia infusions

were administered after completion of each distal anastomoses. Finally, warm

antegrade cardioplegia (37oC) was given three minutes before the removal of the

aortic clamp.

32

7.3.2. Cardioplegia and Diazoxide administration (III, IV)

Patients were allocated into two groups. Eight patients received Diazoxide 50 µg/l

injected into the aortic root at the onset of cross-clamping. Eight other patients

served as Controls and received normal saline as placebo. Likewise, all patients

received routine blood cardioplegia delivered through antegrade route, as

described above.

7.4. Tissue harvesting


Two samples of left ventricular apex were harvested from each patient in both

groups. The first sample was obtained after initiation of CBP and procured

immediately before aortic cross clamp by oblique introduction of Tru-Cut needle

(PRECISA TM 14GX150 mm) into the left ventricle apical wall. The second sample

was taken after surgery adjacent to the location of the first sample by the same

needle before termination of CBP. The puncture sites were secured with small 4-0

Prolene (Ethicon) stitch even when no bleeding occurred. Myocardial tissue (5-10 x

3 mm) was immediately frozen in liquid nitrogen for histological studies.


Two samples of right atrium were procured from each patient. The first sample was

obtained before aortic cross clamping. The second sample was taken after surgery

adjacent to the location of the first sample by the same needle before termination

of CBP. Half of the harvested myocardial tissue (5-10 x 3 mm) was immersed in

phosphate buffered saline and immediately frozen in liquid nitrogen for RNA

analysis. The other half of the sample was procured for electron microscopy or

immersed in formalin and embedded in paraffin for histological studies.

33

7.5. Sample analysis


Assessment of apoptosis

Apoptosis was detected using the TUNEL (terminal transferase mediated dUTP

nick end-labeling) assay, as previously described (Saraste A et al. 1997, Schmitt

JP et al. 2002). Briefly, paraffin-embedded myocardial sections were heated in

sodium citrate solution and digested with proteinase-K to expose DNA. The DNA

strand breaks were then labeled using terminal transferase with digoxigenin-

conjugated ddUTP and visualized using alkaline phosphatase

immunohistochemistry (IHC).

Quantification of expressions of bax and bcl2 genes

The frozen atria samples were homogenized and RNA was extracted using Trizol

reagent (Invitrogen®) according to manufacturer's instructions. The RNA

concentration was determined using spectrophotometry. cDNA was synthesized,

and the quantitative real-time reverse transcriptase polymerase chain reaction

assay was used with primers for bax and bcl-2 (Sequence detection primer,

Applied Biosystems Inc, CA, USA) and probes (TaqMan, TAMRATM Probe,

Applied Biosystems). In order to prevent the amplification of contaminating

genomic DNA, primer pairs were located in different exons. Housekeeping gene

18SrRNA was used as an endogenous control (The TaqMan Ribosomal RNA

Control Reagents, Applied Biosystems). All RT-PCR reactions were run as

duplicate.

Quantification of gene expressions was carried out by using relative standard

curve method. Briefly, standard curves for each plate were generated using serial

dilutions of cDNA syntesized from human tonsil tissue RNA. The amounts of both

target and endogenous control cDNA in each sample were calculated relative to

34

the standards. To eliminate the variation of total cDNA quantity in each sample, the

results were expressed as a ratio of the target gene to the endogenous control.


Light microscopy (LM)

For histology, the 5-μm sections were stained with Hematoxylin and Eosin.

Evaluation was performed blinded to the study protocol and technically unclear

slides were rejected. The following signs of injury were evaluated separately:

presence of myocardial edema and ischemia, presence of hemorrhage,

intramyocardial artery edema and periadventitial inflammation. A histological

damage score was obtained by adding the evaluated signs of injury to allow semi-

quantitative comparison between patients. A relative histological damage score

change was obtained by subtracting the histological damage score during

operation from that before operation.

Quantitative analysis of Aquaporin-7

The frozen atria tissue was homogenized and RNA was extracted using a rotor-

stator homogenizer and NucleoSpin ® RNA II kit (Machery-Nagel GmbH &Co, D ü

ren, Germany) according to the manufacturer' s instructions. The RNA was reverse

transcribed into cDNA, and the quantitative reverse transcriptase polymerase chain

reaction was performed with standard protocols on Abi Prism 7300 instrument

(Applied Biosystems, CA, USA). The PCR reaction was performed with TaqMan ®

Gene Expression assays for Aquaporin-7 (ID Rn00569727 m1) and GAPDH (ID

Rn01462662 g1) (both from Applied Biosystems) with TaqMan ® Universal PCR

Master Mix. All samples were performed as three replicates.

The expression levels of Aquaporin-7 and GAPDH as an internal control/house

keeping gene were evaluated. Ct-values were determined for every reaction and

the relative quantification was calculated using the 2-ΔΔCt method (Livak KJ et al.

35

2002). Briefly, the data was normalized to the expression of housekeeping gene

GAPDH, and values of control samples were used as a calibrator. The expression

of Aquaporin-7 before operation in each patient was settled to obtain the value 1,

while Aquaporin-7 during CABG was expressed as Fold Changes. Aquaporin-7

change was therefore either positive (+1) or negative (-1) during operation as

compared with a base line of 1 before operation.

Electron microscopy (EM)

For 9 patients (4 patients with Diazoxide and 5 Controls), tissue harvesting for EM

included all two samples of right atrium. Muscle specimens were fixed in 2 %

glutaraldehyde in 0.1 M phosphate buffer, washed in 0.1 M phosphate buffer,

dehydrated and embedded in Epoxy resin. The plastic embedded samples were

sectioned for ultrathin sections, stained with Uranyl acetate and lead citrate, and

examined with an Olympus-sis Morada digital camera (Olympus Soft Imaging

Solutions, Munster, Germany). The state of tissue damage before and after CABG

was verified blinded to the study protocol, and technically unclear slides were

rejected. The following signs of injury were noted separately: the presence of

mitochondrial swelling and the presence of preserved mitochondrial integrity before

and during surgery.

Sample collection and Elisa for hyaluronan and syndecan-1

For evaluation of hyaluronan and syndecan-1, blood was collected at induction of

anesthesia (Al-Dadah AS et al. 2007) immediately after aortic clamp removal

(Mizutani S et al. 2006), and 1 hour after surgery and closure of sternal skin wound

(Broadhead MW et al. 2004). Elisa for all three samples at various time-points was

available in 13 patients (6 Controls and 7 in Diazoxide group). All 3 samples at

different time-points were available in 13 patients (6 Controls and 7 in Diazoxide

group). Plasma hyaluronan and syndecan-1 values were determined by enzyme

linked immunosorbent assay (ELISA) by using reagents from R&D Systems

Europe Ltd (Abingdon, UK). Detection limits were 123 pg/ml for hyaluronan and

62.5 pg/ml for syndecan-1.

36

7.5.3. Statistical analyses

Statistical significance was attributed to p-values lower than 0.05. For Study I and

II, statistical analysis was performed using SPSS for Windows software, version

9.0 (SPSS; Chicago, IL, USA). For these studies, sample size estimation was not

performed. The Mann- Whitney U test was used to distinguish demographic

differences between the groups. Continuous variables were analyzed by analysis

of variance (ANOVA) for repeated measures. Logarithmic transformation was

used, as the variables were not normally distributed.

For study III, data is presented as mean standard error of the mean. Statistical

analyses were performed with commercial statistical software (SPSS 17.0, SPSS

Inc, Chicago, IL). Sample size estimation was analysed by Power calculation that

was set to display the 95% confidence interval and performed with statistical

software (PowerAndPrecision 4.0, Biostat, Englewood, NJ).

For Study IV, hyaluronan and syndecan-1 values were adjusted to the base line

value for correlation by Spearman rank rho. Nonparametric data were analyzed

with Kruskal-Wallis for various time points among groups, and Mann Whitney was

used for comparison of two groups for each time points. Statistical analyses were

performed with commercial statistical software (SPSS 19.0, SPSS Inc, Chicago,

IL). Power calculation was set to display the 95% confidence interval and

performed with statistical software (Power And Precision 4.0, Biostat, Englewood,

NJ).

37

8. Results

8.1. Adenosine and TUNEL-positive cells (I)

There were no statistical differences between the study groups in terms of patient

demographics, clinical parameters or CKMB levels. The mean value of TUNEL-

positive cells in Controls was 0.03±0.07 SD and 0.017 ± 0.0 SD in the Adenosine

group (p = 0.73). The different quartiles of TUNEL-positive cells (low, medium and

high) in correlation to the overall positivity suggests for decreased number of

TUNEL-positive cells in the Adenosine group as compared with Controls (p =

0.268).

8.2. Expression of bax and bcl2 genes and Adenosine (II)

After operation, a reduction of tissue expression of bax but not bcl-2 (p = 0.002 and

p = 0.07, respectively) occurred in Controls as compared with preoperative tissue

expression; this was not observed in the Adenosine group (p = 0.45 and p = 0.47).

No differences were observed in the Bax/bcl2 ratio. A tendency towards low bcl-2

and high bax expressions in patients with TUNEL positive cells was observed.

38

Summary of the results of the adenosine study

*Patients classified into quartiles depending on the percentage of the amount of apoptosis (low 25% medium 50% and high 75%) ¥TUNEL positive and negative subgroups (of ventricular samples) in both the Adenosine and Control groups in correlation with the changes of apoptosis- regulating genes in the atrial samples

Control (n= 20) Adenosine (n= 20)

Age, years

63.4

65.5

Man/Woman, n 19/1 17/3 EF mean 63 61 Apoptotic index, % Mean Range Percentile*

Low Medium High

0.034±0.07 0.02-0.33

0.026 0.032 0.097

0.017±0.03 0,01-0.11

0.019 0.025 0.066

Bax/bcl2¥ Before

TUNEL negative TUNEL positive Total

After TUNEL negative TUNEL positive Total

6.02±5.3 5.21±5.2 6.6±15.8

4.38±13.5 7.57±7.5 6.4±20.9

4.94±8.5 2.4±2.4

8.8±13.67

3.88±5.6 5.1±5.1

5.1±15.75

39

8.3. Histopathological changes, Aquaporin-7 expression and Diazoxide (III)

Neither the demographics parameters nor the hemodynamic outcome had

significant differences between the two study groups. Both groups expressed a

significant amount of tissue edema after the operation in comparison to the

baseline samples. Change of Aquaporin-7 expression was mainly positive in

Controls, in contrast to only two patients receiving Diazoxide. Adjustment of

Aquaporin-7 expression to the tissue injury proved that the median of relative

Aquaporin-7 expression was lower in patients with Diazoxide.

8.4. Endothelial glycocalyx integrity and Diazoxide (IV)

Ischemic injury after surgery was further evaluated with EM, revealing

postoperative atrial tissue edema, mitochondrial swelling and ischemic endothelial

cell nuclei. Plasma levels of Syndecan-1 and hyaluronan did not differ at any time

point between Controls and patients receiving Diazoxide. However, significant

decreases of both syndecan-1 and hyaluronan were apparent in patients with

Diazoxide, in contrast to Controls (p < 0.04 and p < 0.04). Syndecan-1 and

hyaluronan plasma levels correlated in patients with Diazoxide but not in Controls.

40

Control (n=8)

Diazoxide (n=8)

Man/Woman, n

3/5

7/1

TNT mean ± sem, mg/L

0.48±0.09 0.69±0.24

Histological changes Base line

Edema, PSU ± sem Arterial vacuolization, PSU ± sem Peridventitial inflammation, n Hemorrhage, n

End of operation Edema, PSU ± sem Arterial vacuolization, PSU ± sem Periadventitial inflammation, n Hemorrhage, n

1.9±0.3 1.4±0.3

0 0

2.3±0.4 2.0±0.4

3 1

1.7±0.6 2.0±0.5

0 0

2.2±0.5 2.0±0.5

1 1

Positive Aquaporin-7 expression, n 7 2 Median Hyaluronan, ng/ml

Induction of anesthesia (Time point 1) Onset of reperfusion (Time point 2) Termination of surgery (Time point 3)

Median Syndecan-1, ng/ml Induction of anesthesia (Time point 1) Onset of reperfusion (Time point 2) Termination of surgery (Time point 3)

16.2±6.6 48.5±13.3 64.3±7.9

6.5±6.6 8.5±2.2 4.2±0.6

18.3±3.5 117.2±22.6 56.1±15.4

3.5±0.7 7.6±0.7 5.6±0.9

Summary of the results of the Diazoxide study

PSU: Point score unit n: number

41

9. DISCUSSION

Abundant data (Lemasters JJ et al. 1996) demonstrates that intraoperative

cardioprotection during CABG has important long-term effects after surgery (Wan

S et al. 1997, Walport M 2001, Eigel BN et al. 2001). Cardioplegia represents an

additional possibility to enhance recovery after CABG; Reversible factors of IRI

due to surgery may be influenced by administration of Adenosine and Diazoxide.

For this study, two sets of hemodynamically stable patients were carefully selected

to minimize confounding bias: 1. Patients without a recent diagnosis of coronary

artery disease (CAD) treated with Adenosine. 2. Patients with a recent diagnosis

of CAD treated with Diazoxide.

Patients receiving Adenosine

We did not find any significant influence on the hemodynamic profile in patients

receiving Adenosine during the early postoperative recovery period as compared

with Controls. In contrast, Adenosine has been shown earlier to enhance cardiac

index (Chauhan S et al. 2000, Chen CH et al. 2005, Germack R et al. 2005). In our

study, rapid pharmacological correction of any hemodynamic changes during early

postoperative recovery may have masked the impact of Adenosine.

We speculate that very fast myocardial arrest induced by Adenosine could

modulate the expression of apoptotic genes and decrease the occurrence of

apoptosis (Walport M 2001). Adenosine had an important effect on the apoptotic

regulation genes bax and bcl-2, while a tendency of reduced apoptotic TUNEL-

positive cells and release of CKMB were observed. Detection of apoptotic

cardiomyocytes is challenging, because the appearance of DNA fragmentation is a

late feature of the apoptotic process (Piper HM et al. 1999). Apoptosis may also

have a patchy tissue distribution (Kunapuli S et al. 2001). Despite the small

sample size in our study, changes in apoptosis-regulating genes served as early

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Germack+R%22%5BAuthor%5D

42

markers of the severity of ischemic myocardial injury. It remains to be seen

whether in hemodynamically unstable patients Adenosine would display

cardioprotection during CABG.

Patients receiving Diazoxide

Immediate postoperative hemodynamic parameters remained comparable in

patients with Diazoxide and Controls, though Diazoxide has been associated with

several different mechanisms of action (Chappell D 2007, Nelson A et al.2008,

Chappell D et al. 2009, Brands J et al. 2010, Johansson PI et al. 2012).

However, Diazoxide administered during CABG attenuated relative Aquaporin-7

expression of the right atrium as compared with its histological damage score.

Excessive Aquaporin-7 expression was dependent on histopathology and

indicates tissue damage (Annecke T et al. 2010). Diazoxide decreased hyaluronan

and syndecan-1 plasma levels, and correlation of these molecules was significant

in patients receiving Diazoxide in contrast to Controls. Decreased degradation of

the endothelial glycocalyx layer after IRI and edema has been shown earlier

(McCully JD et al. 2002, Beresewicz A et al. 2004, Rehm M et al. 2007, Svennevig

K et al. 2008). In future, a relatively simple bed side test -such as evaluation of

peripheral plasma syndecan-1 and hyaluronan levels- (Svennevig K et al. 2008)

may predict early tissue outcome after IRI and CABG. As previously shown,

degradation of glycocalyx components occurs during septic shock and trauma;

increased levels of glycosaminoglycans predicted mortality during septic shock

(Nelson A et al. 2008), while enhanced shock after trauma, sympathoadrenal

activation, tissue damage and coagulopathy were associated with disruption of the

peripheral endothelial layer (Johansson PI et al. 2012). As a marker of endothelial

glycocalyx degradation, increased level of syndecan-1 is associated with

inflammation, protein C depletion, fibrinolysis, and mortality in trauma patients

(Johansson PI et al. 2011). By means of evaluating peripheral glycocalyx

components, it was feasible to identify different coagulopathic states, thereby

facilitating specific treatment strategies (Johansson P et al. 2011). According to

43

our initial experience, Diazoxide may be considered cardioprotective, when

associated with Aquaporin 7 release and suggested stability of the endothelial

glycocalyx layer reflecting reversible injury after IRI. Diazoxide may stabilize the

glycocalyx layer and prevent uncontrolled degradation, including various

components of the layer.

Validity and significance of the results

Limitations of the results must be acknowledged:

1. We recruited a relatively small number of patients. There were 40 patients in

original Study I and II, and 16 patients in Study III and IV. Of these only 29 patients

could be analyzed in Study I, 12 patients in Study III, 9 patients for electron

microscopy in publication IV, and 13 patients for hyaluronan and syndecan-1 in

Study IV. The patients were already at base line susceptible to heterogenous

ischemic insult and edema. It was mandatory to compare the patients' individual

results before and during operation. Importantly, the possibility of type II error, that

is the failure to reject the false null hypothesis, and its potential impact on the

interpretation of the results must be acknowledged.

2. Though the selected patients were hemodynamically stable according to strict

recruitment criteria, the patients without a recent diagnosis of CAD were included

in Studies I and II, whereas Studies III and IV consisted of patients with a recent

diagnosis of CAD. This thesis is based on two entities (Studies I, II and Studies II,

III), and we may unfortunately neither compare nor combine the two sets of patient

cohorts. The aim of these pilot studies was to minimize confounding parameters in

order to avoid interpretation bias based on heterogeneity of demographics. This

was achieved though, at the expense of the number of patients included, and

power analysis was calculated in addition to statistical analysis (III).

44

Conclusion

Taken together, in hemodynamically stable patients undergoing CABG, immediate

administration of Adenosine may enhance cardioprotection as evaluated by

myocardial expression of apoptosis-regulating genes bax and bcl2. Myocardial

expression of Aquaporin-7 and plasma levels of hyaluronan and syndecan-1 may

further elucidate the impact of immediate administration of Diazoxide. According to

our experience in clinical practice, we may now consider to infuse intra-aortic

Adenosine or Diazoxide whether immediate heart stop is desired prior to CPB. The

investigation of long-term outcome of these patients is warranted.

45

10. SUMMARY

1. Myocardial arrest with Adenosine may have a role in modulating the course

of myocardial apoptosis.

2. Adenosine has a positive effect on the down regulation of myocardial

apoptotic genes.

3. Diazoxide administered during CABG attenuates relative Aquaporin-7

expression of the right atrium.

4. Peripheral decreases of plasma syndecan-1 and hyaluronan levels may

reflect the state of endothelial glycocalyx shedding after CABG in patients

with Diazoxide.

46

11. ACKNOWLEDGEMENTS

This study was accomplished in the cardiothoracic surgery department, heart

center Tampere University, Finland, during the years 2005-2013. I wish to express

my sincere gratitude to:

Matti Tarkka , M.D., Ph.D., Professor of cardiothoracic surgery, Head of

cardiothoracic surgical department. He is the team leader and the official

representative of the ethical, scientific and financial issues concerning this work.

He provided all the opportunities to accomplish this work.

Docent Ari Mennander M.D. Ph.D, for skillful and collegial teaching throughout this

study. He has major contribution during the planning, guiding the Diazoxide study

and supervising the final part.

Docent Jari Laurikka, M.D. Ph.D for his precise advice and creative suggestions

during all stages of the work. He and his team took the technical responsibility of

promoting the techniques and safety measures in harvesting the tissue samples.

Docent Antti Saraste M.D. Ph.D, Department of Forensic Medicine, University of

Turku, Finland. He and his team took the responsibility of the apoptosis analysis of

all the tissue specimens.

Docent Niku Oksala M.D. Ph.D, Department of Vascular Surgery, Tampere

University Hospital, Tampere, Finland. He had great contribution during the

planning of the Diazoxide study, guiding and writings of the publications.

Timo Rinne, M.D. Ph.D, leader of the Anesthesiology Unit of the Cardiothoracic

Surgery Center, for providing his experiences since the time of the research

planning and standardizing as well as conducting the research work in the surgical

theater. He and his team took all the technical responsibility of CPB and

cardiolplegia settings during the work.

Vesa Anttila, M.D., Ph.D. Associate Professor Department of Surgery, Oulu

University Hospital and Peter Raivio M.D., Ph.D., Docent of the Department of

Cardiothoracic Surgery, Helsinki University Hospital: I am thankful for their

valuable comments during the revision process of this work.

47

Heini Huhtala, lecturer of statistic, Tampere School of Public Health, Finland. She

carried out the process of the statistical analysis.

Kati Peltomäki, the research coordinator of the Heart Center, Tampere University,

Finland, for her active contributions in handling all the ethical and financial paper

work of the study as well as her sustainable efforts in preparing the appropriate

materials and selecting the candidate patients all through the study.

In fact this work was a collaborative team spirit work. Without it, it would have

been impossible to get it done. I cannot address all the persons behind the efforts

here, but I would like to thank, with great respect and appreciation, all the staff

members in Tampere heart center, who took part in this work in one way or

another.

I would like to mention my warm thanks and appreciation for all co-authors: Otso

Järvinen, Juha Latva-Hirvelä, Kristiina Nuutila, Helena Porkkala, Pekka Saukko

Riikka Aanismaa, Susanna Narkilahti, Timo Paavonen, Tiina Leppanen, Mari

Hamalainen, Sanna Huovinen, Fang Zhao and Eeva Moilanen.

I thank Mr Paul Harrison for reviewing the English language.

Finally, I would like to express my thankfulness to God, my family and to every

teacher, professor, colleague and patient who I have met during my life.

This study was financially supported by the competitive research funding of

Tampere University Hospital, The Finnish Cultural Foundation and The Tampere

Tuberculosis Foundation.

http://www.nextbio.com/b/search/author/Otso%20J%C3%A4rvinen

http://www.nextbio.com/b/search/author/Otso%20J%C3%A4rvinen

http://www.nextbio.com/b/search/author/Juha%20Latva-Hirvel%C3%A4

http://www.nextbio.com/b/search/author/Kristiina%20Nuutila

http://www.nextbio.com/b/search/author/Helena%20Porkkala

http://www.nextbio.com/b/search/author/Pekka%20Saukko

48

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13. ORIGINAL COMMUNICATIONS

SAGE-Hindawi Access to ResearchCardiology Research and PracticeVolume 2009, Article ID 658965, 6 pagesdoi:10.4061/2009/658965

Clinical Study

The Impact of Adenosine Fast Induction ofMyocardial Arrest during CABG on Myocardial Expression ofApoptosis-Regulating Genes Bax and Bcl-2

Ahmed Shalaby,1 Timo Rinne,1 Otso Jarvinen,1 Juha Latva-Hirvela,2 Kristiina Nuutila,2

Antti Saraste,2 Jari Laurikka,1 Helena Porkkala,1 Pekka Saukko,2 and Matti Tarkka1

1 Division of Cardiothoracic Surgery, Heart Center, Pirkanmaa Hospital District, P.O. Box 2000, 33521 Tampere, Finland2 Department of Forensic Medicine, Central Hospital, Turku University, Kiinamyllynkatu 6-8, 20520 Turku, Finland

Correspondence should be addressed to Matti Tarkka, [email protected]

Received 17 May 2009; Revised 11 August 2009; Accepted 19 October 2009

Recommended by Firat Duru

Background. We studied the effect of fast induction of cardiac arrest with denosine on myocardial bax and bcl-2 expression. Methodsand Results. 40 elective CABG patients were allocated into two groups. The adenosine group (n = 20) received 250 μg/kg adenosineinto the aortic root followed by blood potassium cardioplegia. The control group received potassium cardioplegia in blood. Bcl-2and bax were measured. Bax was reduced in the postoperative biopsies (1.38 versus 0.47, P = .002) in the control group. Bcl-2showed a reducing tendency (0.14 versus 0.085, P = .07). After the adenosine treatment, the expression of both bax (0.52 versus0.59, P = .4) and bcl-2 (0.104 versus 0.107, P = .4) remained unaltered after the operation. Conclusion. Open heart surgery isassociated with rapid reduction in the expression of apoptosis regulating genes bax and bcl-2. Fast Adenosine induction abolishedchanges in their expression.

Copyright © 2009 Ahmed Shalaby et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

1. Introduction

Apoptosis has been considered as one of the mechanismsof cell loss during ischemia/reperfusion (I/R) injury [1–3].The presence of cardiomyocyte apoptosis (CA) is evidentearly after cardioplegic arrest and open heart surgery bothin animal models and humans [4, 5]. The mechanismunderlying I/R-induced apoptosis as well as its clinicalsignificance during open heart surgery remains incompletelyelucidated [4]. However, recent studies demonstrate thatcardioplegic arrest immediately activates the myocardialapoptosis signal pathway.

Myocardial upregulation of the proapoptotic gene baxand reduction of both bcl-2 and bcl-2/bax ratios are predis-posing the cardiomyocytes to apoptosis [6, 7]. Their changesin response to transient ischemia have been described inexperimental models and in the border zones of humanmyocardial infarction. Furthermore, inhibition of apoptosisduring reperfusion is associated with reduction in infarction

size, improvement in regional contractile and vascularendothelial functions, as well as augmentation in myocardialblood flow [8–14].

Injection of adenosine into the aortic root followed byblood cardioplegia solution after cross-clamping, producessignificantly faster cardiac standstill in patients with severecoronary artery disease [15, 16]. Adenosine attenuates post-cardioplegic dysfunction in severely injured hearts throughthe operation of receptor-mediated mechanisms [17–20]. Inan experimental animal model, adenosine inhibits apoptosisvia modulation of antiapoptotic bcl-2 and proapoptotic baxgenes and neutrophil accumulation, primarily mediated byan adenosine A2a receptor [21–23].

The main motive behind our present study is basedon the previous findings [22, 23] representing the factthat apoptosis represents a potentially preventable form ofcell death owing to its active nature, specially that thesefindings may have important clinical implications as newcardioprotective treatment strategies are developed.

mailto:[email protected]

2 Cardiology Research and Practice

The aim of this study was to determine whether cardiacsurgery using cardiopulmonary bypass (CPB) is associatedwith changes in the expression of apoptosis-regulating genesbax and bcl-2 in humans. We also studied whether morerapid induction of myocardial arrest by a single bolus injec-tion of adenosine followed by potassium blood cardioplegiamodifies their expression. We hypothesized that analysis ofbax and bcl-2 changes in genes expression might serve amarker of myocardial injury and success of cardioprotectionearly after cardiac surgery with cardioplegic arrest.

2. Material and Methods

2.1. Patient Selection. The main cohort of this study included40 patients who were scheduled for an elective CABGprocedure using on-pump CPB technique. The exclusioncriteria were diabetic patients with sulfonylurea medication,unstable angina, recent myocardial infarction within the lastmonth, redo cardiac operation, preoperative diagnosis ofasthma, chronic obstructive pulmonary disease; the patientsdid not have a history of chronic renal or hepatic diseases,and their values of creatinine (50–90 female, 60–100 male,115 mmol/L) and aP-TT-INR (International normalisedratio; 0.9–1.2 ) were within normal range. Patients withpoor left ventricular function (ejection fraction EF ≤ 30),valvular disease, and those receiving corticosteroids werealso considered not eligible. After institutional approvalby the Ethics Committee of Tampere University Hospital,the protocol for this prospective randomized, double blind,placebo-controlled study was reviewed by National Agencyfor Medicines, Finland. All 40 patients gave their informedconsent.

In our previous publication [24] we had collected thedata from the same study cohort but in the present report wehave added more end points based on more comprehensiveanalysis in order to better understand the mechanisms ofcardioplegic arrest.

2.2. Anaesthesia. A radial artery line and a pulmonaryartery catheter were inserted for haemodynamic monitoring.Anaesthesia was induced with propofol (0.5–1.0 mg/kg),sufentanil (0.6–0.8 μg/kg), and cisatracurium. Sufentanilinfusion was continued with a rate of 0.03–0.05 μg/kg/min.Sevoflurane was used as the main anaesthetic agent through-out the operation, and also provided during the cardiopul-monary perfusion with a vaporiser attached to the fresh gasinlet.

2.3. Operative and Perfusion Techniques. The surgical tech-niques were standardized in all cases, including mediansternotomy, and one internal thoracic artery, along withone to four peripheral veins from the lower extremitiesharvested in each case. Left radial artery was harvestedwhenever indicated. CPB was established with standardcannulation technique, using mild hypothermia (35◦C)with nonpulsative flow with a membrane oxygenator. Thecircuit was primed with 1500 mL of Ringer’s acetate. Theproximal anastomoses were constructed during a singlecross-clamping period.

2.4. Cardioplegia and Adenosine Administration. Patientsrandomization was between January 2006 and May 2007.Patients were allocated into two groups. In the adenosinegroup, 20 patients received 250 μg/kg adenosine into theaortic root just after cross-clamping. This dose was chosenin view of a pilot study to be the lowest effective dose tostop the myocardium. The plan is to use Adenosine as asingle bolus dose to allow the evaluation of adenosine notonly as a pretreatment during the reperfusion but also as amaintenance dose of myocardial protection during ischemiaand reperfusion.

20 patients in the control group received normal salineas placebo. All patients received standard blood cardioplegiadelivered through antegrade route. The basal CP concen-trations were identical in both groups, except for the studymedication. Concentrations of potassium and magnesiumdepend on the mixing ratio of cardioplegia rollers. Ratio 1 : 4is used for the induction, 1 : 8 for the subsequent infusionsand also for the warm terminal infusion.

Total volume of cardioplegia (CP) was not recorded.Cardioplegia was administered by means of time, pressure,and flow (according to the guidelines by Dr. Buckberg).CP was infused via antegrade route with pressure of 60–80 mmHg. Maximal flow was 300 mL/min. Duration of theinduction infusion was three minutes. Additional infusionsafter each distal anastomosis were for one minute, and thefinal warm infusion was given for three minutes. The effectof adenosine is known to be dependent on the temperature[2]. Therefore, the first cardioplegia infusion was given atnormothermia (36-37◦C) until asystole of the heart wasacquired, or at least for one minute if asystole was acquiredsooner. Thereafter, the cooling device setting was 10◦C whichyielded CP of 12-13◦C. Subsequent one-minute antegradecardioplegia infusions were administered after completionof each distal anastomosis. Final warm CP was given atnormothermia; that is, 36-37◦C.

2.5. Tissue Harvesting. From each patient, we harvested twosamples from the right atrium at two different times. Thefirst one was obtained before cross-clamping as the baselinebiopsy and the second from the same location just beforeCPB was discontinued. Myocardial samples (5–10 × 3 mm)were immediately frozen in liquid nitrogen for assessment ofgene expression.

Two samples of left ventricular apex were harvestedin both groups. The first sample was taken immediatelyafter CPB was established by oblique introduction of Tru-Cut needle (PRECISA TM 14G X 150 mm) into the leftventricle apical wall. The second sample was taken fromthe same location by the same needle before weaningfrom CPB. The puncture sites were secured with small 4-0prolene (Ethicon�) stitches even when no bleeding occurred.Myocardial tissue (5–10× 3 mm) was immediately frozen inliquid nitrogen for histological studies.

2.6. Total RNA Extraction and Complementary DNA Syn-thesis. Atria samples were homogenized and total RNA wasextracted with Trizol reagent (Invitrogen�) according tomanufacturer’s instructions. The RNA concentration was

Cardiology Research and Practice 3

determined using spectrophotometry. Total RNA (1 μg)from each sample was incubated with equal amount ofrandom hexamers (PromegaTM) at 70◦C for 10 minutesand then cooled on ice for 5 minutes. ComplementaryDNA (cDNA) was synthesized in a mixture containing M-MLV Reverse Transcriptase RNase H Minus-enzyme (200 U),Recombinant RNasin Ribonuclease Inhibitor (40 U), dNTPmix (25 mM each), M-MLV RT 5x buffer, and Nuclease-free water (all purchased from Promega Corp. WI, USA).Mixtures were incubated at room temperature for the initial10 minutes, then at 50◦C for the final 50 minutes. Finally, thereaction was inactivated by heating for 5 minutes at 95◦C.

2.7. Primers and Probes. Sequences of the oligonucleotideprimers and probes used in real-time quantitative reversetranscriptase polymerase chain reaction (RT-PCR) assaywere the same that used for bax and bcl-2 [25] (Sequencedetection primer, Applied Biosystems Inc, CA, USA) andprobes (TaqMan� TAMRATM Probe, Applied Biosystems)used in real-time quantitative reverse transcriptase poly-merase chain reaction (RT-PCR) assay. In order to preventthe amplification of contaminating genomic DNA, primerpairs were located in different exons. Housekeeping gene 18SrRNA was used as an endogenous control (The TaqMan�

Ribosomal RNA Control Reagents, Applied Biosystems).

2.8. RT-PCR Assay. The quantitative real-time RT-PCRwas performed using Applied Biosystems 7500 RT-PCRsystem and Taqman�-based chemistry. The PCR solution-composed of 5 ng cDNA, forward and reverse primers(400 nM), specific probe (100 nM), and TaqMan� UniversalPCR Master Mix. Primer and probe concentrations for 18Swere 50 nM and 200 nM, respectively. After incubation at50◦C for 2 minutes and denaturing at 95◦C for 10 minutes,PCR was carried out, 50 cycles of 95◦C for 15 seconds and60◦C for 1 minute. All RT-PCR reactions were run as aduplicate.

2.9. Quantification of Gene Expression. Quantification ofgene expression was carried out by using relative standardcurve method. Briefly, standard curves for each plate weregenerated using serial dilutions of cDNA synthesized fromhuman tonsil tissue RNA. The amounts of both target andendogenous control cDNA in each sample were calculatedrelative to the standards. To eliminate the variation of totalcDNA quantity in each sample, the results are expressed as aratio of the target gene to the endogenous control.

2.10. Assessment of Apoptosis in Left Ventricular Tissue.Apoptosis was detected using the terminal transferasemediated ddUTP nick end-labeling (TUNEL) assay, aspreviously described [26, 27]. In brief, paraffin-embeddedmyocardial sections were heated in sodium citrate solutionand digested with proteinase-K to expose DNA. The DNAstrand breaks were then labeled using terminal transferasewith digoxigenin-conjugated ddUTP and visualized usingalkaline phosphatase immunohistochemistry (IHC). To con-firm optimal sensitivity of the assay, it was standardized

with the use of serial sections treated with DNase I toinduce enzymatic DNA fragmentation (positive control ofapoptosis). The apoptotic rate was defined as the positiveTUNEL cardiomyocytes per field, and expressed as % ofcardiomyocytes.

2.11. Statistical Analyses. Statistical analysis was performedusing SPSS for Windows software, version 9.0 (SPSS;Chicago IL, USA). The Mann-Whitney U Test was usedto distinguish demographic differences in means betweenthe groups. Continuous variables were analyzed by analysisof variance (ANOVA) for repeated measures. Logarithmictransformation, median and quarter analysis methods wereused when the variables were not normally distributed.Statistical significance was attributed to P value <.05.

3. Results

3.1. Patient Characteristics

Demographic and Basic Clinical Data. The study cohortincluded 40 patients between the ages of 46 and 73 years, 90%of them were male patients (Table 1). Twenty patients weregiven adenosine and the others served as controls. All thepatients in both groups completed the study protocol. Therewere no adverse effects related to adenosine. There were noadverse postoperative complications in any of the 40 patients.All the patients survived the operation and were dischargedfrom the hospital. There were no adverse effects related toadenosine. There was no significant difference in the baselinecharacteristics of the patients between study groups as shownin Table 1.

As shown in Table 2 and Figure 1, in the control group,expression of bax gene was significantly reduced in thepostoperative samples as compared with the preoperativesamples (P = .002). Expression of bcl-2 showed a similarreduction tendency (P = .07). However, in contrast to thecontrol group, expression of both bax and bcl-2 remainedunaltered before and after the operation in the adenosinetreatment group (P = .45 and .47). There were no significantdifferences in the level of bax/bcl-2 ratio expression in eitherpre- or postoperative samples in the control and adenosinegroups.

As shown in Table 2, (the ventricular samples were usedto detect the apoptotic index and then patients were allocatedinto subgroups accordingly to TUNEL positive and negative,meanwhile the atria samples were used to detect the geneexpression of apoptosis) there were no significant differencesin either expression or bax/bcl-2 ratio between patients whohad or had not TUNEL positive cardiomyocytes in the leftventricular tissue samples. Neither was there correlationbetween gene expression nor the amount of TUNEL positivemyocytes. However, there was a tendency towards lower bcl-2 expression and higher bax expression in patients withTUNEL positive myocytes as shown in Table 2.

Values are presented as median values and standarddeviation. “Before” refers to before cross-clamp applicationand “After” refers to after cross-clamp removal and justbefore CPB discontinuation.


Table 1: Base line Patient Characteristics and operative data.

Adenosine (n = 20) Control (n = 20)

Age (years)∗ 63.4± 6.8 65.5± 7.3

Gender (male/female) 19/1 17/3

New York HearAssociation class III

20 20

EF∗ 61± 5.8% 63± 9.7%

Coronary vessels†

LMA 3 3

50–74%

≥75% 5 3

LAD

50–74% 7 5

≥75% 11 10

CX

50–74% 4 3

≥75% 6 8

RCA

50–74% 5 1

≥75% 8 8

No of Grafted 3 3

vessels∗

CKMB∗ 3 3

CBP time 94± 25 91± 16

Ischemic time 79± 26 72± 18

Weaning time 15± 6.4 19± 8∗

Figures present the mean value, CKMB indicates preoperative value the daybefore the operation.†Vessels classified regarding to the site of lesion and percentage of stenosisand according to the most recent coronary angiography, which is used tomake the decision to operate the patient. Figures present the number ofvessels affected in each category.

4. Discussion

We found a decrease in apoptosis regulating genes inright atria samples obtained immediately after reperfusioncompared to preischemic samples in patients undergoingCABG. Interestingly, expression remained stable in patientsrandomized to adenosine treatment during cardioplegia.Also, in the adenosine group, the bax/bcl-2 ratio decreasedmore than in the control group, although the decrease wasnot statistically significant.

Previously, in a canine model, adenosine reduced apop-tosis induced by I/R injury. This reduction was associatedwith enhanced expression of antiapoptotic bcl2 genes.Adenosine also reduced expression of proapoptotic baxgene in the peri-infarct myocardium [21]. Our results areconsistent with the previous in that the level of antiapoptoticbcl-2 expression decreased after ischemia in the controlgroup while the adenosine treatment prevented this down-regulation [21]. In contrast to the previous study, we foundthat also the expression of proapoptotic bax was reducedin the control group while its expression remained stablewith adenosine treatment. This may be related to very

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

AfterBefore AfterBefore

Bax 18s P = (.002 & .45) Bcl2 18s P = (.07 & .47)

ControlAdenosine

Figure 1: Baseline versus the postoperative expressions of bax andbcl2 in adenosine treated and the control groups. Gene expressionis normalized to the same endogenous gene. Before and After referto the timing of taking the biopsies (Before; before cross clampapplication and After; after cross clamp removal and just beforeCPB discontinuation).

short time reperfusion (versus 6 hours of reperfusion in theearlier study). Cardioplegic protection during ischemia maysuppress proapoptotic signaling, and RNA expression maynot directly reflect protein levels that were measured in theearlier study.

In parallel with the observed changes in the expressionof apoptosis-regulated genes, the amount of apoptoticcardiomyocytes as detected with the TUNEL-assay in the leftventricular samples of the patients appeared to be reducedin the adenosine treated group as reported earlier [28].However, due to small percentage of apoptotic cells andhigh variation in the results, this change was not statis-tically significant. Detection of apoptotic cardiomyocytesis methodologically challenging, because the appearanceof DNA fragmentation is a late feature of the apoptoticprocess and, therefore, TUNEL method may detect onlysmall fraction of eventually apoptotic cells when the samplesare taken very shortly after reperfusion [29]. Therefore, wehypothesized that measurement of changes in apoptosis-regulating genes could serve as an early marker of theseverity of ischemic myocardial injury as well as providemeans to study effects of cardioprotective interventions intissue biopsies obtained in the early reperfusion period.Although there was a difference between the control and theadenosine treated groups, there are several doubts in thisapproach. First, the variation between individual patientsin the baseline and postoperative expression was high sincethis is a clinical study and there is always something cannotbe controlled in small powered trial. Second, we could notdemonstrate relationships either between gene expressionand the amount of apoptosis or the amount TUNEL positivecardiomyocytes. Third, the results need to be interpretedcautiously due to the complex interplay between pro- andantiapoptotic regulators.

Cardiology Research and Practice 5

Table 2: TUNEL positive and negative subgroups (of the ventricular samples) in both the adenosine and control groups in correlation withthe changes of apoptosis-regulating genes in the atria samples.

bax bcl-2 bax/bcl-2

Group Subgroup Before After Before After Before After

TUNEL negative 0.47± 0.83 0.53± 1.17 0.1± 0.07 0.15± 0.7 4.97± 8.5 3.88± 5.6

Adenosine TUNEL positive 0.91± 3.3 0.35± 2.4 0.29± 1.7 0.08± 1.3 2.4± 2.4 5.1± 5.1

Total group 0.52± 16.5 0.59± 2.36 0.104± 0.91 0.107± 0.79 8.8± 13.67 5.1± 15.75

TUNEL negative 1.4± 20.5 0.44± 1.2 0.28± 2.5 0.09± 0.3 6.02± 5.3 4.38± 13.5

Control TUNEL positive 0.92± 4.5 0.64± 0.9 0.23± 0.5 0.12± 0.2 5.21± 5.2 7.57± 7.5

Total group 1.37± 13.07 0.47± 1.04 0.14± 1.64 0.08± 0.25 6.6± 15.8 6.4± 20.9

Therefore, further methodological validation and devel-opment is still needed. Future studies should explore at leastthe use of other genes as potential markers as well as optimaltiming of tissue sampling.

In the overall picture, as expressed in Table 2, the presentstudy has the value to address the facts that CPB (or aorticcross-clamping) has a significant effect on myocardial injurydue to the significant amount of apoptotic cells detected afterweaning from the bypass. Fast induction of myocardial arrestcould be obtained by intraaortic injection of adenosine. Thisinjection was able to modulate the expression of apoptoticgenes decreasing the occurrence of apoptosis significantly.

Some previous studies which were focusing on the effectof fast adenosine induction of myocardial arrest showedthe postoperative clinical improvement in this group ofpatients [20]. As we reported before [24], we could notdemonstrate this clinical effect in our setting but this findingdoes not exclude the effect of adenosine to decrease apoptosisin such patients. Such effect cannot be overlooked duringthe trials to improve the quality of myocardial protection.These results should be confirmed in more highly poweredstudies, possibly with more critical patient cohort. Changingthe timing of adenosine administration during reperfusionshould be considered for further evaluation.

In conclusion, cardioplegic arrest during CABG surgeryresults in changes in apoptosis-regulating genes. The changeis detectable in myocardial tissue samples obtained veryearly after reperfusion and therefore, this method may allowassessment of the quality of myocardial protection.

Acknowledgments

This study was made possible with the research grantsfrom The Pirkanmaa Hospital District Competitive ResearchFunding and from Tampere Tuberculosis Foundation.

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