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Mechanisms Underlying the Pathogenesis of Atrial
Arrhythmias in RGS4-Deficient Mice
by
Alexandra Sorana Mighiu
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Physiology
University of Toronto
© Copyright by Alexandra Sorana Mighiu (2014)
ii
Mechanisms Underlying the Pathogenesis of Atrial Arrhythmias in
RGS4-Deficient Mice
Alexandra Sorana Mighiu
Master of Science
Department of Physiology
University of Toronto
2014
Abstract
Atrial arrhythmias are very common clinically relevant conditions that are strongly associated
with aging and parasympathetic tone. Additionally, ATP-sensitive K+
(KATP) channel activation
has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP
channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here
investigate whether KATP channel activity is increased under decreased RGS4 activity in a
manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction
of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial
tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic
blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of
pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are
consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-
induced re-entrant rotors in the RGS4-deficient mouse model.
iii
Acknowledgements
First and foremost, I would like to extend my gratitude to my supervisor, Dr. Scott Heximer, for
his guidance and encouragement during both my undergraduate and graduate studies. Scott,
thank you for challenging me to be an independent young scientist and for motivating me to
enjoy the research process. I also greatly appreciate your continued support and encouragement
to continue my academic studies. I would also like to thank my advisory committee members,
Dr. Anthony Gramolini and Dr. Peter Backx, for their valuable insight and support over the past
two years and for providing me with excellent letters of reference for all my applications.
Completing this work would have been all the more difficult were it not for the support and
friendship provided by all members of the Heximer lab. Guillaume, Emily, Steph, Joey, and
Joobin – thank you for creating a stimulating, enjoyable, and very productive working
environment. More importantly, thanks for getting me out of the lab every now and then. I am
also very grateful to Jenny for patiently teaching me how to isolate atrial tissues and perform
intra-cardiac experiments, and for breeding and taking care of my animals.
I would also like to thank the Backx lab, especially Rooz, Farzad, and Adam, for their generosity
in sharing the optical mapping, electrocardiogram, and microelectrode equipment, and their
assistance with troubleshooting those experiments. Thank you also to Wallace Yang for teaching
me to analyze the optical imaging data and for sharing his macro-scripts for dominant frequency
analysis.
Most importantly, I would like to express my utmost gratitude to my parents for their endless
support and encouragement. Mom and dad, thank you for helping me navigate all of the ups and
downs of graduate school and for backing me at every stage of my academic career and life.
Without your unwavering support I would not have the courage to pursue my dreams. Finally, to
my dearest sister, Patri - I am indeed your biggest fan. Thank you for being my source of
motivation and my role model, and for supporting me every step of the way. No matter the
distance, I know I can always count on you for anything.
iv
Table of Contents
General Abstract ............................................................................................................................. ii
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ...................................................................................................................................v
List of Figures ................................................................................................................................ vi
List of Abbreviations .................................................................................................................... vii
1 Introduction .................................................................................................................................1
1.1 Atrial Fibrillation ...........................................................................................................1
1.1.1 Normal Electrophysiology of the Heart ..........................................................2
1.1.2 Basic Arrhythmia Mechanisms .......................................................................6
1.2 Regulators of G-protein Signaling ...............................................................................12
1.2.1 RGS proteins in the Heart .............................................................................13
1.2.2 RGS4 .............................................................................................................15
1.3 ATP-Sensitive K+
(KATP) Channels .............................................................................18
1.3.1 KATP Channel Structure and Modulation by Nucleotides .............................18
1.3.2 Regulation of KATP Channel Activity ...........................................................20
1.3.3 KATP Channels in the Heart ...........................................................................21
1.3.4 Role of KATP Channels in Cardiac Arrhythmias ...........................................23
2 Rationale ....................................................................................................................................25
3 Hypothesis ..................................................................................................................................25
4 Materials and Methods .............................................................................................................26
4.1 Experimental Animals .................................................................................................26
4.2 In Vivo Intracardiac Electrophysiology .......................................................................26
4.2.1 Intracardiac Catheterization ..........................................................................26
4.2.2 Electrophysiology Study ...............................................................................27
4.3 High Resolution Optical Imaging and Analysis ..........................................................28
4.3.1 Isolated Atrial Preparation ............................................................................28
4.3.2 Imaging System ............................................................................................29
4.3.3 Experimental Protocols .................................................................................30
4.3.4 Data Processing and Analysis .......................................................................30
4.4 Microelectrode-based Measurement of Atrial Myocyte APD .....................................32
4.5 Quantitative Real-time PCR Reaction and Quantitative Analysis ...............................32
4.6 Statistical Analysis .......................................................................................................34
5 Results ........................................................................................................................................36
6 Discussion...................................................................................................................................56
6.1 Atrial arrhythmias in the absence of RGS4 .................................................................56
6.2 Effect of KATP on atrial arrhythmia vulnerability in RGS4-deficient mice ..................61
6.3 Limitations ...................................................................................................................66
6.4 Conclusions and future directions ................................................................................67
7 References ..................................................................................................................................70
v
List of Tables
Table 5-1. Intracardiac ECG parameters in anesthetized WT and RGS4-/-
mice at baseline and
following bolus carbachol injection ...............................................................................................38
Table 5-2. Incidence and duration of atrial arrhythmias in anesthetized WT and RGS4-/-
mice ..40
Table 5-3. Contingency table showing pacemaker shift is a predictor of baseline arrhythmia
vulnerability in isolated atrial preparations ....................................................................................46
Table 5-4. Effect of Kir6.2 ablation on parasympathetic-mediated arrhythmia inducibility in
isolated atrial examined by optical mapping .................................................................................52
Table 5-5. Effect of Kir6.2 ablation on APD at 70% repolarization .............................................52
Table 5-6. Effect of tolbutamide on parasympathetic-mediated arrhythmias in isolated atria
examined by optical mapping ........................................................................................................53
vi
List of Figures
1 Introduction
Figure 1-1. Re-entry model of atrial fibrillation ...........................................................................11
Figure 1-2. Heterotrimeric G-protein signaling and regulation by RGS proteins ........................17
4 Methods
Figure 4-1. Optical mapping experimental setup……………………………………………….35
5 Results
Figure 5-1. Anesthetized RGS4-deficient mice show episodes of wandering pacemaker at
baseline ..........................................................................................................................................37
Figure 5-2. Induction of atrial arrhythmias in WT and RGS4-/-
mice ...........................................41
Figure 5-3. Increased incidence of rotor formation in WT and RGS4-/-
atria ...............................45
Figure 5-4. Spectral characterization of atrial arrhythmia episodes in WT and RGS4-/-
atria .....47
Figure 5-5. Effect of carbachol on AP duration in WT and RGS4-/-
isolated atria .......................49
Figure 5-6. Effect of tolbutamide on parasympathetic-mediated APD shortening in WT and
RGS4-/-
atria ...................................................................................................................................54
Figure 5-7. Expression of KATP channel subunits in mouse atria ..................................................55
vii
List of Abbreviations
AF Atrial Fibrillation
AERP Atrial Effective Refractory Period
AP Action Potential
APD Action Potential Duration
ATP Adenosine Triphosphate
AV Atrioventricular
β-AR β-Adrenergic Receptor
cAMP Cyclic Adenosine Monophosphate
CCh Carbachol
CV Conduction Velocity
DAD Delayed After-Depolarization
EAD Early After-Depolarization
ECG Electrocardiogram
GAP GTPase Activating Protein
Gαi/o Inhibitory G protein
Gαs Stimulatory G protein
GDP Guanine Diphosphate
GIRK G-protein-Coupled Inwardly Rectifying Potassium Channel
GPCR G-protein Coupled Receptor
GTP Guanine Triphosphate
HCN Hyperpolarization-Activated Cyclic Nucleotide-Gated Cation Channel
HR Heart Rate
viii
ICa,L Inward Ca2+
Current
If Hyperpolarization-Activated Funny Current
IK1 Inward Rectifier Potassium Current
IK,Ach G-protein-Coupled Inwardly Rectifying Potassium Current
IKr Rapidly-Activating Delayed Rectifier Potassium Current
IKs Slowly-Activating Delayed Rectifier Potassium Current
IKur Ultra-Rapid Delayed Rectifier Potassium Current
INa Inward Sodium Current
Ito Transient Outward Current
INa-Ca Sodium-Calcium Exchange Current
ip Intraperitoneal
KATP ATP-Sensitive K+
Channel
LAA Left Atrial Appendage
M2R Muscarinic M2 Cholinergic Receptor
NBF Nucleotide Binding Fold
NCX Sodium-Calcium Exchanger
PIP Phosphatidylinositol
PIP2 Phosphatidylinositol-4,5-Bisphosphate
PLC Phospholipase C
PLN Phospholamban
PKA Protein Kinase A
PKC Protein Kinase C
RAA Right Atrial Appendage
ix
RGS Regulator of G-protein Signaling
RyRs Ryanodine Receptors
SA Sinoatrial
SUR Sulfonylurea Receptor
WT Wild-Type
1
1 Introduction
1.1 Atrial Fibrillation
Atrial fibrillation (AF) is the most common clinically relevant arrhythmia (1) and is
characterized by rapid, uncoordinated atrial electrical activity with consequent deterioration of
ventricular function. On an electrocardiogram, AF is characterized by the replacement of regular
P waves with rapid oscillations or fibrillatory waves and irregular ventricular activation. The
occurrence of AF increases with age, from a prevalence of 0.5% in adults ranging from 50-60
years of age to approximately 10% of adults over 80 years of age (2;3). With the increasing
average age of the human population, the prevalence of AF is expected to double or triple within
the next two or three decades (4-6).
AF is a significant contributor to population morbidity and mortality and is a major
independent risk factor for stroke (7). AF is associated with thromboemboli resulting from blood
stasis and it has been estimated that approximately 20-25% of all strokes can be attributed to AF
(8). Several cardiac and non-cardiac disorders predispose to AF including ischemic heart disease,
coronary artery disease, hypertension, congestive heart failure, and diabetes (9). Many of these
are thought to promote AF by increasing the atrial pressure and/or causing atrial dilation;
however, the precise mechanistic links have not been fully characterized.
Clinically, AF can be divided into paroxysmal (lone), persistent, and permanent (10;11).
Paroxysmal AF, which usually occurs in the absence of underlying heart disease, is defined as
recurrent episodes (≥ 2) of AF that self-terminate within seven days, and can be caused by rapid
focal activity arising from the pulmonary veins. Persistent AF is brought on by the development
of functional (electrical) re-entry substrates and the AF episodes last beyond seven days or
2
require pharmacologic or electrical cardioversion. As the disease progresses, atrial tissues
undergo irreversible structural changes (i.e. fibrosis) and the arrhythmia becomes permanent.
Present pharmacologic therapies for AF have major limitations, including limited
efficacy and significant potential adverse effects due to their effects on ventricular ion currents
(12;13). In view of this, efforts aimed at improving our understanding of the pathogenic
mechanisms responsible for the initiation and maintenance of AF would seem particularly
important. Therefore, the purpose of this thesis is to characterize the role of RGS4 in the atrium
and how deregulated RGS4 function may regulate ATP-sensitive K+ (KATP) channel activity to
modify atrial refractoriness and ultimately contribute to the pathogenesis of AF.
1.1.1 Normal Electrophysiology of the Heart
In the mammalian myocardium the heart beat is generated by specialized, autorhythmic
cells within the sinoatrial (SA) node, the primary pacemaker of the heart. The SA node is located
in the right atrium at the junction of the crista terminalis (a thick band of atrial muscle at the right
atrial appendage) with the superior vena cava (14). From the SA node, the impulse propagates to
the right and left atria through Bachmann’s bundles and the atrial tracts. The electrical wavefront
is then conducted to the atrioventricular (AV) node, located at the junction between the atria and
the ventricle. Because conduction at the AV node is slow, a delay occurs before excitation
spreads to the ventricles which allows for ventricular filling. The impulse then travels rapidly
through the Bundle of His and Purkinje fibre network to the ventricles to cause a coordinated
contraction. The AV node and auto-rhythmic cells of the Purkinje system can also acts as
subsidiary pacemakers, maintaining automaticity in case of failure of the SA node.
3
The specialized cells within the SA node possess the ability to generate spontaneous
action potentials (APs) without the need for chemical or electrical input from other sources. The
mechanism of SA node automaticity has traditionally been attributed to a “voltage clock”
mechanism that is mediated by the hyperpolarization-activated cyclic nucleotide-gated cation
(HCN) channel. The HCN channel becomes activated at the end of the AP (at voltages of
approximately -40 to -50 mV) and allows the passage of the hyperpolarization-activated “funny”
current (If). If is carried predominantly by an influx of Na+ and K
+ ions, which progressively
depolarizes the membrane potential (15). This is known as the diastolic depolarization phase of
the AP and is the basis of SA node automaticity. Once the membrane reaches approximately -30
mV, fast L-type Ca2+
channels are activated and the inward Ca2+
current (ICa,L) generates the
upstroke of the AP (phase 0) (16;17). During repolarization, L-type Ca2+
channels close and
voltage-gated K+
channels open generating an outward K+
current.
More recently, SA node automaticity was also shown to occur via a so-called “Ca2+
clock” mechanism that involves local Ca2+
release from the sarcoplasmic reticulum (SR). This
was first demonstrated by Vinogradova et al. (18), who showed that spontaneous SR Ca2+
release
activates the Na+/Ca
2+ exchanger (NCX). The authors demonstrated that the isolated rabbit SA
node exists in a state of high basal cAMP (cyclic adenosine monophosphate) concentrations and
protein kinase A (PKA) activity. PKA-dependent phosphorylation of phospholamban (PLN) and
ryanodine receptors (RyRs) increases SR Ca2+
uptake and Ca2+
leak through RyRs, respectively.
The increase in intracellular Ca2+
activates the electrogenic NCX, which generates an inward
current (INa-Ca) as Ca2+
is extruded (19;20). INa-Ca is then produced during the late diastolic
depolarization phase which brings the pacemaker potential to threshold (-30 mV) (20).
4
In addition to the spontaneous APs found in the SA and AV nodes, the heart possesses
fast-response APs in the atria and ventricles that are responsible for the coordinated contraction
of the myocardium. These APs are characterized by 5 phases, numbered 0-4. The inward rectifier
K+ current (IK1) is an important background current contributing to the stabilization of the resting
membrane potential between -70 and -80 mV (Phase 4) (21). The resting membrane potential is
also stabilised by other ion pumps/exchangers, most notably, the Na+/K
+ ATPase pump which
actively transports Na+ and K
+ against their electrochemical gradients (22). Myocyte activation is
initiated by the rapid activation of voltage-gated Na+ channels, which generates a large inward
Na+ current (INa) brings the cell from its resting potential to approximately +25 mV (Phase 0) .
Phase 1 is termed early repolarization and occurs immediately after the peak of depolarization.
Na+ channels are inactivated and a small downward deflection or “notch” in the AP is generated
by the activation of a transient outward K+
current (Ito) that partially repolarises the cell (23). In
the atria, the ultra-rapidly activating delayed rectifier K+ current (IKur) also contributes to phase 1
(24). This is followed by phase 2, the ‘plateau phase’, which is maintained by a balance between
inward Ca2+
currents through the L-type Ca2+
channel and outward K+ currents. The main
repolarising current during phase 2 is the rapidly-activating delayed rectifier K+
current (IKr) (25).
Phase 3 represents repolarization and is mediated by outward K+
currents. The two main
repolarising potassium currents that sum to terminate the plateau phase and initiate final
repolarization are IKr and the slowly-activating delayed rectifier K+ current (IKs) (26;27).
Autonomic regulation of pacemaker activity
The autonomic nervous system is the major cardiac-extrinsic determinant of heart rate.
The mammalian SA node is richly innervated by parasympathetic and sympathetic fibers, and
5
enriched with adrenergic and muscarinic receptors, making the SA node susceptible to
autonomic regulation. HR is determined by the balance between vagal and sympathetic input.
Stimulation of the vagus nerve causes a decrease in the SA node firing rate, while sympathetic
stimulation causes an increase in the SA node rate, thereby decreasing and increasing heart rate,
respectively.
Parasympathetic signaling is initiated upon the release of acetylcholine from the vagus
nerve (28). In the SA node, acetylcholine preferentially activates the muscarinic M2 cholinergic
receptors which transduce the vagal signal via intracellular coupling to inhibitory heterotrimeric
G-proteins (Gαi/o). Several effects mediated by both Gαi/o and Gβγ subunits contribute to the
reduction in HR. First, Gβγ heterodimers directly activate the G-protein-coupled inwardly
rectifying potassium channels (GIRK) which generate the hyperpolarizing potassium current,
IK,Ach. This decreases the rate of diastolic depolarization and consequently slows down the heart
rate. Second, Gαi/o inhibits adenylyl cyclase activity to reduce intracellular cAMP levels and
PKA activity, thus leading to decreased depolarizing currents carried by the HCN and L-type
Ca2+
channels, thereby slowing SA node firing rate (29).
Conversely, sympathetic innervation increases HR via β-adrenergic receptors (β-ARs)
which are coupled to stimulatory G-proteins (Gαs) (30;31). β-AR stimulation leads to the
activation of adenylyl cyclase which subsequently increases intracellular cAMP concentrations.
cAMP binds directly to the HCN channel at a C-terminal cyclic nucleotide binding domain (32).
This has been shown to shift the voltage dependence of channel activation to more positive
potentials which enhances channel activity and increases the rate of diastolic depolarization (33).
A faster rate of diastolic depolarization means that the AP threshold will be reached earlier and
6
the AP firing rate will increase. Additionally, cAMP activates PKA which increases ICaL by
opening L-type Ca2+
channels through phosphorylation (34;35).
1.1.2 Basic Arrhythmia Mechanisms
The mechanisms of AF can be generally divided into two major categories: (1) abnormal
automaticity (ectopic activity) and (2) re-entry.
Abnormal Automaticity
Ectopic activity, which is defined as impulse generation outside of the SA node, occurs
when cells develop after-depolarizations. These are membrane depolarizations that can provoke
spontaneous action potentials when they reach the threshold of depolarizing currents. Depending
on when these oscillations occur, “early” and “delayed” after-depolarizations are distinguished.
Early after-depolarizations (EADs) typically occur late in phase 2 or early in phase 3 of the
cardiac action potential and interrupt normal repolarization (36). They occur in the presence of
prolonged action potential duration and bradycardia which can result from various conditions
including hypoxia (37), acidosis (38;39), ventricular hypertrophy (40), and catecholamine
challenge (41).
‘Late phase 3’ EADs have also been described and they interrupt the final phase of
repolarization of the action potential (42;43). In contrast to conventional EADs, the late phase 3
EADs occur when repolarization is dramatically shortened and they are mediated by normal
calcium release from the sarcoplasmic reticulum (SR), instead of spontaneous SR calcium
release. When the AP duration is shortened, intracellular Ca2+
levels would peak during the late
phase of repolarization (phase 3) instead of the plateau phase (phase 2). The triggered AP is
7
believed to be induced by two Ca2+
-dependent currents, INa-Ca, and a Ca2+
-activated Cl- current
(ICl(Ca)) (43). DADs are observed after full repolarization of a cardiac action potential and tend to
occur under conditions that produce intracellular Ca2+
overload, including tachycardia, ischemia,
adrenergic stimulation, low extracellular K+ concentration, and digitalis toxicity (36).
Re-entrant Arrhythmias
Re-entry is a phenomenon that permits the wave of excitation to propagate continuously
within a closed circuit (circus movement). AF-related re-entry can occur in two general forms:
(1) single-circuit re-entry involving one primary driver, and (2) multiple-circuit re-entry,
involving multiple simultaneous re-entry circuits. The persistence of re-entry circuits is
facilitated by the development of a vulnerable atrial “substrate”. Re-entry substrates can be
caused by altered electrical properties or by fixed structural changes. Cardiac tissue exhibits a
discrete refractory period (unexcitable period following a cardiac action potential). In order for a
successful re-entry event to occur, the path length travelled by the impulse in one refractory
period must be longer than the wavelength of re-entry which is the distance travelled by the
impulse in one refractory period and can be derived as the product of the action potential
duration (APD) and conduction velocity (CV) (Figure 1-1).
Under normal conditions when a pro-arrhythmic substrate is absent, the impulse traverses
the circuit and returns to its initiation point in a time that allows it to impinge on tissue that is
refractory and therefore die out. However, when the APD is shortened sufficiently as in some
vulnerable substrates, excitability is recaptured earlier and the premature re-entering impulse can
sustain itself indefinitely within the circuit. Decreased inward Ca2+
currents or increased outward
K+ currents can shorten APD and promote re-entry. Conversely, K
+ current blockade prolongs
8
APD and supresses reentrant AF. Furthermore, slowing of the depolarization wave CV can make
re-entry more likely by increasing the conduction time which allows the tissue to recover
excitability. The main determinants of atrial conduction are sarcolemmal Na+ channels and gap
junction connexin channels. Slowed conduction is favored by reduced Na+ current or gap
junction dysfunction. Decreases in the wavelength are also expected to shorten the size of the re-
entry circuit which increases the number of circuits that can be accommodated by the tissue and
therefore the likelihood of AF.
Atrial Remodeling
An important advancement in the understanding of AF was made when it was observed
that once initiated, AF leads to persistent alterations in atrial function (remodeling) that greatly
increase vulnerability to future fibrillation (“AF begets AF”). In their original goat model,
Wijffels et al. (44) demonstrated that brief bursts of electrical stimulation resulted in an AF
episode that terminated spontaneously within seconds. When AF was repetitively re-induced, the
episodes of AF gradually became longer until they no longer self-terminated. This process is
termed electrical remodeling and involves molecular changes that act to shorten the path required
for a sustained circular re-entry event (rotor) to occur. Characteristic electrophysiologic features
of AF-induced remodeling include shortening of the atrial effective refractory period. These
changes are thought to be due to decreased L-type Ca2+
current. Atrial tachycardia causes atrial
myocyte Ca2+
loading, which triggers short-term functional and long-term gene expression
changes that decrease ICa-L and prevent potentially lethal calcium overload, at the expense of
causing decreased APD and AERP (45-47). Reduced ERP decreases the wavelength and
facilitates the development of multiple-circuit re-entry. In addition to electrical remodeling,
9
structural remodeling can also occur after prolonged episodes of tachycardia (48-50). The most
common structural change is the development of atrial fibrosis which can slow conduction and
create conduction barriers that favor the initiation and maintenance of re-entry.
Parasympathetic Stimulation and Atrial Fibrillation
In addition to electrical remodeling, parasympathetic signaling may play an important
role in the onset of AF. Previous animal studies demonstrated that stimulation of the M2 receptor
with carbachol could facilitate the induction of AF (51), whereas inhibition of Gαi/o-signaling
reduced the incidence of vagal-mediated AF (52;53). Additionally, vagal denervation of the atria
prevented the induction of AF in dogs (54;55). In the clinic, Coumel and colleagues (56)
described clinical cases of AF in which vagal activity preceded the onset of atrial arrhythmias.
Furthermore, among patients with structurally normal hearts, AF is often observed to occur at
night, when vagal tone is relatively high. A number of important electrophysiologic changes
occur in the atrial myocardium in response to an increase in vagal tone. First, acetylcholine
reduces the slope of the diastolic depolarization of the SA node action potential and slows the
heart rate which can increase the risk of developing ectopic triggers in the atrial tissue (57).
Second, acetylcholine shortens the atrial effective refractory period, an important determinant of
re-entry.
Several observations suggest that activation of IK,Ach may be a key component of the
parasympathetic-pathway mediated initiation and maintenance of AF. First, Kovoor and
colleagues (58) demonstrated that activation of IK,Ach is necessary for the pro-arrhythmogenic
action of the M2 receptor agonist, carbachol, because mice lacking the GIRK4 gene (IK,Ach-
deficient knockout mice) were protected from the development of AF in the presence of the
10
cholinergic agonist (58). Moreover, tertiapin-Q, a selective IK,Ach inhibitor, terminates vagally
induced atrial tachyarrhythmias in dogs by prolonging the atrial effective refractory period (59).
Furthermore, Ehrlich and colleagues demonstrated that atrial tachycardia remodeling
promotes agonist-independent IK,Ach activity that shortens atrial APD and increases susceptibility
to AF in dogs (60-62). Importantly, evidence from human studies confirms the presence of
constitutively active (agonist independent) IK,Ach in patients with long-standing AF (63).
Constitutive activity occurred as a result of an increase in the open probability and number of
open channels without changes in other single-channel properties (61). Abnormal
phosphorylation of the IK,Ach channel by PKCε was shown to increase channel activity in both
chronically-paced canine atrial cardiomyocytes (64) and atrial appendages from patients with
chronic AF (65). Clinical studies have also identified a genetic polymorphism in the β3 subunit
of heterotrimeric G-proteins which was associated with reduced IK,Ach activity in atrial myocytes
and protection from AF in humans (66).
Regional heterogeneity in atrial refractoriness, due to regional differences in the
distribution of M2 receptors, GIRK channels, and parasympathetic ganglia, also plays a
considerable role in the initiation and maintenance of AF (67). In the mouse, a greater expression
of Kir3.1 and Kir3.4 subunits of the GIRK channel (68) and greater IK,Ach (69) is present in the
right atria versus the left atria, whereas the opposite has been demonstrated in sheep and humans
(70). Indeed, in the isolated sheep heart, the greater expression of Kir3.1/3.4 subunits and IK,Ach
density in the left atria as compared to the right atrium corresponded to a greater dominant
frequency of AF rotors in the LA (71;72). Collectively, these studies highlight the importance of
identifying endogenous regulators of parasympathetic signaling and IK,Ach activity in the
prevention and treatment of AF.
11
Figure 1-1. Re-entry model of atrial fibrillation. (A) Re-entry requires a trigger for initiation
(i.e. ectopic activity) and a vulnerable “substrate” for maintenance. The fundamental feature is
the wavelength which is the distance travelled by the depolarizing wave in one refractory period
and can be derived as the product of action potential duration (APD) and conduction velocity
(CV). (B) If the path length is smaller than the wavelength of the depolarizing wave, the impulse
will travel the circuit and return to its starting point in a time that allows it to contact tissue that is
refractory and the impulse will die out. However, if either the CV is slow or APD is short
enough, an ectopic trigger will produce a wavelength that travels the circuit and contacts tissue
that is no longer refractory, therefore exciting the tissue and bringing about re-entry.
12
1.2 Regulators of G protein signaling
The activation of the Gαi/o-coupled M2 muscarinic receptor by parasympathetic activity
produces a cascade of physiological changes within the cell. The timing and duration of these
changes are determined by the lifetime of the active (GTP-bound) Gαi/o subunit. In the basal
state, the GDP-bound Gα subunit is coupled to the Gβγ heterodimer and the intracellular surface
of the receptor. Receptor activation involves the exchange of GDP for GTP on the Gα subunit
which leads to the dissociation of the GTP-bound Gα subunit from the Gβγ heterodimer. At this
point, the G protein is in its “ON” state (73;74) and both the Gα and Gβγ subunits are available
to regulate the activity of downstream effector molecules, such as GIRK channels and adenylyl
cyclase (73). Signal termination occurs when GTP is hydrolyzed to GDP on the α-subunit. The
GDP-bound Gα subunit re-associates with the Gβγ heterodimer and the heterotrimeric G-protein
is now considered to be in its “OFF” state (Figure 1-2).
The intrinsic rate of GTP hydrolysis by Gα subunits (the rate-limiting step of for signal
termination) in vitro is too slow to account for the rapid recovery from G-protein-mediated
responses in vivo. Therefore, to produce the rapid ON-OFF kinetic changes needed to modulate
G-protein-mediated activity in vivo, cells require additional factors that increase the rate of GTP
hydrolysis of the Gα subunit. This class of molecules is called GTPase activating proteins
(GAPs). Regulators of G protein signaling (75;76), or RGS proteins, are a mammalian family of
>35 GAPs for Gα subunits (77). RGS proteins are defined by a shared 120-130 amino acid
domain (the RGS domain) that binds directly to the Gα subunit to enhance its intrinsic GTPase
activity by up to 2000-fold over basal levels, which decreases the amplitude and duration of both
Gα and Gβγ-mediated downstream signaling (Figure 1-2a). Binding of RGS proteins to the
activated Gα subunit can also antagonize effector activation and thereby block the generation of
13
G protein-mediated signals (Figure 1-2b). More than 30 RGS and RGS-like (containing a RGS-
homology domain) proteins have been identified which are divided into six distinct subfamilies
(78). These are the A/RZ (RGS17, 19 (GAIP), 20), B/R4 (RGS1-5, 8, 13, 16, 18, 21), C/R7
(RGS6, 7, 9, 11), D/R12 (RGS10, 12, 14), E/RA (Axin, Conductin), and F/RL (RGS-like
proteins including RhoGEFs, GRKs, AKAPs, and sorting nexins (SNXs) subfamilies. RGS
proteins that belong to the A/RZ and B/R4 subfamilies are small proteins (20-30 kDa) with a
short N-terminal domain and a C-terminal RGS domain, while members of the C/R7, D/R12,
E/RA, and F/RL subfamilies are large proteins (up to 160 kDa) that contain several additional
domains which facilitate interaction with various signalling molecules.
1.2.1 RGS proteins in the heart
Virtually all RGS proteins have been identified in the mammalian myocardium as well as
in cultured cardiomyocytes. In the human heart RGS expression was mostly studied in the left
ventricular myocardium or the whole heart. These studies showed high expression of several
RGS proteins including RGS2, RGS3, RGS4, RGS5, RGS6, RGS9, RGS11, RGS19, and
p115RhoGEF (79). Animal studies further characterized differences in expression between
specific regions of the heart. Gene expression studies revealed that RGS2, RGS3, RGS4, RGS6,
RGS10, GAIP and RGSZ2 are endogenously expressed in rat atrial cardiomyocytes while other
RGS proteins such as RGS5, RGS12, RGS16, and RGS18 were identified in atrial tissues but not
in single atrial myocytes (80;81), pointing to non-myocyte sources such as vascular smooth
muscle cells or fibroblasts. The physiological importance of RGS proteins in the heart was
illustrated using RGS-insensitive G protein knock-in cells and mice or RGS-specific knockout
mice.
14
RGS proteins were shown to play an important role in regulating the Gαq/11-mediated
effects on PLC activity, growth, hypertrophy, and contractility. Both clinical and animal studies
have shown that RGS2-deficiency leads to enhanced Gαq/11 signaling. RGS2-knockout mice
develop cardiac hypertrophy and heart failure in response to pressure overload whereas RGS2
overexpression completely abolishes the Gαq/11-induced hypertrophy. Additionally, the
expression of RGS2 is selectively down-regulated in several hypertrophic animal models.
Several other studies have shown that RGS4 inhibits endothelin-1 or PE-induced cardiac
myocyte hypertrophy and inhibits Gαq/11-mediated hypertrophy in mice (82).
Particularly relevant to this thesis is the role of RGS proteins in cardiac pacemaking and
heart rate regulation. Several papers from Neubig and colleagues demonstrated the importance of
RGS proteins in regulating parasympathetic heart rate control in the SA node. Fu et al. (83)
created a glycine to serine point mutation (G184S) in the switch I region of Gαo and Gαi2
subunits that prevents Gα-RGS binding and subsequent Gα deactivation, and introduced it into
embryonic stem cells by homologous recombination. Spontaneously contracting cardiomyocytes
derived from those stem cells were used to study the function of endogenous RGS proteins in
Gαi/o-mediated responses. Cells expressing the RGS-insensitive Gαo (GαoGS/GS) and Gαi2
(Gαi2GS/GS) subunits displayed significantly enhanced sensitivity to carbachol (M2 agonist).
Importantly, the bradycardic responses to carbachol could be completely abolished by the
specific GIRK channel blocker tertiapin-Q, suggesting that enhanced GIRK channel activation in
the absence of RGS regulation is responsible for the enhanced bradycardic response to M2
receptor activation. Similar observations were made in Gαi2GS/GS knock-in mice which
displayed markedly enhanced bradycardia in response to carbachol compared to wild-type
controls (84).
15
In a subsequent study, Fu et al. (85) examined isolated perfused hearts to determine
whether the responses in the intact animal were due to regulation of cardiac pacemakers or to
alterations in central nervous system or vascular responses. In the presence and absence of
isoproterenol, beating rates of homozygous Gαi2GS/GS hearts were more sensitive to inhibition
by carbachol than wild-type control hearts. Furthermore, in addition to the effects on the
sinoatrial node, the Gαi2GS/GS hearts showed evidence of atrioventricular conduction block.
The specific RGS proteins responsible for these effects were not identified in these
studies; however, subsequent RGS-specific knockout models implicated RGS4 and RGS6 as key
regulators of parasympathetic signaling, because their loss was associated with severe
bradycardia in response to parasympathetic signaling in vivo.
1.2.2 RGS4
RGS4, the focus of this thesis, is a member of the B/R4 subfamily of RGS proteins and
has GAP activity for the Gαi/o (Gαi2 and Gαo) and Gαq/11 but not Gαs families of G-proteins
(86;87). The ability of RGS4 to inhibit G protein signaling is tightly coupled to its plasma
membrane localization. The 33 amino acid N-terminus of RGS4 contains a cationic amphipathic
helix that is required for plasma membrane attachment and G protein-inhibitory function (88;89).
Additionally, RGS4 palmitoylation at cysteine residues 2 and 12 drives plasma membrane
localization and function (90).
RGS4 has been identified to be an important regulator of G-protein signaling in the heart.
RGS4 is expressed in isolated atrial myocytes and has been shown to form stable protein
interactions with both the M2 receptor and GIRK channels in pull-down experiments. Doupnik et
al. (91) showed that the on-and-off IK,Ach kinetics were much slower in CHO cells expressing M2
16
receptors (t1/2 ~10 s) compared to IK,Ach kinetics in native atrial myocytes (t1/2<1s). Co-expression
of RGS4 with M2 receptors in CHO cells accelerated the on-and-off IK,Ach kinetics, suggesting
that an RGS protein was necessary for the control of normal GIRK channel function in vivo.
Previous studies in our laboratory have characterized a role for RGS4 as a functionally important
negative regulator of parasympathetic signaling in the SA node (92). RGS4-deficient mice
expressing LacZ under the control of the RGS4 promoter show high expression in the SA node
relative to other cardiac tissues. Functionally, loss of RGS4 did not affect resting heart rate,
however in the presence of carbachol RGS4-deficient mice displayed markedly enhanced
sensitivity to carbachol-mediated bradycardia. Furthermore, anesthetized RGS4-deficient mice
had lower heart rates which were normalized with atropine (M2 blocker), indicating enhanced
vagal tone in these animals. Similar enhancements of muscarinic signaling were observed in
isolated perfused hearts and single SA nodal myocytes.
Importantly, the abovementioned results correlated with alterations in IK,Ach in RGS4-
deficient mice. First, RGS4-deficient SA nodal myocytes showed enhanced membrane
hyperpolarization in response to carbachol. Second, whole-cell patch clamps revealed muscarinic
stimulation had no effect on channel activation or peak current but significantly delayed IK,Ach
deactivation kinetics upon agonist removal or administration of atropine to block M2 receptors,
consistent with a role for RGS proteins in regulating GIRK channel activation and deactivation
kinetics.
More recently, RGS6 was also shown to be an important regulator of M2R-dependent
signaling in the SAN. Specifically, RGS6 ablation was associated with enhanced bradycardic
responses to carbachol in intact animals, isolated hearts, and cultured SAN cells (93;94). As was
the case for RGS4-deficiency, the phenotypes associated with loss of RGS6 appeared consistent
17
with its ability to regulate GIRK channel activity. These studies highlight potential overlapping
roles of RGS proteins.
Figure 1-2. Heterotrimeric G protein signaling and regulation by RGS proteins. G protein
activation is achieved by catalyzing GDP-GTP exchange on the α-subunit which leads to the
dissociation of the Gβγ heterodimer. The G protein is now in its “ON” state and both the Gα and
Gβγ subunits are available to regulate the activity of various downstream effectors. The signal is
turned off when GTP is hydrolyzed to GDP by the Gα subunit, and the Gα subunit re-associates
with Gβγ to generate the inactive form of the heterotrimer. (a) RGS proteins are GTPase-
activating proteins that enhance GTP hydrolysis by Gα thereby rapidly turning off G protein
signaling. (b) RGS proteins can also bind the activated Gα subunit and interfere with interaction
of downstream effectors. Image modified from Zhang and Mende (95).
18
1.3 ATP-sensitive K+
(KATP) channels
KATP channels were first discovered in the heart (96), and have since been identified in a
variety of metabolically active tissues including the brain (97), pancreas (98), smooth muscle
(99), skeletal muscle (100), kidney (101), and cellular organelles like the mitochondria (102).
The key regulatory feature of the KATP channel is strong inhibition of channel activity by
intracellular adenosine 5’-triphosphate (ATP). When intracellular ATP stores are depleted KATP
channels become activated which leads to K+
efflux and hyperpolarization of the cell membrane.
Therefore, KATP channels serve as metabolic sensors that translate changes in cellular metabolism
into changes in electrical activity and are important regulators of a variety of cellular functions
including insulin secretion from pancreatic β cells, K+ recycling in renal epithelia, excitability of
smooth muscle cells and neurons, and cytoprotection in cardiac and brain ischemia.
1.3.1 KATP channel structure and modulation by nucleotides
KATP channels are composed of two distinct subunits, the pore-forming Kir6 (K+ inward
rectifier) subunit and the SUR (sulfonylurea receptor) regulatory subunit, that assemble together
in a 4:4 stoichiometry to form a functional channel. Two genes KCNJ8 (Kir6.1) and KCNJ11
(Kir6.2) encode the pore-forming subunits (103), while two SUR genes, ABCC8 (SUR1) and
ABCC9 (SUR2), encode the regulatory subunits (104;105). Alternative RNA splicing give rise to
two splice variants of SUR2, SUR2A and SUR2B, which have characteristic physiologic and
pharmacologic properties (106). At the genomic level, the genes encoding SUR1 and Kir6.2, and
SUR2 and Kir6.1 are adjacent to each other on 11p15.1 (103) and 12p12.1 (105;107),
respectively, suggesting that there may be a coordinated regulation of SUR and Kir6 subunits at
the gene level.
19
Similar to other members of the inwardly rectifying potassium channel family, the Kir6
subunits have two transmembrane domains (TM1 and TM2) that are linked by a pore loop, and
intracellular NH2 and COOH terminal domains. Binding of ATP to Kir6 inhibits channel activity
by stabilizing the closed state. ATP decreases mean burst duration and increases interburst
intervals without effect on single channel conductance (108). Site-directed mutagenesis has
identified key residues in the N-terminal and C-terminal domains that are required for ATP
binding and therefore inhibition by ATP. Potential ATP-interacting residues include Arg-50 in
the N-terminal and Ile-182, Lys-185, Arg-201 and Gly-334 in the C-terminal (109-112). An
endoplasmic reticulum retention signal is also present on the C-terminal side of Kir6.2 that
prevents trafficking to the plasma membrane in the absence of SUR (113).
SUR is a member of the ATP-binding cassette family and is believed to contain 17
transmembrane helices arranged in three groups (TMD0, TMD1, and TMD2) and connected by
cytoplasmic linkers. The N-terminal domain, TMD0, contains 5 transmembrane helices while
TMD1 and TMD2 each contain 6 transmembrane helices. Additionally, SUR subunits contain an
extracellular NH2 terminal, intracellular COOH terminal, and two nucleotide-binding folds
(NBF-1 and NBF-2). NBF-1 is located between TMD1 and TMD2, while NBD-2 is located after
TMD2. In the presence of Mg2+
, ATP and ADP stimulate channel activity (114-116), an effect
which is mediated by both NBFs that have binding sites for MgADP and MgATP. The SUR
subunit also determines sensitivity of the channel to potassium channel openers and blockers
(117). For instance, all SUR isoforms are inhibited by glibenclamide, while tolbutamide blocks
channels containing the SUR1 but not the SUR2 isoform. Furthermore, diazoxide is an effective
activator of SUR1 and SUR2B, but not SUR2A, while cromakalim and pinacidil can activate
SUR2A and SUR2B, but not SUR1.
20
1.3.2 Regulation of KATP channel activity
In addition to being regulated by intracellular ATP and other nucleotides, accumulating
evidence suggests that KATP channels are modulated by hormones, neurotransmitters, and
intracellular effector molecules such as G proteins, membrane phospholipids, and protein kinases
through direct interaction with Kir6 and SUR subunits. For instance, KATP channel activity was
shown to be enhanced by several G proteins including Gαs, Gαo, Gαi-1, and Gαi-2, however it
remains to be determined which subunit, Gα or Gβγ, mediates this effect. Ito and colleagues
(52;118) first reported the ability of G proteins, especially inhibitory G proteins (Gαi), to activate
KATP channels. Under inside-out patch conditions, application of purified Gαi1 and Gαi2 proteins
increased channel open probability in guinea pig ventricular cells following the application of
acetylcholine (1 µM), an effect which could be reversed by glibenclamide. Application of Gβγ
had no effect on KATP channel activity, but generated a K+
current that could be blocked by
treatment with tertiapin-Q, demonstrating the activation of IK,ACh by Gβγ heterodimers.
Furthermore, Terzic A et al. (119) demonstrated that G protein subunits, Gαo, Gαi1, and Gαi2,
activate cardiac KATP channels by antagonizing ATP-induced inhibition of the channels, but have
no effect in the absence of ATP. Furthermore, Wada et al. (120) demonstrated that intracellular
application of Gβγ-2 decreased ATP-sensitivity of Kir6.2/SUR2-containing KATP channels
through a direct interaction with the C-terminal domain of SUR2.
Protein-kinase mediated phosphorylation of Kir6 and SUR subunits is also an important
mechanism whereby KATP channel activity can be modulated. For example, in COS-1 cells
expressing SUR1 and Kir6.2, the KATP channel is directly phosphorylated by PKA and Gαs-
coupled receptor stimulation which increases channel activity by increasing single channel open
probability and the number of functional channels present at the plasma membrane (121). ATP-
21
sensitivity and channel conductance were not affected. Protein kinase C (PKC) has been shown
to both activate and inhibit KATP channels containing the Kir6.2 subunit depending on the
intracellular ATP concentration. Light and colleagues (122) showed, using single-channel inside-
out recordings, that PKC-mediated phosphorylation of Kir6.2 at threonine-180 has a dual action
on KATP channels, inhibiting when ATP is high and activating when ATP is low. PKC-mediated
phosphorylation of Kir6.2 at serine-372 has been shown to downregulate KATP channels (123).
Finally, trafficking studies reveal that PKC-mediated phosphorylation can cause internalization
of the channel complex, subsequently leading to a decrease in channel activity (124).
Membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP2) and
phosphatidylinositol-4-phosphate (PIP) stimulate KATP channels by binding the Kir6.2 subunit.
Application of PIP2 directly to the intracellular side of excised membrane patches from Xenopus
oocytes resulted in an increase of KATP-mediated currents in the presence of millimolar ATP
concentrations. Conversely, treatment with phospholipase C (PLC) which is known to hydrolyze
PIP2 and thus decrease the concentration of PIP2 inhibited channel activity. Importantly, there
exists a negative coupling between PIP2, KATP channel activity, and ATP-sensitivity, such that
PIP2 increases open probability and decreases ATP-sensitivity.
1.3.3 KATP channels in the heart
Sarcolemmal KATP channels in mouse ventricular myocytes are comprised primarily of
co-assembled Kir6.2 and SUR2A proteins (125). Using mice deficient in the SUR1 gene, Flagg
et al. (126) established that SUR1 and Kir6.2 proteins are the predominant subunits underlying
KATP channels in the murine atria. Consistent with this observation, subsequent pharmacologic
studies revealed that atrial KATP channels were more sensitive to activation by diazoxide (specific
22
to SUR1>SUR2A) than pinacidil (specific SUR2A>SUR1) confirming the predominance of
SUR1 in forming the atrial channel (127). KATP channels have also been described in the SA
node (128), AV node (129), and Purkinje fibers (130), but the molecular composition of these
channels has not been extensively investigated. Han et al. (131) demonstrated that KATP channels
in rabbit SA node pacemaker cells responded to both pinacidil and cromakalim, indicating a
requirement for either SUR2B or SUR2A. A more recent study showed that mRNA expression
of SUR2B is higher than SUR2A in both the murine and canine cardiac conduction system and
the channels were more strongly activated by diazoxide than cromakalim (132).
KATP channels are among the most abundant K+
channels expressed in cardiac tissue and
it has been suggested that activation of only 1% of these channels is sufficient to shorten the
APD by approximately 50% (133). Despite this, KATP channels do not contribute significantly to
the resting membrane potential of cardiac cells and are not major regulators of heart rate or
cardiac contractility under normal physiological conditions. Although the roles of cardiac KATP
channels are incompletely understood, sarcolemmal KATP channels are thought to be important
for cardiac energy homeostasis under various forms of cardiac stress, including ischemia,
exercise, or adrenergic stimulation.
In the ventricles, KATP channels open during ischemia and their activation can be
protective. The increase in K+
conductance during metabolic challenge shortens the APD and
therefore reduces Ca2+
influx which may serve to preserve ATP stores that would otherwise be
consumed during contraction. In support of this, treatment with pinacidil during ischemia has
been shown to increase the levels of ATP and other energy stores such as creatine phosphate
(134). Notably, these effects are abolished in Kir6.2-deficient hearts which lack functional KATP
channels (135). In addition to preserving energy stores, KATP channels also regulate calcium
23
homeostasis. For instance, pinacidil prevents the development of Ca2+
overload following
ischemia/reperfusion injury (136). This is further supported by studies in Kir6.2-deficient mice
which have shown that loss of KATP leads to the development of cellular Ca2+
overload,
contractile dysfunction, and cell death. For instance, treatment with isoproterenol is associated
with increased intracellular Ca2+
, myocardial damage, and increased mortality in Kir6.2-deficient
mice but not WT control mice, due to impaired APD shortening in the absence of KATP.
Moreover, KATP channels are required for the cardioprotective effects of exercise (137). Exercise-
training increases the expression of SUR2A/Kir6.2-containing KATP channels in both rat (138)
and mouse ventricles and shortens APD in response to an increase in heart rate (139).
Furthermore, exercise training leads to enhanced Ca2+
entry, myocardial necrosis, and increased
mortality in Kir6.2-deficient hearts, consistent with a cardioprotective role for KATP channel
activation.
1.3.4 Role of KATP channels in cardiac arrhythmias
Often considered to be cardioprotective, opening of KATP channels can also promote
arrhythmias. Initial studies focussed predominantly on ventricular fibrillation resulting from
myocardial ischemia. Myocardial ischemia induces several changes in the ECG including short
QT interval and ST segment elevation or depression. These effects are ameliorated by KATP
channel blockers glibenclamide and HMR1098 and induced by pinacidil, consistent with KATP
channel activation underlying these changes (140;141). In support, data from Kir6.2-deficient
mice has shown that ST segment elevation is absent in these animals in response to ischemia
(140). Furthermore, pinacidil pretreatment increased the incidence of re-entrant ventricular
arrhythmias following ischemia/reperfusion by reducing the APD and prolonging conduction
24
time (142), while inhibition of sarcolemmal KATP channels reduces the incidence of ventricular
arrhythmias during acute myocardial ischemia and reperfusion in rats (143).
Two recent reports support the hypothesis that KATP channel activation promotes AF.
First, salt-induced hypertension was shown to produce a substrate for AF by activating KATP
channels in a mouse model (144). Atrial arrhythmia inducibility in high salt-fed mouse atria
could be reversed by treatment with glibenclamide and tolbutamide which ameliorated APD
shortening. SUR1 protein expression was also increased in the right and left atrial appendages of
HS mice, but no changes in Kir6.2 expression were observed. Second, Kim et al. (145) recently
demonstrated that opening of KATP channels in the atria during β-adrenoceptor-mediated
metabolic stress generates a pro-arrhythmic substrate. Isoproterenol decreased atrial effective
refractory period, conduction velocity, and wavelength of excitation, and increased arrhythmia
inducibility. Glibenclamide lengthened the wavelength and reduced arrhythmia inducibility in
the presence of isoproterenol. Furthermore, 30 minute perfusion with isoproterenol was shown to
deplete cellular ATP levels in the left atria but not ventricles.
Currently, very little is known about the activity of KATP channels during human AF.
Brundel et al. (146) found variable expression changes in the Kir6.2 subunit in atrial samples of
patients with paroxysmal or chronic AF. In paroxysmal AF, Kir6.2 subunit expression was
increased, whereas a decrease in Kir6.2 expression was observed in chronic AF (146). Wu et al.
(147) showed an increase in IK,ATP amplitude under ischemic conditions in atrial cells from
patients with chronic AF, while another group reported a decrease in KATP channel activation
with rimakalim (KATP opener) in atrial cells isolated from AF patients compared to patients in
normal sinus rhythm (148). These studies underscore the complexity of KATP channel activity in
different forms of clinical AF.
25
2 Rationale
RGS4 is highly and selectively expressed in the sinoatrial node and shows GAP activity towards
Gαi/o and Gαq subunits. Furthermore, RGS4 inhibits M2 muscarinic receptors and GIRK channel
activity and has been shown to regulate heart rate control in the sinoatrial node. Previous studies
from our laboratory have demonstrated that atria isolated from RGS4-deficient mice are more
sensitive to the formation of re-entrant rotors as compared to wild-type littermate controls,
particularly in the presence of parasympathetic stimulation. Furthermore, mRNA expression of
Kir6.2 and SUR1 is up-regulated in elderly (>75 week) RGS4-/-
mice compared to young RGS4-/-
mice and age-matched WT controls. Similarly, in chronic atrial tachypacing in a canine model of
AF caused a down-regulation of RGS4 mRNA expression, while Kir6.2 expression was
increased. These data suggest that up-regulation of Kir6.2 may contribute to enhanced
hyperpolarizing currents in fibrillating atrial myocytes.
3 Hypothesis
In light of the previously described observations, I will test the hypotheses that (1) altered
expression of RGS4 modifies the atrial syncytium in a manner that enhances susceptibility to
atrial fibrillation and (2) KATP channel activity is increased under conditions of decreased RGS4
function and contributes to atrial arrhythmia vulnerability.
26
4 Materials and Methods
4.1 Experimental Animals
The Rgs4tm1Dgen
/J mouse strain as obtained from Jackson Laboratory (Bar Harbor, Maine,
http://jaxmice.jax.org/strain/005833.html) and backcrossed >6 generation onto a C57Bl/6
background. RGS4+/-
mice were intercrossed to generate RGS4-/-
and RGS4+/+
mice. 16-to 20-
week old male and female mice were used for in vivo and ex vivo studies, respectively. In order
to study the contribution of KATP channels to arrhythmia susceptibility, we crossed RGS4-/-
mice
with Kir6.2-/-
mice (obtained courtesy of Dr. Jean-Marc Renaud), generated by targeted
disruption of the KCNJ11 gene, which encodes the pore-forming subunit of the KATP channel
complex. Experimental mice were double transgenic RGS4-/-
or RGS4+/+
with either wild-type
(Kir6.2+/+
) or knockout (Kir6.2-/-
) Kir6.2 expression. All mice were house in standard vented
cages in temperature and humidity controlled rooms with 12-hour light-dark cycles in the
Department of Comparative Medicine animal facility at the University of Toronto. All
experiments conformed to the guidelines set by the Canadian Council on Animal Care.
4.2 In vivo Intracardiac Electrophysiology
4.2.1 Intracardiac catheterization
16-to 20-week old male mice were anesthetized with 1.5% isoflurane inhalation using a
Benson isoflurane chamber and placed on a regulated heating pad to maintain core body
temperature at 36-37 °C. The surface two-lead ECG was continuously recorded from
subcutaneous 30-gauge electrodes (F-E7, Grass Technologies, West Warwick, RI, USA).The
27
parameters derived from the ECG measurements include: heart rate (HR), R-R interval, P-R
interval (beginning of P wave to the beginning of the QRS complex), and P wave duration.
The right jugular vein was then isolated under a dissecting microscope by blunt
dissection and the vein was ligated rostrally using 5-0 silk sutures and a loose ligature was placed
at the caudal end. Micro-dissecting scissors were used to make a small incision in the vessel
between the sutures and a 2F octapolar electrode electrophysiology catheter (CIBer Mouse EPO
Catheter, NuMed Inc., Hopkinton, New York) was guided into the right atrium, through the
tricuspid valve, and into the right ventricle. Each electrode was 0.5 mm in length with a 0.5 mm
interelectrode distance. The distal electrodes were used for sensing the right ventricle and the
proximal electrodes were used for sensing and pacing the right atrium. Correct catheter
placement was confirmed by having a sole ventricular signal in the distal lead, a His bundle
signal in the middle lead, and a predominant atrial signal in the proximal lead. All body surface
and intracardiac ECG signals were recorded and amplified at 5 kHz and filtered between 0.3 and
300 Hz using a Gould ACQ-7700 amplifier and the Ponemah Physiology Platform software
(version 4.60, Data Sciences International).
4.2.2 Electrophysiology study
Inducibility of atrial arrhythmias was assessed using a decremental burst pacing protocol
that consisted of a 20 stimulus drive train at a cycle length of 40 ms with 2 ms decrements to 10
ms. Triple extra-stimulations at a coupling interval of 20 ms were also tested. All protocols were
repeated twice at baseline and 5 minutes after cumulative intraperitoneal (ip) administration of
10 ng/g and 30 ng/g carbachol. AF was defined as rapid and irregular atrial rhythm with irregular
ventricular activation that lasted at least 5s.
28
4.3 High-Resolution Optical Mapping and Analysis
4.3.1 Isolated atrial preparation
16-to 20-week old female mice were administered a 0.2 mL intraperitoneal injection of
heparin (1000 IU/mL, LEO Pharma, Thornhill, Ontario, Canada) 5 minutes before being
anesthetized with 1.5% isoflurane (Halocarbon Laboratories, River Edge, NJ, USA) and killed
by cervical dislocation. The heart was immediately excised and placed in Tyrode’s solution
(35ºC) consisting of (in mmol/L) 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 1.0 MgCl2, 1.8 CaCl2, 5.55
glucose, and 5 HEPES, with pH adjusted to 7.4 with NaOH. The lungs, thymus, and ventricular
tissue were dissected away, leaving a preparation consisting of the interconnected left and right
atria. The superior and inferior vena cavae were cut open and the tissue was pinned to the bottom
of a Sylgard-coated 30-mm petri dish. The dye 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-
1-(3-sulfopropyl)-pyridinium) hydroxide, inner salt (di-4-ANEPPS; Molecular Probes, Eugene,
OR) was added to the Tyrode’s solution and the tissue was incubated for 10 min. The tissue was
then transferred to a Sylgard-coated 3.5 mm petri dish and superfusion with warm Krebs solution
of the following composition: 118.0 mM NaCl, 4.7 mMKCl, 1.2 mM KH2PO4, 1.2 mM MgSO4,
1.0 mM CaCl2, 25.0 mM NaHCO3, 11.1 mM glucose, was then started at a constant flow rate
which was sufficient to maintain the bath temperature to within 1°C of the desired recording
temperature (37°C). The temperature in the bath near the preparation was continually recorded
throughout the experiment using a thermocouple thermometer (Harvard Apparatus).The solution
was continuously bubbled with 95% O2-5% CO2 resulting in pH 7.4. The Kreb’s solution passed
through a glass heating coil (RadnotiGlass Technology, Monrovia, CA) immediately before
entering the superfusion bath.
29
Ex vivo ECGs were recorded simultaneously with optical imaging by placing platinum
needle electrodes (F-E7, Grass Technologies, West Warwick, RI, USA) in close proximity to the
atrial preparation. The positive electrode was positioned at the right atrial appendage (RAA), the
negative electrode was positioned near the inferior vena cava, and the ground electrode was
positioned at the left atrial appendage. ECG data was acquired and analyzed using Axoscope
10.2 software and an Axopatch200B amplifier (Axon Instruments, Foster City, CA). Recordings
were acquired at a sampling rate of 1000 Hz and stored for off-line analysis. A photograph of a
typical experimental setup is shown in Figure 4-1A.
4.3.2 Imaging system
Illuminating light, provided by a 120-W quartz mercury lamp light source (X-cite exacte,
EXFO Life Sciences, Mississauga, ON, Canada) is passed through a selection filter and reflected
off a dichroic mirror (Olympus) onto the atrial preparation. The fluorescence emitted from the
preparation passes through the dichroic mirror and a Schott glass filter (>590 nm; MellesGriot,
Ottawa, ON, Canada) before reaching the camera. A shutter (EXFO) is used to ensure the
preparation is exposed to light only during image acquisition. A high speed, high resolution
camera (model Cascade 128+, Photometrics, Tucson, AZ) was used in its binning mode (64x64
pixels, 16 bit resolution, 1000 frames/s). The camera was equipped with a 25 mm focal length
lens (Computar, Commack, NY) and a 5 mm spacer, resulting in a 10x10 mm field of view. The
image acquisition software PTI ImageMaster 3.0 (Media Cybernetics, Bethesda, MD) and the
camera were connected to a personal computer running the Windows XP operating system
(Microsoft, Redmond, WA) by an image acquisition board (model PCI-1422, National
Instruments).
30
4.3.3 Experimental protocols
Programmed electrical stimulation was performed using a platinum electrode positioned
atop the right atrial appendage, adjacent to the sinoatrial node. Increasing logarithmic doses of
carbachol (CCh), between 0.01 μM and 3 μM, were added to the superfusate Kreb’s bath. After
each dose, arrhythmia induction was attempted using the following decremental burst pacing
protocol: 20 train pulses for 2 ms each separated by 40 ms, repeated with 2 ms decrements to 10
ms. The entire protocol was repeated once and atrial fibrillation was deemed to occur if a rotor
formed and continued following the stimulation for ≥5s. The contribution of IK,ATP to APD and
arrhythmia susceptibility was evaluated in a separate group of wild-type and RGS4-knockout
mice after perfusion with Kreb’s solution containing 300 μM tolbutamide (Sigma-Aldrich),
which was followed by the CCh dose-response.
4.3.4 Data processing and analysis
Data were processed offline using the PTI ImageMaster 3.0 software. The data recorded
for each pixel were processed as follows: 1) subtraction of background fluorescence, 2) linear
trend removal (trend estimated by least-squares fitting a straight line to the data), and 3) sign
change, so that a depolarization corresponded to a positive change in the signal. Regional APD at
70% repolarization (APD70) was measured offline from optically-derived action potentials using
software written in Strawberry Perl. Four-pixel regions were selected and analyzed throughout
the 5 second recording period. Analogous points were used to compare within and between the
same preparations. Action potentials at individual pixel locations affected by remaining motion
artefact or unusually low signal-to-noise ratio were excluded on the basis of the following
criteria applied to the signal-averaged, but unfiltered, signal: 1) the signal must remain at 30% of
31
maximum amplitude until 15 ms before the detected activation (maximum rate of rise) time (to
exclude signals with unstable baseline and/or large noise), 2) the signal must reach ~90% of
maximum amplitude within 10 ms after the activation time (to exclude signals with very slow
upstrokes), and 3) the signal must return (and remain) to > 30% of maximum amplitude by 80
ms after the activation time (to exclude signals with unstable baseline, large noise, or
unreasonably long apparent APDs due to motion artefacts). APDs were then computed as the
intervals between the activation time and the repolarization time detected for each pixel. The
analysis software allows the user to select regions corresponding to anatomic features and
computes summary statistics for action potential durations (APDs) within each selected region.
Two regions were used to facilitate statistical comparisons within and between preparations
throughout the study: 1) the right atrial appendage (RAA), the right atrial tissue anterior to the
sulcus terminalis and 2) the left atrial appendage (LAA), the trabeculated left atrial tissue
anterior to the narrow junction separating the appendage from the smooth-walled part of the left
atrium receiving the pulmonary veins.
Frequency analysis of optical and electrical recordings was performed to assess
arrhythmia dynamics. Fast Fourier transform (FFT) was performed on the electrical recordings to
determine the dominant frequency (DF) and the organization index (OI) of the arrhythmia
episodes. The largest peak of the resulting magnitude spectrum was identified as the DF. The OI
was calculated as the ratio of the area under the dominant peak and the total area of the spectrum.
The total area of the spectrum was calculated from 2 Hz up to 60 Hz. Higher frequencies were
not included because they were deemed to exceed the physiological range of frequencies for AF
rotors. Optical recordings were also used to generate dominant frequency (DF) maps and
evaluate regional differences in DFs. Pixels were selected in both the right and left atrial
32
appendages and the power spectrum of the time series for each pixel was calculated using FFT.
Average DF was calculated as the mean value of all pixels during an arrhythmia episode.
4.4 Microelectrode-based measurement of atrial myocyte APD
The isolated atrial preparation was used as for optical mapping. Action potentials were
gathered using fine tip glass microelectrodes in order to validate the optically-derived action
potentials. Micropipettes were pulled from borosilicate glass capillaries (with filament, 0.50 mm
inner diameter, 1.0 mm outer diameter, Sutter Instruments, Novato, CA) with a Flaming/Brown
micropipette puller (Model P-87, Sutter Instruments). The micropipettes were filled with 3M
KCl and mounted on a silver-silver chloride electrode. A micromanipulator was used to precisely
position micropipettes. Individual atrial myocytes were impaled with microelectrodes and
electrical activity was recorded with a patch-clamp amplifier (Axopatch200B; Axon Instruments,
Foster City, CA). Action potentials were recorded from at least three different regions in each of
the right and left atrial appendages. Recordings were acquired at a sampling rate of 1000 Hz and
stored on a computer with Axoscope 10.2 for off-line analysis. APD was measured offline using
software written in Strawberry Perl as described previously.
4.5 qRT-PCR Reaction and Quantitative Analysis
Hearts were excised from 20-week-old WT and RGS4-/-
male mice which had been killed
by cervical dislocation. The atria were carefully dissected from the ventricles as described above
(Methods 4.3.1) and flash-frozen in liquid nitrogen for further RNA isolation. Total RNA from
three pools of two mice was extracted using TRIzol reagent (Invitrogen Life Technologies). All
quantitative RT-PCR was performed using ABI Prism 7900 HT (Applied Biosystems) and the
33
SYBR Green detection system. Five hundred nanograms of total RNA was reverse transcribed
with random hexamer primers using the Superscript II kit (Invitrogen Life Technologies)
following the manufacturers protocols. cDNA was diluted to a final concentration of 1 ng/µL.
Two microliters of the RT reaction mixture was subsequently used as a template for real time
PCR quantification. The following primers were used for the detection of KATP channel subunits:
SUR1: upstream 5’-GCCTTCGTGAGAAAGACCAG-3’, downstream 5’-
GAAGCTTCTCCGGTTTGTCA-3’; SUR2A/B: upstream 5’-ATCATGGATGAAGCCACTGC-
3’, downstream 5’-AACGAGGCAAACACTCCATC-3’; Kir6.2: upstream 5’-
TTGGAAGGCGTGGTAGAAAC-3’, downstream 5’-GGACAAGGAATCTGGAGAGAT-3’,
Kir6.1: upstream 5’-ACCAGAATTCTCTGCGGAAG-3’, downstream 5’-
GCCCTGAACTGGTGATGAGT-3’. Each cDNA sample was evaluated for KATP channel
subunits, and GAPDH and 18S were used as housekeeping genes to serve as normalizing
controls in independent wells. A no-RT and no template control sample were included for each
primer set. Data obtained from the PCR reaction were analyzed using the comparative CT
method (User Bulletin No. 2, Perkin Elmer Life Sciences). The CT for each sample was
manipulated first to determine the ΔCT [(average CT of sample triplicates for the gene of
interest) – (average CT of sample triplicates for the normalizing gene, 18S)] and second to
determine the ΔΔCT [(ΔCT sample)-(ΔCT for the calibrator sample)]. The internal calibrator
sample (Kir6.2 mRNA from WT atria) was run concurrently on the same plate and designated as
an external control. Values are expressed in log scale and the relative mRNA levels are
established by conversion to a linear value using 2−ΔΔCT. Data represent the relative mRNA levels
for each KATP channel subunit in these tissues.
34
4.6 Statistical Analysis.
Values are expressed as means ± s.e.m. Two-way ANOVA followed by Bonferonni post-
hoc test was used for comparisons between genotypes and multiple carbachol concentrations.
When needed, unpaired student’s t-test was performed. Discrete data was analyzed using
Fisher’s exact test. Statistical significance was defined as P<0.05. Statistical tests were
performed using GraphPad Prism 5.0 software.
35
Figure 4-1. Optical imaging experimental setup. Figure shows photograph of isolated mouse
atrial preparation during a typical experiment (A) and optical imaging system (B). The
preparation was oriented so that the right atrial appendage (RAA) was on the left side of the
image. Anatomical features [the superior (SVC) and inferior (IVC) vena cava and crista
terminalis] are marked by dashed lines; LAA, left atrial appendage. Arrhythmia induction was
performed using a platinum electrode positioned on the RAA adjacent to the SA node. Ex vivo
ECGs were acquired simultaneously with optical mapping by placing electrodes in close
proximity to the atrial preparation. The positive electrode was positioned near the RAA and the
negative electrode was placed at the IVC.
36
5 Results
5.1 In vivo phenotype of RGS4-deficient mice
RGS4 is highly and selectively expressed in the murine SA node compared to other
cardiac tissues and has been shown to be an important regulator of parasympathetic signaling and
heart rate control (92). To characterize the effect of RGS4 ablation on cardiac function,
electrophysiology experiments were conducted on anesthetized RGS4-knockout (RGS4-/-
) and
wild-type (WT) mouse hearts at baseline and following cholinergic stimulation with carbachol.
Electrocardiographic parameters were measured using surface ECG and intracardiac
electrograms which were obtained by passing an octapolar catheter into the right ventricle
through the jugular vein.
5.1.1 Altered atrial conduction in RGS4-deficient mice
According to the representative surface ECG and intracardiac electrogram traces depicted
in Figure 5-1, mice lacking RGS4 (3 of 14, 21%) showed evidence of a wandering pacemaker,
either absent or negative P waves in lead II, and beat-to-beat changes in the P-R interval under
control conditions, otherwise absent in WT mice. The presence of a wandering pacemaker
indicates that the leading pacemaker site shifts between different regions in the atria, and may
indicate the presence of sinus node dysfunction in some animals lacking RGS4. Given that RGS4
has been shown to be a modulator of parasympathetic signaling, we investigated the effect of
cholinergic stimulation with carbachol (CCh) on ECG morphology. The average R-R interval
was not different between WT and RGS4-/-
mice under control conditions. However, in the
presence of CCh (30 ng/g), R-R interval was prolonged (P<0.01) in RGS4-/-
(133 ± 1.8 ms)
37
compared to WT (122 ± 3.3) controls, indicating slower heart rate in RGS4-/-
mice in response to
parasympathetic stimulation (~10% decrease in HR with carbachol). CCh injection, over the
dose range used in this study, had no effect on P-R interval or P wave duration. A detailed
summary of the ECG parameters obtained from WT and RGS4-/-
mice at baseline and following
CCh stimulation is provided in Table 5-1.
Figure 5-1. Anesthetized RGS4-deficient mice show episodes of wandering pacemaker at
baseline. (A) 3 of 14 RGS4-deficient (RGS4-/-
) mice displayed evidence of wandering
pacemaker, either absent or negative P waves, and shortened PR intervals. Representative
surface ECG and intracardiac electrogram recordings of anesthetized WT and RGS4-/-
mice are
shown. (∆, P wave; ▼, QRS complex). Magnified view of short PR interval is shown in the
inset. (B) Summary of PR interval variance at baseline in WT and RGS4-/-
mice (n = 3 per group,
*P<0.05 compared to WT determined by unpaired Student’s t test). (C) Average PR intervals at
baseline during wandering pacemaker activity in anesthetized mice. (n = 3 per group, *P<0.05
compared to WT littermates). Values are mean + SEM.
38
Table 5-1. Intracardiac ECG parameters in anesthetized WT and RGS4-/-
mice at baseline
and following bolus carbachol injection.
RR, R-R interval; HR, heart rate (bpm); PR, P-R interval during sinus rhythm measured from
start of P wave to start of QRS complex; Pwidth, P wave duration. Repeated measures two-way
ANOVA with a Bonferonni’s post-hoc test was used to determine statistical significance of ECG
parameters between groups at baseline and with carbachol. Values are mean (ms) ± SEM. n = 8
for wild-type (WT); n = 9 for RGS4-knockout (RGS4-/-
). *P<0.05 vs. WT + carbachol (30 ng/g)
controls, †P<0.05 vs. baseline, ‡P<0.05 vs. WT + carbachol (10 ng/g).
Baseline Carbachol (10 ng/g) Carbachol (30 ng/g)
WT RGS4-/-
WT RGS4-/-
WT RGS4-/-
RR 121.3 ± 2.6 121.5 ± 1 124.8 ± 3 134.8± 3.2† 122 ± 3.3 133 ± 1.8†*
PR 39.3 ± 0.6 39.0 ± 1 39.6 ± 1 39.7 ± 0.8 38.0 ± 1.3 38.8 ± 0.5
Pwidth 16.5 ± 0.3 15.2 ± 1 16.6 ± 1 16.0 ± 1 16.3 ± 1 15.1 ± 1
HR 494 ± 10 494 ± 5 479 ± 11 449 ± 10.3† 490 ± 13 449 ± 5†*
39
5.1.2 Cholinergic stimulation in vivo provokes atrial arrhythmias in RGS4-/-
mice
Using an octapolar EP catheter, brief, non-sustained episodes (<5s) of atrial arrhythmias
could be induced in WT (3 of 12) and RGS4-/-
(4 of 13) mice at baseline. CCh injection (30 ng/g,
ip) increased atrial arrhythmia susceptibility with decremental burst pacing, which provoked
sustained arrhythmias in 10 of 13 RGS4-/-
and 2 of 12 WT mice (P=0.0048). Furthermore, CCh
injection prolonged arrhythmia duration with only 2 of 14 RGS4-/-
mice developing sustained
atrial arrhythmias at baseline, whereas 10 of 13 mice had sustained atrial arrhythmias after CCh
administration (P=0.0018). In the presence of CCh, mean arrhythmia duration in RGS4-/-
mice
was 122 ± 38 sec compared to 83.5 ± 57 sec in WT mice. A detailed summary of the incidence
and duration of atrial arrhythmias is provided in Table 5-2. The arrhythmias were characterized
by a loss of regular P waves and increased R-R interval variance on the surface ECG, while the
intracardiac electrograms displayed rapid and irregular atrial activity (Figure 5-2). Furthermore,
spectral characterization of intracardiac atrial electrograms revealed an arrhythmia with an
intrinsic dominant frequency (DF) of 48.2 ± 1.3 Hz in RGS4-/-
mice. In contrast, atrial
arrhythmias in WT atria were characterized by a lower DF (40.9 ± 4.2 Hz). These data confirm
our previous observation that loss of RGS4 activity is associated with increased atrial arrhythmia
vulnerability in vivo under parasympathetic challenge (149).
40
Table 5-2. Incidence and duration of atrial arrhythmias in anesthetized WT and RGS4-/-
mice.
Values are mean ± SEM. *P = 0.0048 vs. WT + carbachol (30 ng/g); †P = 0.0018 vs. baseline,
determined using Fisher’s exact test.
Baseline Carbachol (10 ng/g) Carbachol (30 ng/g)
< 5s > 5s < 5s > 5s < 5s > 5s
WT Incidence 3/13 0/13 4/13 0/13 2/12 2/12
Duration (s) 1.36±0.8 n/a 2.4±0.4 n/a 1.74±0.4 83.5±57
RGS4-/-
Incidence 4/14 2/14 2/14 4/14 2/13 10/13*†
Duration (s) 2.4±1 7.7±0.5 2.1±1 85.6±54 1.46±0.4 122±38
41
Figure 5-2. Induction of atrial arrhythmias in WT and RGS4-/-
mice. (A) Representative
surface ECG and intracardiac electrogram recordings showing normal sinus rhythm at baseline
(∆, P waves; ▼ QRS complex). (B) Induction of sustained atrial arrhythmia following bolus
carbachol injection (30 ng/g ip) and decremental burst tachypacing (ATP, solid bar). Note the
rapid atrial rate seen on the intracardiac electrogram and the irregular ventricular response seen
on the surface ECG. Magnified view of surface ECG is shown in the inset. iECG, intracardiac
electrogram; sECG, surface ECG.
42
5.2 Ex vivo phenotype of RGS4-deficient atria
To further examine the effects of RGS4 ablation on cardiac conduction and further
explore the mechanism underlying atrial arrhythmogenesis ex vivo, I performed optical mapping
studies of isolated atrial preparations using the fluorescent potentiometric dye, di-4-ANEPPS.
This dye allows for the visualization of the spread of membrane depolarization as well as the
spatiotemporal properties of repolarization to map spontaneous pacemaker activity.
5.2.1 Spontaneous pacemaker activity is altered in RGS4-/-
atria
Under control conditions, atrial activation in WT tissues originated from a site in the right
atrium near the superior vena cava (SVC), as expected from the known anatomic location of the
SA node in the mouse heart (14). The activation wavefront then spread quickly toward the right
and left atrial appendages. This pattern of activation was very consistent between WT
preparations (14 of 17); however, in 3 of 17 (18%) preparations, multiple competing pacemakers
were observed resulting in beat-to-beat changes of the leading pacemaker site between the SA
node and the atrioventricular junction (AVJ) region. Consistent with previous reports, addition of
CCh (≥ 100 nM) to WT atria shifted the leading pacemaker site inferiorly within the SA node or
to a position in the inferior right atrium at the junction with the inferior vena cava (IVC).
Under control conditions, the majority of RGS4-/-
atria (9 of 15, 60%) had a primary
pacemaker that had already migrated toward the AVJ region. The addition of CCh did not induce
a further change in the pacemaker site of those atria. Superfusion with the M2 receptor
antagonist, atropine, did not reverse the nodal shift phenotype (n=3, data not shown) observed in
the RGS4-/-
atria.
43
5.2.2 Cholinergic stimulation promotes rotor formation in RGS4-/-
atria
To induce arrhythmias in isolated atria, rapid electrical stimulation was applied via a
platinum electrode positioned adjacent to the SA node on the RAA. Arrhythmias were identified
on the optical images by the appearance of altered waves of depolarization that behaved as self-
sustaining rotors (Figure 5-3A), and a high frequency atrial signal on the ECG (Figure 5-3B).
Arrhythmia inducibility was assessed at baseline and following the administration of increasing
logarithmic doses of CCh. Figure 5-3C shows the number of atria that developed re-entry rotors
lasting more than 5 seconds following atrial tachypacing, under control conditions and following
superfusion with CCh. Rapid atrial pacing induced re-entrant rotors in 1 of 9 WT and 3 of 10
RGS4-/-
atria at baseline. Importantly, pacemaker shift was observed to occur in 100% of atrial
preparations that developed arrhythmias at baseline. Furthermore, we found an association
between pacemaker migration and baseline arrhythmia susceptibility, irrespective of mouse
genotype (Table 5-3). These data may suggest an important role for pacemaker dysfunction in
atrial arrhythmia induction in the absence of parasympathetic challenge. CCh increased
arrhythmia inducibility in both groups; however, significantly more (P=0.0198) atrial
arrhythmias could be induced in RGS4-/-
preparations (9/10) compared to WT preparations (3/9)
at 300 nM CCh.
In RGS4-/-
atria, the arrhythmia is maintained by re-entrant rotors which are exclusively
anchored in the right atrium with fibrillatory conduction spreading toward the left atrium.
Therefore, we assessed arrhythmia dynamics using dominant frequency maps calculated from
optical recordings. Spectral characterization of single pixel recordings revealed an intrinsic
periodic behaviour with a DF of 37 ± 3.6 Hz in the RAA, while the LAA was being activated at a
mean frequency of 23.2 ± 3.1 Hz. An example of a right to left frequency gradient as observed in
44
RGS4-/-
atria is illustrated in Figure 5-4 which shows optical recordings and representative DF
maps of the LAA and RAA in which the different colors represent DF domains, together with
their corresponding values. These results are consistent with the presence of altered
refractoriness leading to conduction block in regions of the SA node that fail to allow 1:1
conduction from the dominant rotor in the right atrium.
45
Figure 5-3. Incidence of rotor formation in WT and RGS4-/-
atria. (A) Time sequence
showing one cycle of a sustained re-entrant rotor (red arrow) event obtained by optical mapping
of a di-4-ANEPPS-loaded atrial preparation isolated from an RGS4-deficient mouse. The image
is oriented so that the right atrial appendage is on the right side of the image. (B) Ex vivo
electrogram recording showing induction of re-entrant rotor following burst atrial pacing (solid
bar). (C) Table shows percentage of atria that developed re-entry rotors lasting at least 5 seconds
following rapid atrial pacing at baseline and following carbachol administration in wild-type
(WT) and RGS4-deficient (RGS4-/-
) atria. Note that 1 of 9 WT and 3 of 10 RGS4-/-
atria
developed rotors in the absence of carbachol. WT, n = 9; RGS4-/-
, n = 10. *P=0.0198 vs. WT
controls.
46
Table 5-3. Contingency table showing pacemaker shift is a predictor of baseline
arrhythmia vulnerability in isolated atrial preparations.
Pearson Chi-square test was used to verify the association between baseline pacemaker shift and
atrial arrhythmia inducibility.
Observed Atrial Arrhythmia
Yes No Total
Predicted Atrial
Arrhythmia (SA node shift)
Yes 9 6 15
No 0 18 18
Total 9 24 33
P value = 0.0001
47
Figure 5-4. Spectral characterization of atrial arrhythmia episodes in WT and RGS4-/-
atria. The frequency of atrial activation in RGS4-/-
was higher in the right compared to the left
atrial appendages, while the frequencies were similar across those regions in WT atria. Figure
shows representative dominant frequency maps of the left and right atrial appendages with
values of dominant frequencies (in Hz) [left panel], depolarization/repolarization kinetics of a
four pixel region of interest in the left and right atrium [middle panel], and spectral frequency
plot of the dominant frequency in the right atrium. RA, right atrium; LA, left atrium.
48
5.2.3 Abbreviated AP durations in RGS4-/-
atria
The maintenance of re-entrant activity as observed in RGS4-/-
atria requires an
appropriate atrial tissue substrate with an electrical wavelength that is smaller than the physical
dimensions of the tissue involved in supporting the re-entrant path. Previous work has shown
that parasympathetic signaling increases susceptibility to AF by abbreviating atrial
refractoriness. Using conventional microelectrodes, we determined whether cholinergic
stimulation altered regional APDs in a manner consistent with increased rotor susceptibility.
Although baseline AP durations measured at 70% repolarization (APD70) were not different
between WT and RGS4-/-
atria (16.9 ± 0.49 ms vs. 17.0 ± 0.65 ms (RAA) and 14.8 ± 0.47 ms vs.
14.7 ± 0.26 ms (LAA), respectively), CCh (300 nM) significantly reduced APD70 in RGS4-/-
atria
but not in WT control atria (Figure 5-5B). In line with previous studies showing heterogeneity of
M2 receptor signaling in atria (68), the APD reductions varied across different regions of the
preparation with abbreviation of APD being most evident in the RAA. Taken together, these data
indicate that loss of RGS4 increases parasympathetic-mediated effects on the APD of atrial
myocytes and this may reduce the wavelength of excitation to facilitate arrhythmia induction.
49
Figure 5-5. Effect of carbachol on AP duration in WT and RGS4
-/- isolated atria. (A)
Overlay of representative microelectrode-derived action potentials selected from the specified
regions under control conditions (●) and after perfusion with CCh (○, 300 nM). (RAA, right
atrial appendage; LAA, left atrial appendage). (B) Summary data for action potential durations at
70% repolarization times (APD70) under control conditions and following superfusion with
carbachol at 100 nM and 300 nM concentrations. Two-way ANOVA with Bonferonni’s post-hoc
test was used to determine statistical significance of baselines and APD70 with CCh between
groups. Values are mean + SEM, n = 3 for WT atria; n = 4 for RGS4-/-
atria. *P<0.001 vs.
baseline, †P<0.01 vs. WT controls, ‡P<0.01 vs. baseline.
50
5.3 Effect of KATP on atrial arrhythmia vulnerability in RGS4-deficient mice
Once initiated, a consequence of fibrillating atrial tissues is increased intracellular Ca2+
load (150), a condition which may be resolved by KATP channels. Activation of IKATP shortens the
APD which decreases Ca2+
influx, contractile force of atrial tissues, and possibly the ATP
demand. Previous work in our laboratory has implicated KATP channels in the progression of AF
in RGS4-/-
mice. Notably, mRNA expression of Kir6.2 and SUR1 is up-regulated in elderly (> 75
week) RGS4-/-
mice and chronically paced canine atria. These data suggest that up-regulation of
Kir6.2 is induced in response to high atrial rates and may contribute to enhanced hyperpolarizing
currents in fibrillating atrial myocytes. In order to investigate the contribution of KATP channels
to arrhythmia susceptibility and rotor dynamics, we generated mice with knockdown of both
RGS4 and Kir6.2 and determined susceptibility to CCh-induced rotor formation using optical
imaging studies.
5.3.1 Loss of the Kir6.2 subunit confers protection against pacing-induced re-entry
In a second cohort of animals rapid atrial pacing induced re-entrant rotors in 2 of 8 WT
and 2 of 10 RGS4-/-
preparations under control conditions. In contrast, no atrial arrhythmias were
observed in Kir6.2-knockout (Kir6.2-/-
) or RGS4-Kir6.2 double knockout (RGS4-/-
Kir6.2-/-
) atria
at baseline and there was no evidence of pacemaker shift (n = 7 and n = 11, respectively).
Pacemaker shift was only observed to occur at CCh doses ≥ 100 nM. Table 5-4 shows the
percentage of atria that developed arrhythmias in each of the four groups of animals. CCh
administration increased arrhythmia inducibility in all groups; however, higher doses were
required to induce arrhythmias in Kir6.2-/-
and RGS4-/-
Kir6.2-/-
atria. In contrast to RGS4-/-
atria
which developed reentrant rotors when challenged with CCh and decremental burst tachypacing,
51
both Kir6.2-single and double-knockout atria predominantly developed focal tachycardias (i.e.
non-reentrant) that fired repetitively from the same location.
We next investigated the effect of Kir6.2 ablation on APD at baseline and following CCh
stimulation at 300 nM. APD70 was measured from action potentials obtained using optical
imaging. Consistent with a previous study (127), baseline AP durations were prolonged in both
the right and left atrial appendages of Kir6.2-/-
(RAA, 19.33±1.1 ms; LAA, 22.45 ± 0.74 ms) and
RGS4-/-
Kir6.2-/-
(RAA, 20.0 ± 1.2 ms; LAA, 21.7 ± 1 ms) atria compared to WT (RAA, 15.83 ±
0.65 ms; LAA, 16.68 ± 0.65 ms) and RGS4-/-
(RAA, 15.89 ± 1.1 ms; LAA, 16.15 ± 0.38 ms)
atria. Following CCh treatment, AP durations in the right atria were significantly shorter
(P<0.05) in RGS4-/-
(12.12 ± 0.61 ms) compared to WT (15.25 ± 0.77 ms) controls. A similar
effect of CCh was observed in the left atria, but this did not reach significance. Furthermore,
CCh decreased right and left atrial APD values in both Kir6.2-/-
and RGS4-/-
Kir6.2-/-
atria;
however, no differences were observed between the two groups. A detailed summary of average
APD70 values obtained from the right and left atria at baseline and following CCh administration
is shown in Table 5-5.
52
Table 5-4. Effect of Kir6.2 ablation on parasympathetic-mediated arrhythmia inducibility
in isolated atria examined by optical mapping.
WT, wild-type; RGS4-/-
, RGS4-knockout; Kir6.2-/-
, Kir6.2-knockout; RGS4-/-
Kir6.2-/-
, RGS4-
Kir6.2 double knockout; CCh, carbachol. *P=0.0055 vs. RGS4-/-
Kir6.2-/-
; ‡P=0.043 vs. WT;
†P=0.0237 vs. RGS4-/-
Kir6.2-/-
.
Table 5-5. Effect of Kir6.2 ablation on APD at 70% repolarization.
AP durations at 70% repolarization were calculated from optically-derived action potentials.
Two-way ANOVA with Bonferonni’s post-hoc was used to determine statistical significance of
APD70 at baseline and with CCh between groups. Values are shown as mean (ms) ± SEM. n = 9
for WT, n = 9 for RGS4-/-
, n = 5 for Kir6.2-/-
, n = 5 for RGS4-/-
Kir6.2-/-
. *P<0.05 vs. WT +
carbachol, **P<0.05 vs. WT control, ***P<0.01 vs. RGS4-/-
control, #P<0.01 vs. RGS4-/-
+
carbachol, †P<0.05 vs. WT control, ‡P<0.001 vs. WT control, ≠P<0.05 vs. control.
n/N (%) WT RGS4-/-
Kir6.2-/-
RGS4-/-
Kir6.2-/-
Baseline 2/8 (25) 2/10 (20) 0/7 (0) 0/11 (0)
CCh (100 nM) 3/8 (38) 8/10 (80)* 0/7 (0) 1/11 (9)
CCh (300 nM) 3/8 (38) 9/10 (90)‡† 2/7 (29) 4/11 (36)
Right atria Left atria
Control Carbachol Control Carbachol
WT 15.83 ± 0.65 15.25 ± 0.77 16.68 ± 0.65 15.4 ± 1.1
RGS4-/-
15.89 ± 0.66 12.12 ± 0.61* 16.15 ± 0.38 13.44 ± 0.48
Kir6.2-/-
19.33 ± 1.1† 15.96 ± 1.1≠ 22.45 ± 0.74‡ 16.13 ± 1.1≠
RGS4-/-
Kir6.2-/-
20.0 ± 1.2*** 16.6 ± 1.2# 21.7 ± 1*** 17.4 ± 0.89#
53
5.3.2 Effect of tolbutamide on arrhythmia vulnerability and APD
Finally, the KATP channel blocker tolbutamide was used to investigate the contribution of
KATP channels to atrial arrhythmia vulnerability in WT and RGS4-/-
atria. Tolbutamide blocks
SUR1-containing KATP channels (151) and was shown to prevent atrial tachyarrhythmia (145)
and reverse APD shortening induced by metabolic stress in rat hearts (152). During optical
imaging of atrial preparations, tolbutamide decreased arrhythmia inducibility of RGS4-/-
atria in
the presence of CCh. Superfusion with tolbutamide and CCh (300 nM) resulted in atrial
arrhythmias being induced in 2/7 (29%) atria compared with 9/10 (90%) in atria treated with
CCh alone (Table 5-6, P=0.0345). Arrhythmia inducibility of WT atria was unaffected by
tolbutamide. Consistent with results obtained from Kir6.2-deficient mice, pharmacologic
blockade of KATP channels with tolbutamide prolonged baseline APD in both WT and RGS4-/-
atria. Furthermore, in the presence of tolbutamide, CCh decreased APD in both WT and RGS4-/-
atria, with no differences observed between the two groups (Figure 5-7).
Table 5-6. Effect of tolbutamide on parasympathetic-mediated arrhythmias in isolated
atria examined by optical mapping.
Tolbutamide, 300 µM. †P = 0.0345 vs. RGS4-/-
-Tolbutamide; **P = 0.0198 vs. WT-
Tolbutamide.
- Tolbutamide + Tolbutamide
n/N (%) WT RGS4-/-
WT RGS4-/-
Baseline 1/9 (11) 3/10 (30) 0/5 (0) 0/7 (0)
CCh (100 nM) 3/9 (33) 8/10 (80) 0/5 (0) 2/7 (29)
CCh (300 nM) 3/9 (33) 9/10 (90)** 1/5 (20) 2/7 (29)†
54
Figure 5-6. Effect of tolbutamide on parasympathetic-mediated APD shortening in WT and
RGS4-/-
atria. Average APD70 values obtained from the right (A) and left (B) atrial appendages
using conventional microelectrodes. Values are mean + SEM. n = 5 for WT mice; n = 6 for
RGS4-/-
mice. *P<0.05; †P<0.01; ns, not significant. Dashed line indicates APD in RGS4-/-
atria
treated with CCh alone. RAA, right atrial appendage; LAA, left atrial appendage.
55
5.3.3 KATP channel subunit expression
Atrial tissues from young (< 20 weeks) WT and RGS4-/-
hearts were analyzed to
determine mRNA levels of KATP channel subunits using qRT-PCR. Consistent with previous
reports, we observed a greater level of expression of SUR1 compared to SUR2A/B subunits
(P<0.001), supporting the claim that SUR1 subunits are the predominant sulfonylurea receptors
forming KATP channels in the murine atrial tissues (126;127). However, no significant differences
in SUR1, SUR2A/B, Kir6.2, or Kir6.1 mRNA expression were observed in atrial samples from
young WT and RGS4-/-
mice (Figure 5-8).
Figure 5-7. Expression of KATP channel subunits in mouse atria. Relative mRNA expression
of SUR1, SUR2A/B, Kir6.2, and Kir6.2 in atria isolated from young (<20 week-old) WT and
RGS4-/-
mice was assessed by quantitative real-time PCR. KATP channel subunit expression is not
different between the two groups. Values are mean + SEM from 3 pools of two atria.
56
6 Discussion
6.1 Atrial arrhythmias in the absence of RGS4
Susceptibility to AF is strongly influenced by parasympathetic signaling. Vagal nerve
stimulation activates IK,Ach which hyperpolarizes atrial APs and decreases the APD of atrial
cardiomyocytes, an effect which is believed to be important in AF promotion. Because of its
potent inhibitory actions on parasympathetic-dependent IK,Ach, recent work has shown that RGS4
acts as an intrinsic protective mechanism against the development of AF (149). First, in an
attempt to confirm these findings, we investigated whether RGS4 ablation increases
susceptibility to AF by performing intracardiac experiments on anesthetized RGS4-/-
mice and
WT littermate controls. Indeed, we demonstrated that RGS4-/-
mice are more vulnerable to the
initiation of atrial arrhythmias, particularly in the presence of parasympathetic stimulation.
Previously, it was reported that anesthetized RGS4-/-
mice had lower baseline heart rates
compared to WT (92). Our results differ from that study in two ways. First, our heart rate results
for anesthetized WT mice are significantly lower (~500 beats/minute compared to ~600
beats/minute). Second, we did not observe any differences in the heart rate of WT and RGS4-/-
under anesthesia. We cannot explain the observed differences with respect to heart rate under
anesthesia. However, we did observe a significant reduction in the heart rate of RGS4-/-
mice
following ip carbachol injection. Thus, the enhanced susceptibility to atrial arrhythmias observed
in the absence of RGS4 might be mediated in part by parasympathetic-dependent reductions in
heart rate.
Parasympathetic activity has also been shown to modulate AV conduction. The P-R
interval, the time from the beginning of the P wave to the beginning of the QRS complex,
reflects atrial and AV nodal conduction. Our results of surface ECG recordings showed no
57
difference in P-R interval between RGS4-/-
and WT mice at baseline, and there were no
differences in the effects of CCh on the P-R interval between the two groups. This may reflect
the fact that RGS4 is not important for AV conduction in mice, consistent with its high
expression in the SA but not the AV node. However, some of the RGS4-/-
mice (21%) displayed
ECG characteristics that were consistent with a wandering pacemaker showing periodic episodes
of shortened P-R intervals and increased P-R variability in the absence of carbachol challenge.
The mechanism whereby a wandering pacemaker might contribute to atrial arrhythmia
inducibility in intact animals is currently unclear but may be of interest in future studies,
particularly in light of data from optical mapping studies discussed below. In humans, first
degree AV block causing prolonged PR intervals on the ECG has previously been associated
with increased risk of developing AF (153); however, until recently very few studies have
characterized the effect of short P-R intervals on arrhythmia vulnerability. In a follow-up to the
Copenhagen ECG study, Nielsen and colleagues (154) found that female patients with short P-R
intervals (<121 ms) were at an increased risk of developing AF; however, this risk was not
observed for male patients. Another cohort study showed a significant association between short
P-R interval and recurrent AF after adjustment for age, male sex, hypertension treatment, and
higher body mass index (155).
Our results in anesthetized mice are supported by optical mapping studies that revealed a
migration in the leading pacemaker site in excised RGS4-/-
atria. In our study we show that
pacemaker shift correlates with atrial arrhythmia induction in the absence of CCh treatment.
Specifically, atrial tissues with a leading pacemaker located at the superior cavoatrial junction
were resistant to pacing-induced arrhythmias. Moreover, 100% of atrial tissues that developed
arrhythmias at baseline showed an inferior shift in the leading pacemaker site towards the AVJ
58
region. One hypothesis for this increased susceptibility is that sinus node function is altered
when RGS4 activity is low and therefore may contribute to arrhythmia induction. However, we
cannot rule out the possibility that a migration of the leading pacemaker site is associated with
changes in the refractoriness, spontaneous depolarization, or conduction velocity in atrial
myocytes in a manner that increases arrhythmia susceptibility. Paroxysmal AF can be caused by
rapidly firing ectopic beats originating from the pulmonary veins (156;157). A study of the
pulmonary vein cardiomyocytes of patients with AF demonstrated that these cells possess altered
electrophysiologic properties (158). For instance, APDs of pulmonary vein cardiomyocytes are
shorter than left atrial APDs in patients with AF, while the opposite was observed in control
subjects, with longer APDs in the pulmonary veins. In support, rapid atrial pacing of canine
pulmonary vein cardiomyocytes decreased APD and increased spontaneous pacemaker activity
originating from those structures (159).
Alternatively, it can be argued that electrical stimulation of the right atrium (near the SA
node) may enhance the release of acetylcholine from cholinergic nerve terminals, and ultimately
contribute to the genesis of AF at baseline. Previously, Godoy et al. (160) demonstrated that
electrical stimulation of the isolated rat right atrium could reproducibly induce arrhythmias, an
effect which was completely abolished by treatment with atropine. Furthermore, stimulation of
the left atrium, which has fewer parasympathetic nerve terminals, could not induce AF in
isolated atrial preparations. Therefore, the enhanced susceptibility to AF observed at baseline
may be due to the release of acetylcholine by electrical stimulation of the right atrium.
Fibrillatory conduction in the atria is thought to be driven by (1) a single, stable re-entrant
circuit, (2) multiple, unstable re-entrant circuits, (3) or a single, rapidly firing focus. Previous
work in isolated hearts of various animal models demonstrated that re-entrant rotors were
59
consistently detected in vagally-mediated AF. In an in vitro canine right atrial preparation,
Schuessler et al. (161) demonstrated that acetylcholine-mediated AF was driven by a single re-
entrant circuit. Subsequent studies in isolated sheep atria demonstrated that a single, stable re-
entrant circuit in the left atrium could cause AF. Our optical imaging studies in RGS4-/-
atria
similarly support a single-circuit model of AF.
The appearance of rotors in vagal AF is promoted by decreases in the wavelength of the
cardiac impulse. The wavelength, given by the product of APD and CV, is the shortest path-
length that can sustain re-entry and defines the size of the re-entrant circuits. APD abbreviation
and slowing of the depolarization wave conduction velocity facilitate re-entry by shortening the
wavelength and therefore the size of the re-entrant circuit. In this study we demonstrated that in
the absence of RGS4, parasympathetic stimulation significantly abbreviates the APD.
Consequently, the abbreviated repolarization in the presence of parasympathetic modulation may
create a vulnerable pro-arrhythmic substrate. Additionally, vagal activation of IK,Ach also
generates regional differences in atrial refractoriness due to the spatially variable distribution of
vagal nerve endings and cholinergic receptors, and has been shown to render atrial tissues more
vulnerable to re-entry (67). In the present study, the APD shortening observed in RGS4-/-
atria
was more pronounced in the RAA compared to the LAA.
Previous studies have suggested that sites of DF may constitute driver regions responsible
for AF perpetuation. Therefore, we performed DF analysis in hopes that it would provide further
insight into the mechanisms of arrhythmia maintenance. Atrial arrhythmias were generally
characterized by higher DFs in RGS4-/-
atria compared to WT atria. Furthermore, power
spectrum analysis of different regions of the atrial preparation revealed the frequency of atrial
activation during periods of arrhythmia was higher in the RAA compared with the LAA. Thus,
60
the arrhythmias observed in RGS4-deficient mice appear to be sustained by a high frequency
rotor (atrial tachycardia) in the right atrium maintains the overall activity of the arrhythmia.
Since the rotors are operating at a very high frequency, and there are regional differences in
APD, it is likely that other regions of the atria cannot follow in a 1:1 fashion and intra-atrial
electrical conduction block results in a decrease in AP frequency in regions farther from the site
of re-entrant rotor origin. These results are supported by the observation that expression of
murine M2 receptors and GIRK channels is higher in the SA node and RAA than the LAA which
facilitates a greater abbreviation of refractoriness that allows this tissue to support the
development of high frequency rotors in the RAA (68). The concept of a dominant frequency
gradient has been described previously in various animal and human models. In isolated sheep
hearts treated with acetylcholine, AF was sustained by high frequency rotors in the LA with
fibrillatory conduction toward the RA (71). Importantly, this gradient was also observed in
patients with paroxysmal AF and was due to a decrease in Kir3.1/3.4 expression in the RA
compared to the LA (162;163).
Ours is not the first study to implicate altered RGS protein activity in the genesis of atrial
arrhythmias. It has previously been shown that RGS2-deficient mice are more susceptible to
pacing-induced atrial arrhythmias in the absence of cholinergic stimulation (164). AERPs were
significantly lower in RGS2-deficient mice compared to WT controls and this difference was
eliminated by the selective M3 muscarinic receptor blocker, darifenacin. Since RGS2 is an
inhibitor of Gαq but not Gαi/o-signaling, these results suggest that enhanced arrhythmia
susceptibility in the absence of RGS2 is driven by Gαq-coupled M3 receptors. Another member
of the RGS protein family, RGS6, has been shown to control M2 receptor activation of IKAch
61
(93;94) and may play a role in the regulation of vagally-mediated AF. In fact, it was recently
shown that RGS6-deficient mice are more sensitive to the induction of AF with CCh and burst
pacing compared to WT control mice (165). It is worth noting that the dose used in that study,
0.5 mg/kg CCh, was much higher than our CCh dose (0.03 mg/kg ip). Finally, atrial electrical
remodeling and increased AF inducibility in a rat model of endurance exercise has been shown
to be associated with increased vagal tone and enhanced sensitivity of IKAch to cholinergic
stimulation (149). Expression studies revealed decreased mRNA levels of several RGS proteins,
including RGS3, RGS4 and RGS10, suggesting that augmented RGS protein activity may
contribute to the enhanced sensitivity of IKAch to vagal stimulation. Collectively, these studies
along with the findings reported in this thesis highlight the importance of RGS proteins in
controlling the effects of parasympathetic signaling on atrial refractoriness and subsequent
arrhythmogenesis.
6.2 Effect of KATP on atrial arrhythmia vulnerability in RGS4-deficient mice
KATP channels serve as metabolic sensors that translate changes in cellular metabolism
into changes in electrical activity. In cardiac myocytes, KATP channels are normally closed by
high ATP levels but open in response to various forms of cardiac stress, including metabolic
impairment and Ca2+
overload. KATP channel activation can reduce excessive Ca2+
influx and may
conserve intracellular ATP stores that would otherwise be depleted during the contractile cycle.
For instance, treatment with the KATP channel opener pinacidil during ischemia increased
intracellular ATP levels and other energy stores such as creatine phosphate (134). In support,
Kir6.2-knockout mice develop contractile dysfunction during ischemia, which likely reflects
ATP depletion in the absence of KATP channels.
62
Once initiated, a consequence of fibrillating atrial tissues is increased intracellular Ca2+
load. The atrial myocardium has been shown to remodel in response to AF in order to reduce
excessive Ca2+
entry and toxicity. The well-established cellular mechanisms that have been
associated with AF-induced remodeling include a reduction in ICaL (166), Ito (167), and IK,Ach
current density, decreased expression of ICaL, and up-regulation of IK1 and IK,Ach (168;169). In
addition to these changes, KATP channel activation may be an adaptive response to the arrhythmia
induced Ca2+
overload. Activation of KATP channels hyperpolarizes the cell membrane and
reduces Ca2+
influx. This in turn decreases the contractile force of atrial tissues and reduces the
consumption of ATP to protect the cell from metabolic impairment and Ca2+
overload. However,
an important consequence of KATP channel activation is APD shortening which is a critical
determinant of re-entry. Previous work in our laboratory has implicated KATP channel activation
in the progression to chronic AF observed in RGS4-/-
mice. Notably, the pore-forming subunit of
the KATP channel, Kir6.2, is up-regulated in elderly (>75 weeks) RGS4-/-
mice and chronically
paced canine atria. These data suggest that up-regulation of Kir6.2 is induced preceding chronic
AF and may contribute to the development of a substrate supporting the maintenance of AF. In
light of these observations, we investigated the contribution of KATP channels to arrhythmia
susceptibility and rotor dynamics in the absence of RGS4 activity.
We here show that suppression of KATP channel activity, whether by genetic deletion of
the Kir6.2 subunit (RGS4-/-
Kir6.2-/-
) or through the use of channel antagonists (tolbutamide),
decreases susceptibility to atrial arrhythmias that are caused by re-entry. Atrial arrhythmias were
observed to occur in the absence of KATP channel activity; however, these occurred at much
higher doses of CCh than in RGS4-deficient atria and the majority of sustained arrhythmia
episodes were non-reentrant focal tachycardias. Our data are consistent with previous reports
63
suggesting that KATP channel activation increases susceptibility to re-entrant arrhythmias, while
KATP channel inhibition is expected to increase susceptibility to arrhythmias that are caused by
abnormal automaticity or triggered activity. Loss of Kir6.2 was previously associated with
triggered activity by modulating Ca2+
handling. Liu et al. (170) reported that Kir6.2-knockout
mice develop early phase 3 EADs when challenged with isoproterenol and EAD-induced
triggered activity occurred more frequently in Kir6.2-knockout mice compared to WT controls.
Conversely, several studies reported that activation of KATP channels abbreviates the atrial APD
and may contribute to the development of re-entrant atrial arrhythmias. First, Glukhov et al.
(127) studied the effect of KATP channel openers (diazoxide and pinacidil) and blocker
(glibenclamide) in isolated mouse atrial preparations. The authors reported that diazoxide caused
shortening of the atrial APD and glibenclamide restored the APD to baseline values. In a
subsequent study, the effects of diazoxide and pinacidil were investigated in failing and non-
failing human hearts (171). The authors reported that pinacidil significantly decreased APD in
both failing and non-failing atria and facilitated the induction of AF with burst pacing. Another
group demonstrated that β-adrenergic receptor stimulation with isoproterenol depletes ATP
levels in rat left atria and creates a pro-arrhythmic substrate by activating KATP channels (145).
The authors proposed that the isoproterenol-induced atrial arrhythmias may involve a re-entrant
mechanism since they observed a decrease in APD, CV, and wavelength of excitation, which are
important determinants of re-entry. In support, in a rat isolated atrial preparation, pinacidil
caused action potential shortening and enhanced arrhythmia induction, both of which were
reversed by glibenclamide (172).
In the present study, we show that Kir6.2 ablation and KATP channel inhibition with
tolbutamide prolonged APD in the right and left atrial appendages at baseline. These data
64
suggest the presence of basal KATP activation in excised atrial tissues under control conditions.
The observed effects on atrial repolarization could have resulted from non-specific effects of
sulfonylureas on other cardiac channels. For instance, tolbutamide has been reported to inhibit
the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel (173) and
activation of the CFTR Cl- channel is thought to prevent excessive prolongation of APD.
Therefore, inhibition of these channels by tolbutamide may explain the prolonged APD observed
at baseline. However, this hypothesis seems unlikely since repolarization was similarly
prolonged in Kir6.2-deficient mice.
Alternatively, KATP channels may play a role in regulating Ca2+
homeostasis under
normal physiological conditions. This was recently reported by Storey et al. (174) who showed
that Kir6.2 knockdown in isolated rat ventricular myocytes leads to the development of higher
intracellular Ca2+
concentrations compared to control myocytes, even in the absence of metabolic
inhibition. Therefore, basal Ca2+
homeostasis may be perturbed in the absence of KATP channels,
which may subsequently prolong the APD. Another possible explanation is that alterations in
KATP channel activity/expression may be induced by the experimental preparation of the atrial
tissues, perhaps as a result of hypoxic stress. For instance, decreased oxygen concentrations up-
regulates hypoxia inducible factors and HIF1-α has been shown to increase expression of several
KATP channel subunits including Kir6.1, SUR2, and SUR1 (175;176). Performing in vivo
intracardiac arrhythmia assessment experiments on these animals might eliminate some of these
concerns.
We next investigated the effect of CCh on atrial repolarization in the absence of KATP
channels by measuring APD at 70% repolarization times. CCh decreased APD70 of both Kir6.2-/-
and RGS4-/-
Kir6.2-/-
atria with no differences between groups. Similar results were obtained
65
using tolbutamide, with CCh causing a decrease in APD70 in both WT and RGS4-/-
atria and no
differences were observed between the two groups. These data might suggest that the in atrial
myocytes, the predicted inhibitory effect of RGS4 on M2R-Gαi/o-IKAch signaling may be less
important than the inhibitory effect of RGS4 on KATP channel activity. This effect may or may
not involve RGS4-mediated inhibition of Gαi/o-proteins. Although the differences in APD that
we found between CCh-treated WT and RGS4-/-
atria and Kir6.2-/-
and RGS4-/-
Kir6.2-/-
atria
would suggest that RGS4 predominantly modulates KATP channels instead of GIRK channels,
deletion of both RGS4 and Kir6.2 could have indirectly caused changes in the expression of
other ion channels that would support alternative explanations for our results. Furthermore, there
may be other adaptations in these mice, such as changes in post-translational modifications or
intracellular trafficking events, that could explain our APD data. These questions have not been
examined in the present study. Taken together, our data suggest that in the absence of KATP -
channel activity, APD is not sufficiently shortened in RGS4-/-
atria (even during parasympathetic
stimulation) to allow for the induction of a functional re-entry circuit.
Finally, we investigated the expression of KATP channel subunits, SUR1, SUR2A/B,
Kir6.2 and Kir6.1, in atrial tissues from WT and RGS4-/-
mice. We observed a greater expression
of SUR1 compared to SUR2A/B, consistent with the notion that SUR1 is the predominant
sulfonylurea receptor in the mouse atria (126). However, we did not observe any differences in
KATP subunit expression between WT and RGS4-/-
tissues. These results suggest that there may
be a decrease in ATP-sensitivity through interaction with G-protein subunits in the absence of
RGS4.
Another possible explanation is that tachypacing results in sufficient metabolic
impairment, particularly in the absence of RGS4, that results in KATP activation/up-regulation
66
which further abbreviates the APD to sustain the arrhythmia. There is a growing body of
evidence suggesting that energy metabolism is modulated during AF. Cha et al. (177) have
shown that failing hearts have lower concentrations of high-energy phosphates and decreased
activity of creatine kinase. Importantly, these changes correlated with an increased risk of
developing AF. Likewise, in a goat model of AF, decreased phosphocreatine levels were
observed within one week of AF which returned to baseline after 16 weeks of AF, suggesting
that compensatory mechanisms (i.e. KATP) may be activated to restore the cardiac energy state
(178). In line with these findings, a recent study demonstrated that β-adrenergic stress depletes
cellular ATP levels, decreases the atrial refractory period and conduction velocity, and increases
AF induction via activation of KATP channels (145). Moreover, Kir6.2 and SUR2 subunits are up-
regulated in mouse and rat ventricles in response to exercise (139), raising the possibility that
KATP channel expression may be up-regulated in myocytes in response to tachycardia. Further
experiments will be required to fully address these possibilities.
6.3 Limitations
While our studies used both genetic and pharmacologic approaches to characterize the
role of KATP channels in atrial arrhythmia vulnerability, we acknowledge that there are
limitations to our study design. First, the mouse strains used in our experiments are global
knockouts of the rgs4 and KCNJ11 genes, therefore the observed effects are not specific for the
atria because both RGS4 and Kir6.2 are expressed not only in cardiac muscle but many other
tissues including pancreatic β-cells, neurons, and smooth muscle. Although the role of RGS4 and
Kir6.2 in the atria was demonstrated by performing studies in isolated atrial tissues, the effects of
RGS4-/-
and Kir6.2-/-
on other tissues may have affected the results of our studies. For instance,
67
RGS4 is highly expressed in the adrenal glands (179) and RGS4-/-
mice have increased
circulating levels of catecholamines (180). This may have confounding effects particularly in our
double knockout mouse cohort since catecholamine challenge predisposes to the development of
triggered arrhythmias in the absence of KATP channels.
Another important limitation concerns the use of optical mapping to measure action
potential duration. Motion artifacts can distort optical action potentials and prevent accurate
measurements of the repolarization phase of the action potential. Therefore, in a subset of atrial
preparations simultaneous recordings of fluorescence and microelectrode action potential
recordings were obtained in order to validate the optically-derived action potentials. Another
limitation involves the use of isolated atrial tissues to investigate the function of KATP channels.
KATP channels are normally closed at physiological concentrations of ATP (~5 mM) but these
channels can open even in the presence of ATP. KATP channel activity is stimulated by a decrease
in the ATP-to-ADP ratio, a decrease in the intracellular pH, and cellular hypoxia. Therefore,
changes in KATP channel activity may have been induced at the time of animal sacrifice or during
the atrial isolation procedure and this may have an effect on the interpretation of the importance
of KATP channels on arrhythmia induction and maintenance in vivo.
6.4 Conclusion and Future Directions
In this MSc thesis, I have demonstrated that RGS4-deficient mice and isolated atrial
tissues are more susceptible to the development of re-entrant atrial arrhythmias in the presence of
cholinergic stimulation. Furthermore, our data suggests that the observed pro-arrhythmic
phenotype may be caused by changes in atrial refractoriness since we observed a greater
abbreviation of the APD in the absence of RGS4. However, the mechanisms responsible for the
68
initiation of the observed arrhythmias were not characterized. Burashnikov et al. (181)
demonstrated that late-phase 3 EAD-induced triggered activity may be responsible for the
initiation of AF in the presence of vagal stimulation. In coronary-perfused canine atrial
preparations, acetylcholine was used to abbreviate the APD. This was followed by rapid atrial
pacing that increased SR calcium release, which in the setting of decreased APD, led to the
development of late-phase 3 EADs and extra-systoles that initiated AF. Since we observed a
marked abbreviation of the APD in RGS4-deficient atria in the presence of carbachol (300 nM),
we can investigate this possibility by rapidly pacing atrial tissues while simultaneously recording
action potentials using conventional microelectrodes.
Moreover, both Kir6.2 genetic knockout and pharmacologic blockade of sarcolemmal
KATP channels with tolbutamide reduced the extent of AP shortening and decreased the number
of inducible arrhythmias in the absence of RGS4. Our data indicate that increased activity and/or
expression of KATP channels might be part of the mechanism that leads to the development of re-
entry atrial arrhythmias. However, these conclusions should be interpreted within the context of
the research design. We studied the effect of KATP channels on arrhythmia susceptibility in the
RGS4-/-
mouse model using atrial tachypacing and parasympathetic stimulation. Therefore,
additional studies must be carried out in order to determine whether KATP channels are direct
effectors of RGS4, independent of parasympathetic signaling, or whether KATP channels are
remodeled (increased IKATP or Kir6.2/SUR1 expression) as a consequence of the rapid atrial rate
that is observed during the arrhythmia.
In order fully elucidate the underlying mechanisms, we propose the following
experiments. First, KATP channel blockers, glibenclamide and tolbutamide, can be administered
during a sustained arrhythmia episode in order to investigate whether KATP channel blockade can
69
promote spontaneous termination. Second, in order to confirm the effect of RGS4 ablation on
KATP activity, experiments can be performed to assess the effect of potassium channel opening
drugs, diazoxide (specific to SUR1>SUR2A) and pinacidil (specific to SUR2A>SUR1), on APD
and arrhythmia susceptibility in atria isolated from WT and RGS4-deficient mice. If KATP
channel activity is increased under conditions of decreased RGS4 activity, we would expect to
see a greater abbreviation of APD and increased susceptibility to arrhythmia induction in RGS4-
deficient atria compared to WT controls. Furthermore, we would expect to see a more
pronounced effect in the presence of diazoxide, since it is an effective activator of SUR1-
containing KATP channels that are thought to be present in murine atrial tissues (126). Another
important question that needs to be addressed is whether KATP channels are more sensitive to
activation under metabolic inhibition when RGS4 levels are decreased. It is possible that in the
absence of RGS4, tachypacing causes sufficient metabolic impairment, that results in KATP
activation which further abbreviates the APD to sustain the arrhythmia. Therefore, we would
expect KATP channels to be more sensitive to activation under metabolic inhibition, particularly
under conditions of decreased RGS4 activity.
70
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