Remodeling of Myocardial Passive Electrical Properties: Insights Into the Mechanisms of Malignant Arrhythmias and Sudden Cardiac Death

Remodeling of Myocardial Passive Electrical Properties: Insights into the Mechanisms of Malignant Arrhythmias and Sudden Cardiac Death.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Carlos Luis del Rio, M.S.

Graduate Program in Electrical and Computer Science

The Ohio State University

2015

Dissertation Committee:

Professor Bradley D. Clymer, Ph.D., Advisor

Professor George E. Billman, Ph.D., Co-Advisor

Professor Furrukh S. Khan, Ph.D.

Carlos Luis del Rio

2015

Abstract

Despite extensive research, sudden cardiac death (SCD) resulting from ischemia-induced malignant arrhythmias, such as ventricular fibrillation (VF), remains a leading cause of death, particularly following myocardial infarction (MI). Furthermore, SCD is generally the first and most common manifestation of the disease, as current risk-stratifying tools are inaccurate and insufficient. Acute/chronic changes in the passive electrical properties governing electrotonic coupling in the myocardium have been proposed as a potential mechanism mediating both the onset and maintenance of arrhythmias, as the loss of homogenizing electrotonic coupling can exacerbate intrinsic pro-arrhythmic electrical heterogeneities within the ventricle, especially during repolarization. However, no study to date has assessed the ability of indices reflective of electrotonic changes to stratify intrinsic arrhythmic susceptibility in vivo.

Leveraging a well-established in vivo post-MI canine model of SCD and lethal arrhythmias, this research work investigates the pro-arrhythmic role of changes in the passive electrical properties of the myocardium, as measured by its complex electrical impedance spectrum

(MEI). The studies were performed under the general hypothesis that the loss of electrotonic coupling accompanies and facilitates the development of malignant arrhythmias in the setting of ischemia and post-MI ventricular/autonomic remodeling.

Specifically, acute/chronic electrotonic changes/heterogeneities in the setting of three well- ii established risk factors for arrhythmias/SCD, namely acute ischemia, myocardial infarction, and/or autonomic imbalances, were evaluated. Furthermore, since the experimental post-MI model used yields two well-defined populations of animals, one prone and the other resistant to ischemia-induced lethal arrhythmias, the prognostic value of electrotonic changes to detect intrinsic arrhythmic susceptibility was also assessed.

In short, both infarcted and remote (i.e., non-infarcted) myocardium were shown to undergo chronic electrotonic remodeling following a sustained ischemic insult (coronary artery ligation). Overall, the healing infarction was shown to have lower impedance than remote myocardium, with animals prone to malignant arrhythmias showing a wider electrotonic gradient (i.e., impedance difference) between these two regions. In the electrotonically-remodeled post-MI myocardium, acute β-adrenoceptor activation (either during bouts of exercise or via a direct pharmacological challenge) acutely increased passive (electrotonic) coupling in the remote myocardium, with animals susceptible to

SCD showing the greatest electrotonic responsiveness. Meanwhile, electrotonic uncoupling induced by an acute ischemic insult (coronary artery occlusion) to the non- infarcted myocardium of post-MI animals susceptible to malignant arrhythmias, was shown to facilitate/uncover (i.e., predict) pro-arrhythmic ventricular electrical oscillations and repolarizarion abnormalities (such as TWA). Moreover, acute electrotonic uncoupling during ischemia was shown to be modulated by interventions affecting autonomic balance, as passive electrical derangements were blunted by complete/partial β-adrenoreceptor blockade and/or vagal nerve stimulation, but enhanced by exercise.

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In conclusion, these experiments establish post-MI passive electrical heterogeneities and their modulation by autonomic changes and/or acute ischemia, as a risk/prognostic factor for SCD/arrhythmic susceptibility in vivo.

A mis padres, Pedro Luis y María de los Ángeles.

A mi hijito Javier, and to Sarah, my soulmate.

Acknowledgments

While a single paragraph cannot fully express my appreciation for their support, I would nonetheless like to document my gratitude to Dr. Bradley Clymer, Dr. George Billman, Dr.

Furrukh Khan, Dr. Michael Howie, and Mr. Roger Dzwonczyk. Their friendship and invaluable guidance as professors, scientists, and engineers have taught me that passion, collaboration, and dedication can make the unthinkable a reality. Additionally, I will always treasure Dr. Robert Hamlin’s brilliance, eagerness, and endless desire to learn.

To all of you, my dear professors, and to all of the other friends and collaborators that I have had the pleasure of meeting, please receive my most sincere and eternal gratitude.

Vita

1998...... B.S. (summa cum laude), Electrical

Engineering, Universidad Simón Bolívar

(Caracas, Venezuela)

2005...... M.S. Electrical & Computer Engineering,

The Ohio State University (OH, USA)

Publications

Dzwonczyk R, del Rio C, Brown DA, Michler RE, Wolf RK, Howie MB. Myocardial

Electrical Impedance Responds to Ischemia and Reperfusion in Humans. Comput Cardiol.

2002; 29: 541−543. del Río CL, Dzwonczyk R, Clymer BD, McSweeney TD, Awad H, Czerwinski P, Howie

MB. Use of Myocardial Electrical Impedance to Assess the Efficacy of Preconditioning.

Comput Cardiol. 2002; 29: 489−492.

Dzwonczyk R, del Rio CL, Brown DA, Michler RE, Wolf RK, Howie MB. Myocardial

Electrical Impedance Responds to Ischemia and Reperfusion in Humans. IEEE Trans

Biomed Eng 2004; 51:2206-09.

vii del Río CL, Dzwonczyk R, McConnell PI, Clymer BD, Howie MB, Billman GE. Beta-

Adrenergic Receptor Blockade Attenuates the Electronic Uncoupling Induced by Coronary

Artery Occlusion. Comput Cardiol 2004; 31: 405−408.

Dzwonczyk R, del Rio CL, Sun B, Michler RE, Howie MB. Devices used to expose the posterior coronary artery in OPCABG surgery may cause ischemia. Proc. IEEE Northeast

Bioengineering Conference, April 2005: 148 – 149.

McConnell PI, del Rio CL, Jacoby DB, Pavlicova M, Kwiatkowski P, Zawadzka A,

Dinsmore JH, Astra L, Wisel S, Michler RE. Correlation of Autologous Skeletal Myoblast

Survival with Changes in Left Ventricular Remodeling in Dilated Ischemic Heart Failure.

J Thorac Cardiovasc Surg. 2005 Oct; 130(4):1001. del Rio CL, McConnell PI, Clymer BD, Dzwonczyk R, Michler RE, Billman GE, Howie

MB. Early Time Course of Myocardial Electrical Impedance During Acute Coronary

Artery Occlusion in Pigs, Dogs And Man. J Appl Physiol. 2005 Oct; 99(4):1576-81.

Dzwonczyk R, del Rio CL, Sai-Sudhakar C, Sirak JH, Michler RE, Sun B, Kelbick N,

Howie MB. Vacuum-assisted apical suction devices induce passive electrical changes consistent with myocardial ischemia during off-pump coronary artery bypass graft surgery.

Eur J Cardiothorac Surg. 2006 Dec; 30(6):873-876.

Dzwonczyk R, del Rio CL, Sun B, Howie MB. Myocardial Electrical Impedance

Correlates with Ischemic ECG ST-Segment Changes in Humans. Conf Proc IEEE Eng

Med Biol Soc. 2006; 1: 2568-2569.

viii

McConnell PI, del Rio CL, Kwiatkowski P, Farrar DJ, Sun BC. Assessment of cardiac function during axial-flow left ventricular assist device support using a left ventricular pressure-derived relationship: comparison with pre-load recruitable stroke work. J Heart

Lung Transplant. 2007 Feb; 26(2):159-66. del Rio CL, McConnell PI, Kukielka M, Dzwonczyk R, Clymer DB, Howie MB, Billman

GE. Electrotonic Remodeling following Myocardial Infarction in Dogs Susceptible and

Resistant to Sudden Cardiac Death. J Appl Physiol. 2008 Feb;104(2):386-93. del Rio CL, Dawson TA, Clymer BD, Paterson DJ, Billman GE. Acute Vagal Nerve

Stimulation Attenuates Early Passive Electrical Changes Induced by Myocardial Ischemia:

Heart Rate Mediated Attenuation. Exp Physiol. 2008 Aug; 93(8):931-44.

Dzwonczyk R, del Rio C, McSweeney TD, Zhang X, Howie MB. Myocardial electrical activity does not affect myocardial electrical impedance measurements. J Clin Monit

Comput. 2009 Aug; 23(4):217-22.

Howie M, del Río C, Khan F, Lopez L, Dzwonczyk R, Bergese S. (2009). A Secure and

Expandable Electronic Patient Record System Using Web-based Technology. Ibnosina

Journal Of Medicine And Biomedical Sciences, IJMBS. 2009; 1(3): 73-9.

Kijtawornrat A, Panyasing Y, Del Rio C, Hamlin RL. Assessment of ECG interval and restitution parameters in the canine model of short QT syndrome. J Pharmacol Toxicol

Methods. 2010 May-Jun;61(3):231-7.

Hamlin RL, del Rio C. An approach to the assessment of drug-induced changes in non- electrophysiological properties of cardiovascular function. J Pharmacol Toxicol Methods.

2010 Jul-Aug;62(1):20-9.

Zwijnenberg RJ, del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs when given as a pre-anesthetic IV,

IM or SQ and in combination with butorphanol. Am J Vet Res. 2010 Jun;71(6):604-9.

Zwijnenberg RJ, del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel, butorphanol and a butorphanol-perzinfotel combination on the minimum alveolar concentration of isoflurane in cats. Am J Vet Res. 2010 Jun;71(11):1270-6.

Panyasing Y, Kijtawornrat A, del Rio C, Carnes C, Hamlin RL. Uni- or bi-ventricular hypertrophy and susceptibility to drug-induced torsades de pointes. J Pharmacol Toxicol

Methods. 2010 Sep-Oct;62(2):148-56

Zwijnenberg RJ, del Rio CL, Cobb RM, Ueyama Y, Muir WW. Evaluation of oscillometric and vascular access port arterial blood pressure measurement techniques versus implanted telemetry in anesthetized cats. Am J Vet Res. 2011 Aug;72(8):1015-21.

Kranstuber AL, del Rio C, Biesiadecki BJ, Hamlin RL, Ottobre J, Gyorke S, Lacombe VA.

Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling. Front Physiol. 2012

Jul 19;3:292.

Hamlin RL, del Rio C. dP/dt(max)--a measure of 'baroinometry'. J Pharmacol Toxicol

Methods. 2012 Sep;66(2):63-5.

Brown DA, Hale SL, Baines CP, del Rio CL, Hamlin RL, Yueyama Y, Kijtawornrat A,

Yeh ST, Frasier CR, Stewart LM, Moukdar F, Shaikh SR, Fisher-Wellman KH, Neufer

PD, Kloner RA. Reduction of early reperfusion injury with the mitochondria-targeting

Peptide bendavia. J Cardiovasc Pharmacol Ther. 2014 Jan;19(1):121-32.

Kijtawornrat A, Ueyama Y, del Rio C, Sawangkoon S, Buranakarl C, Chaiyabutr N,

Hamlin RL. Test of the Usefulness of a Paradigm to Identify Potential Cardiovascular

Liabilities of Four Test Articles With Varying Pharmacological Properties in Anesthetized

Guinea Pigs. Toxicol Sci. 2014 Feb;137(2):458-68.

McConnell PI, Anstadt MP, del Rio CL, Preston TJ, Ueyama Y, Youngblood BL. Cardiac function after acute support with direct mechanical ventricular actuation in chronic heart failure. ASAIO J. 2014 Nov-Dec;60(6):701-6.

Muir WW, Ueyama Y, Pedraza-Toscano A, Vargas-Pinto P, del Rio CL, George RS,

Youngblood BL, Hamlin RL. Arterial blood pressure as a predictor of the response to fluid administration in euvolemic nonhypotensive or hypotensive isoflurane-anesthetized dogs.

J Am Vet Med Assoc. 2014 Nov 1;245(9):1021-7.

Muir WW, del Rio CL, Ueyama Y, Youngblood BL, George RS, Rausch CW, Lau BS,

Hamlin RL. Dose-Dependent Hemodynamic, Biochemical, and Tissue Oxygen Effects of

OC99 following Severe Oxygen Debt Produced by Hemorrhagic Shock in Dogs. Crit Care

Res Pract. 2014;2014:864237. xi del Rio CL, Clymer BD, Billman GE. Myocardial Electrotonic Response to Submaximal

Exercise in Dogs with Healed Myocardial Infarctions: Evidence for β-Adrenoceptor

Mediated Enhanced Coupling during Exercise Testing. Front. Physiol. 2015 6:25.

Fields of Study

Major Field: Electrical and Computer Engineering

xii

Table of Contents

Abstract ...... ii

Acknowledgments...... vi

Vita ...... vii

Table of Contents ...... xiii

List of Tables ...... xv

List of Figures ...... xviii

Chapter 1: Introduction ...... 1

Chapter 2: Early Electrotonic Changes during Acute Ischemia ...... 12

Introduction ...... 13

Methods ...... 15

Results ...... 19

Discussion ...... 20

Chapter 3: Parasympathetic Effects on Early Ischemic Electrotonic Changes ...... 29

Introduction ...... 30

Methods ...... 34

Results ...... 40

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Discussion ...... 45

Chapter 4: Electrotonic Remodeling following Myocardial Infarction ...... 63

Introduction ...... 64

Methods ...... 66

Results ...... 72

Discussion ...... 74

Chapter 5: β-Adrenoceptor Mediated Enhancement of Electrotonic Coupling ...... 89

Introduction ...... 90

Methods ...... 92

Results ...... 100

Discussion ...... 104

Chapter 6: Ischemia-Induced Electrotonic Uncoupling in the Surviving post-MI

Myocardium and SCD ...... 124

Methods ...... 126

Results ...... 132

Conclusion ...... 134

Chapter 7: Summary ...... 142

References ...... 146

xiv

List of Tables

Table 2.1: MEI parameters during acute coronary occlusion in various species...... 25

Table 3.1: Hemodynamic and myocardial electrical impedance (MEI) response to acute

LCX coronary artery occlusion (CAO) and subsequent (overlapping) acute bilateral cervical vagotomy (VAG) in intact animals (CTRL group, n = 7)...... 55

Table 3.2: Hemodynamic and myocardial electrical impedance (MEI) response to LCX coronary artery occlusion (CAO) and subsequent (overlapping) vagal nerve stimulation

(VNS) in previously vagotomized animals (i.e., post bilateral vagotomy) with (VAG/P) and without (VAG) atrio-ventricular-pacing...... 56

Table 3.3: Electrocardiographic response to acute LCX coronary artery occlusion (CAO) and subsequent (overlapping) vagal nerve stimulation (VNS) in previously vagotomized animals (i.e., post bilateral-vagotomy) (VAG group, n = 11)...... 57

Table 4.1: Myocardial electrical impedance (MEI) of ischemic/infarcted and remote (non- infarcted) myocardium following left-anterior descending (LAD) coronary artery ligature

(myocardial infarction, MI). Acute MI measurements (shaded) were obtained in anesthetized animals (at the end of the surgical preparation/LAD ligation)...... 83

Table 4.2: Myocardial electrical impedance (MEI) data recorded from infarcted and remote

(non-infarcted) myocardium after left-anterior descending (LAD) coronary artery ligation.

Data are grouped according to subsequent susceptibility to ischemia-induced ventricular fibrillation...... 84

Table 5.1: Myocardial electrical impedance (MEI), heart-rate (HR) and ECG-derived indices of heart rate variability in awake-unsedated dogs with healed left-anterior descending (LAD) myocardial infarcts, both before (baseline) as well as during a submaximal exercise test...... 114

Table 5.2: Exercise-induced changes in both the myocardial electrical impedance (MEI) and the heart-rate (HR) of awake-unsedated dogs with healed left-anterior descending

(LAD) myocardial infarct; comparative effects of the underlying susceptibility to malignant arrhythmias (S vs. R), and of β-adrenoceptor blockade (+BB)...... 115

Table 5.3: Electrocardiographic response(s) to submaximal exercise. Data collected immediately after the discontinuation of exercise...... 116

Table 6.1: Changes in the myocardial electrical impedance (MEI) induced by acute ischemia (LCX coronary artery occlusion, CAO) in conscious dogs with a healed myocardial infarct; comparative effects of either β-adrenoceptor blockade (+BB) or autonomic activation (via submaximal exercise, +SMT) as well as underlying susceptibility to malignant arrhythmias (S vs. R)...... 135

xvi density (PSDratio) of inter-beat electrical events; comparative effects of either β- adrenoceptor blockade (+BB) or autonomic activation (via submaximal exercise, +SMT) on MEI are also shown...... 136

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List of Figures

Figure 2.1: Sample MEI data (Z[n], open circles) measured on a dog (top panel, from

Howie at al., 2001) and a swine (bottom panel, from del Rio et al., 2002) showing the MEI biphasic rise after coronary artery occlusion (at 0 min): early increase immediately following the ischemic insult (I), a plateau phase (II), and rapid increase thought to mark cellular uncoupling (III) ...... 26

Figure 2.2: Sample data analysis procedure on swine data (from del Rio et al., 2002). TOP:

Original MEI measurements (z[n], open circles) indicating moving analysis window (WN) and best (mean-square) linear fit (L) at sample n. BOTTOM: MEI first derivative (ΔZ[n]) estimated from the linear fit (L[n]), indicating ischemic plateau threshold (ΔZp, i.e., 3% of the maximal derivative) and value at ni (ΔZ[n]). Occlusion marker (no) and ischemic plateau sample (np) are used to calculate baseline and ischemic plateau MEI values (Zb and Zp respectively). NOTE: The fact that ΔZ[n] appears to increase before coronary artery occlusion (no) is an artifact of the non-causal windowing technique used...... 27

Figure 2.3: Sample normalized MEI data (Z[n]) after coronary artery occlusion: Original

MEI measurements on representative pig (open circles, from del Rio et al., 2002), dog

(open triangles, from Howie et al., 2001) and human (open stars, from Dzwonczyk et al.,

2004) indicating occlusion marker (at 0 min). Inserts: Estimated MEI first derivatives

(ΔZ[n]) for each subject, indicating ischemic plateau threshold (ΔZp, i.e., 3% of its

xviii maximum value). Observe that ΔZ[n]) in the dog does not fall below the ischemic plateau threshold (ΔZp), i.e., MEI does not reach ischemic plateau...... 28

(reactance, Xo) components, is given by the Vo/Io ratio; and 4) a single value (MEIo), representing the mean MEI modulus (|Zo|) over frequency, is reported. RIGHT: MEI samples were taken every 3s (circles), and 30s averages were studied (squares). Over time,

MEI values remain stable (i.e., low variance, σ), until the onset of ischemia (coronary artery occlusion, CAO; at t = 0 min)...... 58

CAO (VAG/S group). The response to VNS after CAO was only studied in animals vagotomized prior to ischemia onset (i.e., VAG, VAG/P and VAG/S groups). Seven vagotomized dogs (VAG n = 5; VAG/P n = 1; and VAG/S n = 1) developed ventricular xix fibrillation (VF) during CAO prior to VNS. (*) In VAG/P dogs: after VNS (shown), pacing was discontinued and stimulation was repeated (not shown). Pacing was established after the CAO onset in two dogs in the VAG/P group...... 59

(overlapping) vagal nerve stimulation (VNS). Note early ST-segment elevation and progressive QRS widening induced by myocardial ischemia that was attenuated during overlapping VNS...... 60

(overlapping) vagal nerve stimulation (VNS) when A) heart-rate was allowed to change freely (top, VAG group) or B) held-constant by atrio-ventricular pacing (bottom, VAG/P group) during stimulation. Inserts show mean responses during VNS for the respective groups (i.e., VAG and VAG/P)...... 61

Figure 3.5: A (top): Representative arterial blood pressure (BP) and heart rate (HR) tracings showing characteristic responses to a sodium nitroprusside (SNP) induced decrease in BP before (left) and after (right) bilateral stellectomy. B (bottom): Representative myocardial electrical impedance (MEI) recording showing the response to the left circumflex (LCX) coronary artery occlusion (CAO) and subsequent (overlapping) vagal nerve stimulation

(VNS) following bilateral stellectomy in a previously vagotomized animal (i.e., post bilateral-vagotomy). Inserts show mean responses for the group (VAG/S)...... 62

Figure 4.1: Representative (top) and aggregated (bottom, mean ± SD) acute myocardial electrical impedance (MEI) changes following a 2-stage left-anterior descending (LAD) coronary artery occlusion (at t = 0 min). Note the early MEI increase, but subsequent recovery, following the initial ischemic insult (constriction stage I) and the sustained rise after complete coronary occlusion (ligation, stage II)...... 85

Figure 4.2: Early time-course of myocardial electrical impedance (MEI) following acute left-anterior descending (LAD) coronary artery ligature (myocardial infarction, MI). Note that in contrast to acutely ischemic tissue (shaded area), healing infarct (chronically ischemic) has lower impedance than remote (non-infarcted) myocardium, resulting in an electrotonic gradient/dispersion (ΔMEI). Remote values were measured on the distal left- circumflex (LCX) coronary artery distribution. Chronic data (days 1, 2 and 7) were collected in awake-unsedated animals at rest...... 86

Figure 4.3: A (top): Myocardial electrical impedance (MEI) of infarcted and remote (non- infarcted) myocardium one-week after left-anterior descending (LAD) coronary artery ligation. Note the wider electrotonic dispersion, i.e., larger impedance difference between remote and ischemic myocardium (ΔMEI), in animals later found susceptible to ischemia- induced malignant arrhythmias. B (bottom): Individual ΔMEI values 7 days post-MI.

Remote values were measured on the distal left-circumflex (LCX) coronary artery distribution...... 87

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Figure 4.4: Empirical (gray) and bi-normal (fitted, black) receiver-operator characteristic

(ROC) curves generated for the myocardial electrical impedance (MEI) differences between infarcted and remote (non-infarcted) myocardium (i.e., ΔMEI) 7 days after left- anterior descending (LAD) coronary artery ligation; the area under the ROC curve (AUC) provides a measure of diagnostic accuracy (48). Inset: Percent correct classification of susceptible (sensitivity, %) and resistant (specificity, %) animals as a function of ΔMEI; the optimal classification threshold (i.e., ΔMEIo~145 Ω) was selected at the point of intersection of the two curves (~83%), providing positive (PPV) and negative (NPV) predictive values of 82.8%...... 88

Figure 5.1: Schematic representation of the six-level submaximal exercise test (SMT).117

Figure 5.2: Exercise-Induced changes in the heart-rate (HR, top-left) and ECG-derived vagal-tone index (bottom-left) and well as in the myocardial electrical impedance (MEI, bottom-right with representative response in top-right) of awake-unsedated dogs with healed left-anterior descending (LAD) myocardial infarcts (n = 25, except at recovery where n = 14)...... 118

Figure 5.3: Relationship(s) between the exercise-induced changes in the heart-rate (top) and ECG-derived vagal-tone index (bottom) with the concomitant reductions in myocardial electrical impedance (MEI); relationships were “centered”, i.e., deviations from each animal’s mean values (over the whole exercise bout) were studied...... 119

Figure 5.4: Effects of (non-selective) β-adrenoceptor blockade (+BB, propranolol) in the myocardial electrical impedance (MEI, right), and heart-rate (HR, left) response(s) to

xxii exercise; β-adrenoceptor blockade blunted the MEI response to exercise. SMT = submaximal exercise test...... 120

Figure 5.5: Representative (top) and overall/mean (bottom) myocardial electrical impedance (MEI) response to direct β-adrenoceptor stimulation at rest (via escalating-dose infusion of isoproterenol), showing dose-dependent MEI decrease...... 121

Figure 5.6: Comparative myocardial electrical impedance (MEI) response to either submaximal exercise (white) or acute rate-matched ventricular pacing (black); only exercise decreased MEI...... 122

Figure 5.7: Effects of underlying arrhythmic susceptibility of each animal in the myocardial electrical impedance (MEI) response to exercise; animals prone to ischemia-induced VF

(i.e., S; n = 12) had a significantly larger MEI response to submaximal exercise, when compared those that were resistant (i.e., R; n = 9)...... 123

Figure 6.1: Representative electrocardiogram (ECG) and respective Power-Spectral-

Density (PSD) taken before/during an acute left-circumflex (LCX) coronary artery occlusion (CAO) in a conscious post-MI dog shown to be susceptible to ischemia-induced ventricular fibrillation. Both the standard deviation of the T-wave amplitude within each analysis epoch (a surrogate-marker of T-wave alternans, TWA) and the power ratio

(PSDratio) between the spectral-density at the heart beat frequency (fbeat) and its first harmonic (fHIGH = 2*fbeat) were used in order to quantify temporal repolarization variability before and during ischemia...... 137

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Figure 6.2: Representative action potentials (AP) and Power-Spectral-Densities (PSD) recorded from myocytes isolated from dogs either susceptible (S, bottom) or resistant (R, top) to malignant arrhythmias (from Sridhar et al., 2009); a marked increase in the relative power-density of higher-order frequencies (PSDratio) can be observed in (susceptible) cells presenting of premature depolarization...... 138

Figure 6.3: Representative myocardial electrical impedance (MEI) increases induced by acute ischemia (LCX coronary artery occlusion, CAO) in conscious dogs with a healed myocardial infarct; complete β-AR blockade (+BB, top) blunted the ischemic MEI increase, with the independent blockade both β1- (middle) and β2-ARs (bottom) appearing to mediate such electrotonic protection...... 139

Figure 6.4: Relationship(s) between the ischemia-induced electrotonic uncoupling, as reflected by myocardial electrical impedance changes (i.e., ΔMEI), and the ECG-derived

TWA amplitude, in conscious post-MI dogs known to be either susceptible (S, top) or resistant (R, bottom) to malignant arrhythmias...... 140

Figure 6.5: In an in silico model, a non-uniform cell-to-cell coupling resistivity (Ri) reflecting the heterogeneous electrotonic properties of the interface(s) between remote

(non-infarcted), ischemic, and infarcted (scar) myocardium (A, top left) results in altered propagation: electrotonic uncoupling during modeled ischemia (i.e., coronary artery occlusion, CAO) results in unidirectional block (C, bottom left), while its attenuation by

β-AR blockade prevents it (D, bottom right). Panels B-D show propagating action potentials in four cable segments (cells # 10, 20, 26 and 32)...... 141

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Chapter 1: Introduction

Sudden cardiac death (SCD) is one of the primary causes of natural death in the industrialized world, accounting for over 300,000 deaths—or half of all heart disease mortality—in the United States each year (Mozaffarian et al., 2015). In particular, most

SCD episodes are attributable to the acute onset of malignant arrhythmias, such as ventricular tachycardia and fibrillation (Huikuri et al., 2001; Goldberger et al., 2014;

Wellens et al., 2014). However, despite significant advances in the understanding of the physiological substrate(s) mediating/facilitating these arrhythmias, risk stratification for

SCD remains difficult and insufficient (Goldberger et al., 2014; Wellens et al., 2014;

Zaman and Kovoor, 2014). Indeed, the majority of SCD episodes occur in patients with either low, intermediate, or no known risk factors (e.g., Wellens et al., 2014), with cardiac arrest being the first symptom of disease in over 20% of the cases (Müller et al., 2006;

Mozaffarian et al., 2015).

Malignant arrhythmias leading to SCD are generally accepted to have a reentrant nature that is established by the ill-timed combination of both an initiating “trigger” (e.g., a premature after-depolarization) and a supporting “substrate” (e.g., conduction slowing/unidirectional block) (Janse and Wit, 1989; Coronel et al., 2010). For instance,

SCD victims and patients at risk commonly present genetic/acquired cellular abnormalities affecting active electrical/ionic properties (e.g., impulse initiation, calcium handling, and/or repolarization), which favor increased myocyte automaticity and after- 1 depolarization (i.e., the “trigger”), particularly when exacerbated by autonomic imbalances/enhanced sympathetic drive (e.g., Chen et al., 2007; Pokorný et al., 2011;

Wellens et al., 2014; Marsman et al., 2014; Shen and Zipes, 2014). Similarly, acute ischemic events (e.g., see Mehta et al., 1997; Myerburg and Junttila, 2012; Tikkanen et al.,

2012) and myocardial infarction (MI) are well-established risk factors for SCD (e.g.,

Adabag et al., 2010; Zaman and Kovoor, 2014), as both provide favorable milieus for the onset/maintenance of arrhythmias by increasing myocardial heterogeneity and conduction abnormalities (i.e., the “substrate”).

Indeed, the exacerbation of intrinsic myocardial repolarization heterogeneities, be it in time (e.g., temporal dispersion) and/or space (e.g., transmural dispersion), has been established as a common mechanistic pathway that can both trigger and sustain arrhythmias both in genetic and acquired disease (e.g., see Antzelevitch, 2005b). Emori and

Antzelevitch (2001), for instance, provided direct evidence linking heterogeneous transmural repolarization to the onset of early after-depolarizations (EADs) and triggered beats precipitating ventricular tachycardia. Similarly, after-depolarizations resulting from spontaneous diastolic sarcoplasmic reticulum (SR) calcium release can leverage spatial differences in repolarization in order to propagate (e.g., Huffaker et al., 2007; Sato et al.,

2014), leading to temporal stochastic oscillations in both intracellular calcium and action potential duration/repolarization that are also pro-arrhythmic (e.g., Salama, 2006; Myles et al., 2011; Karagueuzian et al., 2013). As such, risk-stratification techniques focusing on the evaluation of repolarization abnormalities, particularly during states of autonomic activation (such as exercise) and hindered repolarization reserve have proven helpful

(albeit far from perfect) in identifying patients prone to SCD (Goldberger et al., 2014;

Wellens et al., 2014).

For instance, substantial clinical/laboratory evidence suggests that electrocardiographic indices reflective of changes in temporal and spatial (e.g., transmural) repolarization gradients, such as the duration of the T-wave’s terminal portion (i.e., Tpeak- to-Tend or TPE) or the short-term QT variability, are sensitive markers of arrhythmogenic liabilities (e.g., Lubinski et al., 1998; Medina-Ravell et al., 2003; Antzelevitch, 2005b;

Arteyeva et al., 2013; Porthan et al., 2013; Niemeijer et al., 2014). Oosterhoff and colleagues (2011), for example, showed that the short-term dispersion of repolarization, an index reflective of both stochastic and beat-to-beat oscillation in active/passive electrical properties (Heijman et al., 2013), was a good predictor of sudden arrhythmic death in a cohort of patients with structural heart disease. Similarly, temporal changes in the T-wave amplitude resulting from alternans in both action potential duration and/or intra-cellular calcium have been linked to an increase in mortality in patients with heart disease (e.g.,

Rosenbaum, 2001 and Narayan, 2006). As such, the assessment of microvolt T-wave alternans (TWA or MTWA) during low-intensity exercise has been shown to predict not only arrhythmic events in post-MI patients, but also arrhythmia-free survival in patients with ventricular dysfunction (Narayan, 2006; Cantillon et al., 2007; Amit et al., 2010;

Verrier et al., 2011; Calò et al., 2011; Merchant et al., 2012; Shizuta et al., 2012).

Notably, concomitantly with the recognition of myocardial conduction/repolarization heterogeneities as a primary mediator of malignant arrhythmias

(e.g., Bishop et al, 2013), the understanding of the intrinsic electrotonic, or passive

3 electrical, forces that homogenize propagation in the myocardium has also gained relevancy. Indeed, since Engelmann (1875) postulated that myocytes were joined to each other, living and working together (“healing over” principle as referenced by Janse et al.,

2002), vast evidence has established the crucial role that electrotonic forces play in the determination of arrhythmic propensity in both the normal and pathological myocardium.

Toyoshima and Burgess (1978), for example, demonstrated that electrotonic interactions, particularly during ventricular repolarization, are a key factor in the intracellular distribution of ectopic/pro-arrhythmic potentials.

Electrotonic Coupling

This is, perhaps, an opportune time to provide a brief overview of the concept of electrotonic coupling, and therefore also to recognize the role of Dr. Silvio Weidmann, who laid the “engineering” foundations of modern cardiac electrophysiology. By his own admission, Dr. Weidmann’s work provided the quantitative basis to Engelmann's “healing over” concept (Niggli et al., 2006), recognizing the importance of passive myocardial electrical properties to cardiac excitation/conduction (Weidmann, 1952). Weidmann

(1952) applied earlier theoretical principles (Hodkings and Rushton, 1946) to model a cardiac (purkinje) fiber as a core-conductor/cable, having “a well-conducting protoplasm and by a thin surface membrane having a high resistance (rm) and a large capacity (cm) per unit area”. Such a “cable model” considers that the intracellular and extracellular potentials vary along the longitudinal axis only, and that both the cytoplasm and the extracellular spaces can be approximated as ideal ohmic conductors (with ri and re respective resistances

4 per unit length). Hence, propagating cardiac action potentials along a fiber can be described by the following second-order partial differential equation (PDE):

 1   2    V  c  V  I V ,t   2 m m m ionic   (1)  re  ri  x t

2 where Iionic is the nonlinear membrane ionic current density (µA/cm ), defined by the active/stochastic electrical properties of the cell. Alternatively, multiplying by rm, equation

(1) can be re-written as

2  2 V  V V V,t (2) x2 m t m ionic

r where   m is the length (and/or space) constant, a parameter that indicates how far re  ri a stationary current will “electrotonically” influence the voltage along the fiber, and

  rmcm is the trans-membrane time-constant (e.g., Plonsey, 1969).

Under this theoretical framework, Weidmann (1952) showed that the electrical length/space constant was much larger than the cell length (defining the basis for the

“electrotonic” modulation/homogenization of potentials across adjacent cells) and observing that the internal longitudinal resistance (myoplasm in series with cell-to-cell contact) was much smaller than the membrane resistance. This result suggested the existence of low-resistance connections between neighboring cells, which Weidmann later demonstrated by studying and modeling (via the use of analog electrical circuits) the diffusion of potassium (Weidmann, 1960 and 1966), showing that permeability of the intercalated disk to this ion was far greater than of the cell membrane. Since then, both electrotonic coupling and these inter-cellular junctional connections/channels, or “gap 5 junctions” (as well as their conforming proteins), have become vital to the understanding of propagation and arrhythmogenesis. Indeed, numerous reviews have been written on the subject, extensively summarizing/presenting the importance of cell-to-cell passive electrical coupling/uncoupling to the onset/maintenance of malignant arrhythmias (e.g., for review see Kleber, 1992; De Mello, 1999; Cascio, 2001; De Groot and Coronel R, 2004;

Cascio et al., 2005, Wit and Peters, 2012; Dhein et al., 2014; Kleber and Saffitz, 2014). For instance, both Dhein et al. (2014) and Kleber and Saffitz (2014) recently discussed/described how electrotonic interactions, mediated both by junctional coupling proteins and geometrical/physiological factors, modulate source-sink phenomena in order to trigger/sustain normal cardiac rhythm and arrhythmogenesis.

In addition, it is also important to note that electrotonic interactions among neighboring myocytes, modulated by the combined passive electrical properties of the intra-, extra- and intercellular (i.e., junctional) pathways, serve to orchestrate cardiac propagation. At rest, myocardial cells are depolarized (i.e., have a negative membrane potential) due to the different ionic composition of the intracellular and extracellular spaces, and the selective permeability of the cell membrane to the various types of ions

(e.g., see Sperelakis, 1995; Delmar, 2006). Upon excitation, the membrane capacitance charges, triggering an abrupt change in the cell’s permeability to sodium (Na+) ions (i.e., opening of the sodium channels), which move following the electrochemical gradient into the cell, resulting in depolarization (and the generation of an action potential characterized by the specific active electrical properties of the cell). As these action potentials propagate, the depolarization (i.e., positively or non-negatively charged) wave-front acts as an

6 electrotonic source of depolarizing current for the adjacent repolarized tissue (the sink)

(Joyner and Sugiura, 1991; Shaw and Rudy, 1997; Xie et al., 2010).

Not surprisingly, electrotonic coupling is a well-established factor modulating both repolarization disturbances and arrhythmic mechanisms; in short, poorly-coupled cells have limited electrotonic influence over their neighbors (i.e., lower space constant facilitating heterogeneities) and therefore, are more likely to exhibit source-sink mismatches and pro-arrhythmic behaviors (e.g., Joyner and Sugiura, 1991; Shaw and

Rudy, 1997; Pastore and Rosenbaum, 2000; de Groot and Coronel, 2004; Wit and Peters,

2012; Saffitz and Kléber, 2012). For instance, consistently with its clear involvement in

SCD, myocardial ischemia has been shown to acutely depress electrotonic coupling (e.g.,

Kléber et al., 1987; Cascio et al., 1990; Smith et al., 1997), facilitating and coinciding with the onset of arrhythmias (e.g., de Groot and Coronel, 2004). Indeed, cell-to-cell uncoupling, occurring either acutely (e.g., ischemia) and/or chronically (e.g., due to electrophysiological/structural remodeling) (Kessler et al., 2014), is thought to be a requirement for the propagation of source-sink (Xie et al., 2010) and chaotic/stochastic

(Sato et al., 2009) after-depolarizations leading to arrhythmias, particularly in the setting of local autonomic stimulation (Myles et al., 2012). Similarly, Myles et al. (2010) and

Storm et al. (2010) showed that pro-arrhythmic transmural gradients of repolarization are determined and modulated by electrotonic interactions between cells. Moreover, reductions in passive electrical coupling/loading can lead to both localized refractory period heterogeneity (i.e., a substrate for reentry) (Bishop et al., 2014) as well as to the

7 exacerbation of intrinsic/extrinsic pro-arrhythmic temporal (e.g., beat-to-beat) dispersion of repolarization (Pueyo et al., 2011; Walmsley et al., 2015).

Conversely, enhanced/preserved electrotonic coupling, e.g., via pharmacological gap-junction modulation (Hennan et al., 2006; Kjølbye et al., 2008), has been shown to blunt myocardial ionic heterogeneities (e.g., Quan et al., 2007; Huelsing et al., 2000; Himel et al., 2013), masking and even reducing pro-arrhythmic risk. Indeed, electrotonic coupling can suppress EADs (Huelsing et al., 2000; Zaniboni et al., 2000; Himel et al., 2013), and reduce spatial/temporal dispersion of repolarization (Zaniboni et al., 2000; Quan et al.,

2007; Heijman et al., 2013). Zaniboni and colleagues (2000), for example, showed that stochastic temporal (beat-to-beat) repolarization variability and EAD formation were limited by electrotonic interactions. Similarly, electrotonic interactions have been shown to modulate pro-arrhythmic oscillations/alternans in both repolarization and intracellular calcium in silico, and more recently, also in vitro/vivo (Pastore and Rosenbaum, 2000;

Watanabe et al., 2001; Cherry and Fenton, 2004; Sato et al., 2006; Kjølbye et al., 2008; Jia et al., 2012). For instance, Jia et al. (2012) recently demonstrated in vitro and in silico that the spread of cellular calcium instabilities/alternans is directly (albeit not monotonically) mediated by cell-to-cell electrotonic coupling, with lower coupling enhancing calcium- alternans amplitude.

Furthermore, while the classic “ingredients” of a reentrant arrhythmia (i.e., trigger and substrate) have been commonly treated as independent (but co-existing) phenomena, recent literature demonstrates marked electrotonic interdependences between them (e.g.,

Poelzing and Rosenbaum, 2009). Indeed, several groups have demonstrated, both in the

8 laboratory (e.g., Laurita et al., 1996; Laurita and Rosenbaum, 2000; Myles et al., 2010;

Walton et al., 2013; Cabo, 2014) and the clinic (Hanson et al., 2009), the ability of well- timed premature stimulus to electrotonically modulate the pro-arrhythmic properties of the myocardium (substrate), particularly in the setting of remodeled ventricles with altered coupling (e.g., post-MI). For instance, Cabo (2014) recently showed in silico that premature stimuli propagate dynamically across structural heterogeneities post-MI, altering both their nature (i.e., prematurity/propensity) as well as the passive myocardial electrical properties that not only modulate such propagation but determine the ability of these premature stimuli to trigger an arrhythmia. Similarly, Walton et al. (2013) showed that tissue geometry and action potential morphology determine the degree of modulation that electrotonic coupling exerts on the dispersion of myocardial repolarization.

Interestingly, evidence also suggests that electrotonic remodeling could trigger, by itself, pro-arrhythmic adaptive processes affecting active electrical properties. For example, Libbus et al. (2004) showed that electrotonic loading acutely reduced early action potential repolarization in the epicardium, indicating remodeling (attenuation) of the transient outward potassium current (Ito). Down-regulation of Ito provides the cell with the capacity to deliver greater electrotonic current to partially uncoupled cells downstream

(Wang et al., 2006; Spitzer et al., 2006; He et al., 2015), thereby acting as an intrinsic

“impedance matching” (or load balancing) mechanism. However, it is also accepted that slowing of early repolarization prolongs action potential duration and increases repolarization heterogeneities, especially in remodeled myocardium (Lue and Boyden,

1992; Qin et al., 1996; Liu et al., 2004; He et al., 2015; Long et al., 2015), which can predispose to the development of potentially lethal reentrant arrhythmias.

Remarkably, despite the unequivocal mechanistic relationship(s) between passive electrical changes and arrhythmias, no study to date has directly assessed the ability of indices reflective of electrotonic coupling to stratify arrhythmic susceptibility in vivo. Thus, the studies described/presented within this dissertation/document leveraged both a well- established in vivo model of SCD/lethal arrhythmias post-MI (e.g., see Billman, 2006), and measurements of passive electrical properties via complex electrical impedance spectrum

(e.g., Kléber et al., 1987; Howie et al., 2001; Dzwonczyk et al., 2004), in order to establish the relevance and arrhythmic prognostic value of electrotonic changes/heterogeneities.

In particular, the remaining of this document/dissertation is divided into five different chapters that both describe and discuss independent experiments that aimed to probe/address electrotonic interactions and remodeling under various clinically-relevant settings, such as those of acute ischemia, MI, and autonomic activation. Notably, all of the results/observations presented have been published, either completely or partially, as independent manuscripts. For instance, the first two chapters (2 and 3 – del Rio et al., 2005; del Rio et al., 2008a), describe early passive electrical changes triggered by acute coronary occlusion, both in humans and in laboratory models—in particular, chapter 3 (del Rio et al., 2008a) further explores acute ischemic electrotonic uncoupling, and its interaction with the autonomic nervous system (i.e., electrical parasympathetic stimulation), in the setting of normal cardiac physiology. Meanwhile, chapter 4 (del Rio et al., 2008b) describes electrotonic changes following a sustained ischemic insult leading to MI—more

10 importantly, the experiments described in this chapter establish/demonstrate a relationship between post-MI passive-electrical remodeling and arrhythmia susceptibility. Finally, chapters 5 (del Rio et al., 2015) and 6 (del Rio et al., 2004) test the interrelationship(s) between acute sympathetic autonomic activation and electrotonic coupling in the post-MI heart, both at rest and/or during a concomitant ischemic insult; in these two chapters, the link between electrotonic changes and SCD susceptibility is further addressed. Overall, these experiments successfully established passive electrical chronic remodeling and acute heterogeneities in vivo as a risk/prognostic factor for SCD/arrhythmias susceptibility.

Chapter 2: Early Electrotonic Changes during Acute Ischemia

Electrotonic derangements, as reflected by changes in myocardial electrical impedance (MEI) and physiological end-points have been correlated during acute ischemia. However, the importance of MEI’s early time course is not clear. This multi- specie study, published (in part) in the Journal of Applied Physiology (del Rio et al., 2005) evaluated such significance by comparing the temporal behavior of MEI during acute total occlusion of the left anterior descending coronary artery (LADa) in anesthetized humans, dogs and pigs. Here, interspecies differences in three MEI parameters (baseline, time to plateau onset, and plateau value normalized by baseline) were studied, and noteworthy differences in the MEI time to plateau onset were observed: in dogs, MEI ischemic plateau was reached after 46.3±12.9 min of occlusion, a significantly longer period when compared to that of pigs and humans (4.7±1.2 min and 4.1±1.9 min, respectively). However, no differences could be observed between both animal species regarding the normalized MEI ischemic plateau value (15.3±4.7 % in pigs, vs. 19.6±2.6 % in dogs). For all studied MEI parameters, only swine values resembled those of humans. The severity of myocardial supply ischemia, resulting from coronary artery occlusion, is known to be dependent on collateral flow. Thus, as dogs possess a well-developed collateral system (unlike humans or pigs), they have shown superior resistance to occlusion of a coronary artery. Here, the early MEI time course after LADa occlusion, represented by the time required to reach ischemic plateau, was proven to reflect such interspecies differences. 12

Introduction

Despite the impressive amount of literature on the subject, a clear definition of myocardial ischemia remains elusive (Hearse, 1994). However, the term is generally used to describe an imbalance between myocardial oxygen supply and demand that leads to a shift from oxidative to anaerobic metabolism (Ferrari, 1995; Hearse, 1994). These metabolic disturbances affect the high energy stores of the myocardium, causing ionic, functional, and ultrastructural changes, which may ultimately lead to cellular injury and infarction (Jennings et al., 1978; Reimer and Jennings, 1981; Jennings et al., 1990). Thus, since it has been proven that the extent and severity of such changes are modulated by the duration of the oxygen supply/demand imbalance and by the level of myocardial collaterization present in the region at risk (Jennings and Reimer, 1983; Maxwell et al.,

1987; Schaper and Pasyk, 1976; Schaper et al., 1987), any system aiming to detect ischemia correctly should be sensitive to these factors.

Electrical impedance, a passive electrical property, has been shown to reflect the myocardial response to ischemia, being correlated not only to the associated functional abnormalities (Howie et al., 2001; Sasaki et al., 1994), but also with the underlying ionic

(Cascio et al., 1990; Smith et al., 1995; Owens et al., 1997; Cascio et al., 2001) and metabolic (Ellenby et al., 1987; Gebhard et al., 1987) changes in the myocardium.

However, despite the general agreement that myocardial electrical impedance (MEI) increases significantly with supply ischemia (induced by coronary artery occlusion), the relevancy of its early time course to the understanding of the ischemic process is not clear.

Using arterially blood-perfused rabbit papillary muscles, Kléber and colleagues (1987) found that MEI increases in a biphasic manner during ischemia: an early increase immediately following the ischemic insult, a plateau phase, and thereafter, a rapid increase thought to mark cellular uncoupling (Cascio et al., 1990; Cinca et al., 1997; Owens et al.,

1996; Smith et al., 1995).

We, like other investigators (Ellenby et al., 1987; Gebhard et al., 1987; Smith et al.,

1995; Cinca et al., 1997), have observed (unpublished) similar patterns in dogs (Howie et al., 2001) and pigs (del Rio et al., 2002) (see Fig. 2.1), two representative large-animal models traditionally used to study myocardial supply ischemia as can occur in humans

(e.g., during beating-heart coronary artery revascularization). However, these species have well-documented physiological differences, especially regarding collaterals (Maxwell et al., 1987): while dogs possess a well-developed collateral system, pigs (like humans) generally lack it (White and Bloor, 1981; Maxwell et al., 1987), and therefore are more susceptible to supply ischemia (Harken et al, 1981; Schaper et al., 1987; Fujiwara et al.,

1989). Thus, we hypothesize that a formal comparison between the MEI response of dogs and pigs to coronary artery occlusion should highlight those MEI parameters which are able to differentiate between subjects with (e.g., dogs) and without (e.g., pigs) resistance to supply ischemia. To date, such comparison is lacking.

Additionally, we have presented in vivo and in situ measurements of MEI during beating-heart coronary artery bypass surgery in humans, where MEI was shown to respond to coronary artery occlusion/reperfusion during the grafting process (Dzwonczyk et al.,

2004). The availability of such data, collected under similar conditions to those from pigs

14 and dogs, provides a unique opportunity: the side-by-side validation of two large-animal

(supply) ischemia models, canine and swine, against their clinical counterpart. From such a comparison, a better understanding of how well these two models approximate (both in magnitude and temporal behavior) the passive electrical changes seen in human myocardium early during coronary artery occlusion could be gained, further testing the argument that pigs constitute a better model of human disease than dogs (Maxwell et al.,

1987). Hence, this study was designed in order to compare the early temporal behavior of

MEI during acute occlusion of the LAD coronary artery in humans with that of dogs and pigs.

Methods

MEI ischemic data were obtained from three previously presented studies

(performed by this laboratory with prior institutional approval), and divided (by species) into 3 groups:

• Dogs (D): Taken from work performed by Howie et al. (2001), who recorded MEI during acute coronary artery occlusion and reperfusion in anesthetized male dogs. For this interspecies validation study, all animals (n = 10, 22.8 ± 2.0 kg) from the 120 minute LADa occlusion group were chosen.

• Pigs (P): Collected by us as part of a study examining the preconditioning effects of high-dose adenosine on MEI (del Rio et al., 2002). Here, seven (3 male, 4 female) juvenile pigs (n = 7, 28.1 ± 1.0 kg) from the control (placebo) group, subjected to a 10 minute acute LADa occlusion, were selected.

• Humans (H): Measured intra-operatively (with prior informed consent) during elective beating-heart left anterior myocardial revascularization (Dzwonczyk et al., 2004).

At our institution, such procedure involves complete acute occlusion of the coronary artery being grafted (LADa), in order to achieve a clean operating field. In this study, we report on the MEI response to such procedural complete occlusion (13.2 ± 3.96 min) of the LADa in seven (5 male, 2 female) patients (n = 7, 91.4 ± 13.6 kg) with moderate pre-operative

(angiographically-demonstrated) LADa stenosis (70% to 80%, inclusive).

It should be noted that both canine and swine animal protocols were approved by the Institutional Lab Animal Care and Use Committee (ILACUC) at this institution, and adhered to the statutes of the Animal Welfare Act and the guidelines of the Public Health

Service. The human protocol was approved by the Biomedical Sciences Institutional

Review Board (IRB), and complied with the Codes of Federal Regulations Title 45-Part 46 of National Institutes of Health (NIH) and Title 21-Part 50 of the Food and Drug

Administration (FDA).

Despite different aims, all experiments (for the selected groups) shared the same structure: a baseline period of at least 3 minutes, where no interventions were made, followed by complete LADa occlusion. In all cases, isoflurane was used for anesthesia maintenance, hemodynamic variables (including temperature) were maintained within their normal physiologic range, and MEI was measured using identical equipment and technique.

As previously described (Howie et al., 2001; Dzwonczyk et al., 2002; Dzwonczyk et al., 2004), in open-chest conditions, two standard (commercial) temporary pacing wires

(Medtronic Streamline™ 6500 and/or A&E Medical MYO/WIRE™ M-25, 8 – 13 mm2 of exposed surface area) were sutured completely into the mid-myocardial wall, approximately 1 cm apart in the LADa distribution, and distal to the occlusion site (and stenosis, on the human case). From these leads, confirmed (visually) to be in the center of the region rendered ischemic by LADa occlusion, an MEI monitor developed at this laboratory was used to measure the complex myocardial electrical impedance spectrum

(Howie et al., 2001; Dzwonczyk et al., 2002; Dzwonczyk et al., 2004), a combination of the true electrical impedance spectrum of the myocardium and that of the electrode-tissue interface. In short, a computer controlled circuit stimulated the myocardium with a sub- threshold zero-mean bipolar current, consisting of two alternating rectangular pulses

(±5µA, 100µs wide) generated 200ms apart. The complex MEI spectrum was calculated as the ratio (at each frequency) of the current and voltage spectra resulting from the ensemble averages of ten (positive) stimulus pulses and their respective responses. The frequency-domain resolution was either 5.4 (pigs/humans) or 100 (dogs) Hz (Howie et al.,

2001; Dzwonczyk et al., 2002; del Rio et al., 2002; Dzwonczyk et al., 2004). Regardless, all studies reported (every 3s) on the mean MEI modulus over the studied non-uniform frequency range (0.27 – 5.90 kHz, i.e., the spectrum’s main lobe).

Analysis: As MEI has been shown to be sensitive to temperature, electrode system’s geometry and its location in the myocardium (Rush et al., 1963; Robillard and Poussart,

1979; van Oosterom et al., 1979; Steendijk et al., 1993 and 1994; Tsai et al., 2000 and

2002), in order to reduce inter-subject variability we studied MEI changes expressed as

17 percentages from baseline (normalized MEI, Z[n]). Here, baseline MEI (Zo) was defined as the average of the 15 samples immediately preceding the coronary artery occlusion marker (no, inclusive).

Normalized data were analyzed by a moving window technique. A rectangular window of length N (WN[n], n = 30 or 1.5min) was used and successive windows overlapped by N-1 samples; within each window the best linear fit (L[n], in a least-squares sense) to the MEI data was calculated. The slope of the fitted line was used as an estimate of the MEI first derivative at the center of the window (ΔZ[n]). The estimated MEI first derivative served to detect the ischemic plateau onset time sample (np) as defined in (del

Rio et al., 2002); i.e., the sampled time point where ΔZ[n] has fallen to 3% of its maximum value (ΔZp) (see Fig. 2.2). Once np was determined, the MEI ischemic plateau value (Zp) was defined as the average of the 15 MEI measurements immediately following it

(inclusive).

MEI baseline, time to plateau onset and normalized plateau value are presented as means with standard deviations (mean ± sd). Mean intergroup (cross-species) differences in these parameters were evaluated using the non-parametric Kruskal-Wallis analysis of variance (ANOVA) test. If significant differences were observed, then post hoc pair-wise comparisons between all groups were made using Dunn’s method. On the other hand, for each individual, MEI measurement uncertainty is dominated by measurement noise

(Gaussian) (Tsai et al., 2002), thus mean differences between baseline and plateau values were evaluated using a paired Students t-test assuming unequal variances (two-tailed). In all cases, P<0.05 was considered statistically significant.

Results

As it has been previously shown (Howie et al., 2001; del Rio et al., 2002;

Dzwonczyk et al., 2004), MEI increased immediately and significantly (P<0.05) from baseline after LADa occlusion, reaching an ischemic plateau value subsequently. This held true for all subjects and species studied. However, significant interspecies differences, both in the magnitude and timing of such changes, were observed (see Table 2.1 and Fig. 2.3), with the time to MEI plateau onset showing the most remarkable variation.

In dogs, MEI ischemic plateau was reached after 46.3 ± 12.9 min of LAD occlusion.

A significantly (P<0.05) longer period when compared to that of pigs (4.7 ± 1.2 min), and humans (4.1 ± 1.9 min). Similarly, baseline MEI values on pre-ischemic (pre-occlusion) myocardium differed (P<0.05) between canines (781 ± 39 Ω) and the other species studied

(swine: 444±67 Ω, humans: 489±135 Ω).

Although baseline measurements showed significant differences between dogs and pigs, no differences could be observed between these two species regarding their normalized ischemic plateau value, a parameter traditionally used to correlate MEI with ischemia (Kleber et al., 1987; Ellenby et al., 1987; Gebhard et al, 1987; Smith et al., 1995;

Cascio et al., 1990; Owens et al., 1996; Cascio et al., 2001; Howie et al., 2001) and other diseased states of the myocardium (Grauhan et al, 1996; Pfitzmann et al., 2000).

Normalized MEI reached 19.6 ± 2.6 % and 15.3 ± 4.7 % at plateau in dogs and pigs, respectively. Interestingly, humans, who also showed baseline differences with canine data, had a lower normalized ischemic plateau value (11.0 ± 6.0 %, P<0.05). It should be

19 noted that regarding all MEI parameters studied (time to plateau onset, baseline and normalized ischemic plateau value), swine and human groups were only distinguishable by the wider distribution of values observed in humans (as expected due to the intrinsic variability of coronary artery disease).

Discussion

The degree of myocardial collaterization is recognized as an important interspecies differentiating factor (Harken et al., 1981; Fujiwara et al., 1989), and has been shown to influence the extent and severity of myocardial ischemic injury directly (Jennings and

Reimer, 1983; Schaper et al., 1987). Thus, as canines possess a well-developed collateral system, unlike humans or pigs, they have been shown to have remarkably higher resistance to supply ischemia as reflected by reduced rates of ATP depletion (Schaper et al., 1987), and smaller infarcts (Fujiwara et al., 1989). Here, the MEI ischemic time course, represented by the time from coronary artery occlusion to MEI ischemic plateau onset, was shown to reflect such interspecies differences in collaterization.

Although originally attributed to a rapid collapse of the intravascular and interstitial spaces (“vascular collapse”) (Kleber et al., 1987), the exact mechanism determining the early behavior of MEI under acute ischemia remains unknown. Kléber et al (Fleischhauer et al., 1995; Yan et al., 1996) demonstrated that initial impedance rise during zero-flow ischemia is sensitive to osmotically-induced cell swelling (i.e., to changes of the extra/intracellular volume relationship). As such, the time-dependent concentrations of intra/extra-cellular ions (such as calcium, [Ca2+]i, hydrogen, [H+]i, and potassium, [K+]e)

(Smith et al., 1995; Owens et al., 1996; Cascio et al., 2001), anerobic byproducts and ATP

(Ellenby et al., 1987; Gebhard et al., 1987; Sugiura et al., 1990) found on the ischemic myocardium have been shown to play an important role. For example, extracellular potassium ([K+]e) accumulation during ischemia is documented to follow a tri-phasic time course (Hill and Gettes, 1990; Kleber, 1984) similar to that of MEI. Furthermore, the secondary rise (i.e., third phase) of both parameters is closely coupled in time (Cascio et al., 1990; Smith et al., 1995; Owens et al., 1996). Indeed, Owens et al. (1996) demonstrated that such terminal (secondary) rise signals ischemic electrical cell-to-cell decoupling, and only occurs after a significant accumulation in [Ca2+]i and [H+]i (as initially suggested by

Cascio and colleagues, 1990).

However, using paired ventricular myocytes, Sugiura et al. (1990) demonstrated that (electrical) junctional conductance changes with ATP concentration, independently of intracellular free gap-closing ions (such as [Ca2+]i), thus suggesting also a direct relationship between active ionic transport and the MEI. In other words, they proposed that the depletion of myocardial high energy stores (e.g. during ischemia) leads to increases in intercellular electrical impedance (the reciprocal of electrical conductance) by means of modifying the ATP-mediated ionic conductivity through the cellular membranes.

Interestingly, the activation of ATP-dependent potassium (KATP) channels has been shown not only to be mediated by myocyte swelling (Priebe and Beuckelmann, 1998), but also to affect the timing and magnitude of early [K+]e accumulation (Rodriguez et al.,

1002), a well-established cause of early electrical disturbances during ischemia (Morena et al., 1980). The role of these channels as part of the underlying mechanism behind changes

21 in the passive electrical properties of ischemic myocardium is further strengthened by the work of Bollensdorff et al. (2004); they demonstrated that KATP-channels mediate [Na+]e influx to the myocytes, and therefore, [Ca2+]i overload leading to cell-to-cell uncoupling.

Hence, a slower MEI progression to plateau after coronary artery ligation (as observed on canine myocardium), is not only suggestive of delayed breakdown in cellular (ionic) homeostasis, but also, of slower ATP depletion rates (i.e., of a less damaging ischemic process), and likely, to delayed cell-swelling. This is in good agreement with the classic results of Shaeper and colleagues (1987) and with the slower [K+]e accumulation in ischemic canine myocardium reported by David et al. (1988) (evident when compared to results from swine [Hill and Gettes,1980; Smith et al., 1995]).

As a result, the MEI timing similarities observed between swine and human data indicate that the passive electrical properties of human myocardium early during coronary artery occlusion are closely modeled by those in pigs. Furthermore, given MEI’s metabolic ties, this supports the argument that pigs are a better model than dogs for acute myocardial supply ischemia as can occur in humans (Maxwell et al., 1987), at least for moderately diseased patients (as those studied here) expected to have a poorly developed collateral system (Cohen et al., 1989; Pohl et al., 2001).

On the other hand, while close MEI time courses may reflect comparable myocardial metabolisms during ischemia, the observed interspecies differences in MEI baseline and normalized plateau values (the other parameters studied) could be suggestive of specialization in the myocardial tissue ultrastructure, as suggested in the literature (van

Oosterom et al., 1979; Sasaki et al., 1994; Steendijk et al., 1994). However, as stated above,

MEI measurements have been shown to be affected by temperature (Tsai et al., 2002), electrode separation (Robillard and Poussart, 1979; Steendijk et al., 1993), depth (van

Oosterom et al., 1979; Tsai et al., 2000) and orientation (relative to the muscle fibers) (Rush et al., 1963; van Oosterom et al., 1979; Steendijk et al., 1993 and 1994). For instance,

Steendijk and colleagues (1994) reported significantly different MEI values for measurements made longitudinally and transversely across canine myocardial fibers

(313±49 and 487±49 Ω•cm @ 5 kHz, respectively). Hence, as the spacing and orientation of the temporary pacing wires (used to acquire MEI data) on the beating heart are difficult to control, conclusions based on the absolute difference among baseline values between the species are not possible.

Furthermore, as the normalized MEI plateau value indicated coronary artery occlusion precisely but failed to differentiate between the (clearly different) ischemic processes on dogs and pigs, this study emphasizes the relevance of the early ischemic MEI time course. Here, the MEI temporal behavior following coronary artery occlusion

(parameterized as the time to reach MEI ischemic plateau) reflected the higher collateral density of canine myocardium, acting perhaps, as a direct indicator of ischemic resistance and metabolism. Therefore, MEI timing parameters could be a valuable tool during surgical myocardial revascularization procedures, where cardio-protective techniques that seek to enhance the myocardial endurance to ischemia (e.g., cardioplegic arrest, preconditioning, etc.) are currently performed blindly, i.e., with minimal on-line indication of their success.

In conclusion, this study not only confirms MEI as a valid on-line myocardial ischemia monitor, but highlights the importance of including parameters sensitive to the

MEI time course in the ischemia monitoring process.

Table 2.1: MEI parameters during acute coronary occlusion in various species†.

Pigs Dogs Humans Parameter P<0.05* (P, n=7) (D, n=10) (H, n=7)

Baseline () 444±67 781±39 489±135 P/D, D/H

Time to Plateau Onset (min) 4.7±1.2 46.3±12.9 4.1±1.9 P/D, D/H

Normalized Plateau (%) 15.3±4.7 19.6±2.6 11.0±6.0 D/H

*: P/D: Pigs vs. Dogs; P/H: Pigs vs. Humans; D/H: Dogs vs. Humans. †: Collected at 37.1±0.2 °C (pigs), 38.0±0.3 °C (dogs), and 36.3±0.5 °C (humans).

1000

950 I II III

) 900



] ] ( n

[ 850

z , ,

800 MEI

750

700 0 10 20 30 40 50 60 Time (min)

650

600 I II III

) 

550

] ] (

[ z

, , 500 MEI

450

400 0 10 20 30 40 Time (min)

Figure 2.1: Sample MEI data (Z[n], open circles) measured on a dog (top panel, from Howie at al., 2001) and a swine (bottom panel, from del Rio et al., 2002) showing the MEI biphasic rise after coronary artery occlusion (at 0 min): early increase immediately following the ischemic insult (I), a plateau phase (II), and rapid increase thought to mark cellular uncoupling (III)

)

zp 

L[ni]

] ] (

n [

z WN[ni] , ,

zb MEI

no ni np

] ] (%) Δz[ni] n [ Δz

z p Δ

no ni np Time (sample)

Figure 2.2: Sample data analysis procedure on swine data (from del Rio et al., 2002). TOP: Original MEI measurements (z[n], open circles) indicating moving analysis window (WN) and best (mean-square) linear fit (L) at sample n. BOTTOM: MEI first derivative (ΔZ[n]) estimated from the linear fit (L[n]), indicating ischemic plateau threshold (ΔZp, i.e., 3% of the maximal derivative) and value at ni (ΔZ[n]). Occlusion marker (no) and ischemic plateau sample (np) are used to calculate baseline and ischemic plateau MEI values (Zb and Zp respectively). NOTE: The fact that ΔZ[n] appears to increase before coronary artery occlusion (no) is an artifact of the non-causal windowing technique used.

16 Pig (P)

14 Dog (D) ](%)

Human (H) n

[

Δ )

12 (P)

 ] (

n 10

](%)

[



z Δ , 8 (H)

](%)

[

z Δ 4 (D)

2 Normalized MEI Normalized 0

-2

-1 0 1 2 3 4 5 Time (min)

Figure 2.3: Sample normalized MEI data (Z[n]) after coronary artery occlusion: Original MEI measurements on representative pig (open circles, from del Rio et al., 2002), dog (open triangles, from Howie et al., 2001) and human (open stars, from Dzwonczyk et al., 2004) indicating occlusion marker (at 0 min). Inserts: Estimated MEI first derivatives (ΔZ[n]) for each subject, indicating ischemic plateau threshold (ΔZp, i.e., 3% of its maximum value). Observe that ΔZ[n]) in the dog does not fall below the ischemic plateau threshold (ΔZp), i.e., MEI does not reach ischemic plateau.

Chapter 3: Parasympathetic Effects on Early Ischemic Electrotonic Changes

Parasympathetic activity during acute coronary artery occlusion (CAO) can protect against ischemia-induced malignant arrhythmias; nonetheless, the mechanism mediating this protection remains unclear. During CAO, early myocardial electrotonic uncoupling

(described in Chapter 2) is associated with autonomically mediated 1A-arrhythmias and can modulate pro-arrhythmic dispersion of repolarization. Thus, the effects of acutely enhanced or decreased cardiac parasympathetic activity on early electrotonic coupling during CAO, as measured by myocardial electrical impedance (MEI), were investigated in this study, published (in part) in the Experimental Physiology journal (del Rio et al.,

2008a). In short, anesthetized dogs were instrumented for MEI measurements, and left- circumflex occlusions were performed in intact (CTRL) and vagotomized (VAG) animals.

CAO was followed by either vagotomy (CTRL) or vagal nerve stimulation (VNS, 10 Hz,

10V) in the VAG dogs. VNS was studied in two additional sets of animals. In one set heart rate (HR) was maintained by pacing (220 bpm), while in the other set bilateral stellectomy preceded CAO. MEI increased after CAO in all animals. A larger MEI increase was observed in vagotomized animals (+85 ± 9Ω from 611 ± 24Ω, n = 16) when compared to intact controls (+43 ± 5Ω from 620 ± 20Ω, n = 7). Acute vagotomy during ischemia abruptly increased HR (from 155 ± 11 bpm to 193 ± 15 bpm) and MEI (+12 ± 1.1 Ω, from

663 ± 18 Ω). In contrast, VNS during ischemia (n = 11) abruptly reduced HR (from 206 ±

6 to 73 ± 9 bpm) and MEI (-16 ± 2 Ω, from 700 ± 44 Ω). These effects of VNS were 29 eliminated by pacing but not by bilateral stellectomy. VNS during CAO also attenuated

ECG-derived indices of ischemia (e.g., ST-segment: 0.22 ± 0.03 vs. 0.15 ± 0.03 mV) and of rate-corrected repolarization dispersion (TPEc: 84.5 ± 4.2 vs. 65.8 ± 5.9 ms, QTc: 340

± 8 vs. 254 ± 16 ms). VNS during myocardial ischemia exerts negative chronotropic effects, limiting early ischemic electrotonic uncoupling and dispersion of repolarization, possibly via a decreased myocardial metabolic demand.

Introduction

Decreased cardiac parasympathetic control in the setting of abnormally augmented sympathetic drive, as can occur acutely during myocardial ischemia (Malliani et al. 1969;

Minisi 2005), increases the risk of sudden cardiac death due to malignant arrhythmias

(Zipes & Rubart 2006). For instance, in dogs with decreased vagal nerve activity (due to congestive heart-failure) ventricular arrhythmias are preceded by elevated left stellate ganglion nerve activity (Ogawa et al. 2007). Similarly, indices of cardiac vagal activity, such as heart rate variability and/or baroreceptor reflex sensitivity (Task Force of the

European Society of Cardiology and the North American Society of Pacing and

Electrophysiology 1996) are also markedly attenuated in animals prone to ischemia- induced ventricular fibrillation both before (Schwartz et al. 1988; Vanoli et al. 2008) and after myocardial infarction (Billman et al. 1982; Billman 2006). Comparable results have been reported in the clinic. For example, depressed baroreflex sensitivity is a strong predictor of cardiac mortality (La Rovere et al. 1998) and life-threatening arrhythmias (La

Rovere et al. 2001) in patients surviving myocardial infarction. More recently, Passariello

30 et al. (2007) reported that an acute decrease in heart rate variability preceded (within minutes) the onset of ischemic sudden death in patients with ECG Holter monitoring.

Consequently, interventions that increase parasympathetic control during myocardial ischemia are known, both clinically and experimentally, to have potent anti-arrhythmic effects. Numerous studies demonstrate that vagus nerve stimulation increases the electrical stability of the ventricle, preventing arrhythmias during ischemia (Ando et al. 2005; Corr

& Gillis 1974; Kent et al. 1973; Vanoli et al., 1991). This potent cardio-protection can be mimicked by direct pharmacological cholinergic-receptor activation (Billman 1990; De

Ferrari et al. 1993) or by the enhanced cardiac parasympathetic balance induced by aerobic exercise training (Billman 2002; Billman & Kukielka 2006, 2007). However, the mechanisms responsible for this vagally mediated modulation of arrhythmias during acute myocardial ischemia remain largely to be determined.

Decreased electrotonic coupling has been proposed as a possible mechanism that provokes malignant arrhythmias during ischemia (e.g., de Groot & Coronel 2004; Cascio et al. 2005). Passive electrical changes in the myocardium, as measured by myocardial electrical impedance, occur in a biphasic manner immediately following coronary artery occlusion (Kléber et al. 1987), coinciding temporally with the well-established ischemic time course for extracellular potassium ([K+]o) accumulation (Cascio et al. 1990; Owens et al. 1996; Smith et al. 1995) and the bimodal distribution of arrhythmias (Cinca et al.

1997; Smith et al. 1995). For example, spontaneous ventricular arrhythmias during the first

30 minutes of ischemia are known to occur in two distinct phases (Kaplinsky et al. 1979).

The first phase, called “immediate” or “1A”, occurs 2 - 12 minutes after coronary occlusion

31 and is associated with ST-segment changes, as well as with a reversible myocardial electrical impedance increase (Cinca et al. 1997; Smith et al. 1995). The second phase or

“1B”occur later during ischemia after a period of relative electrical stability (reflected by

[K+]o and impedance plateaus) and is associated with a secondary larger and generally irreversible myocardial electrical impedance increase due to decreased conductance though gap junction channels (Cascio et al. 2005; De Groot & Coronel 2004).

Notably, a close mechanistic relationship exists between ischemic autonomic activation, passive electrical changes, and the arrhythmias that arise within minutes (type

1A arrhythmias) after a coronary occlusion (Zaza & Schwartz 1985; de Groot & Coronel

2004). Both vagal stimulation (Redwood et al. 1972) and reduced sympathetic activity

(Cinca et al. 1987) produce significant reductions in the magnitude of ST-segment deviations associated with myocardial ischemia. Meanwhile, these ST-segment shifts are closely associated with the metabolic derangements that increase extracellular resistivity

(initial impedance rise) early after coronary occlusion (Yan et al. 1996; Smith et al. 1995;

Cinca et al. 1997). Myocardial ischemia causes –within seconds– a shift from oxidative to anaerobic metabolism (Reimer et al. 1981), where osmotic load is rapidly increased due to the intracellular accumulation of metabolic byproducts. The increased osmolarity triggers cell swelling, shrinkage of the extra-cellular space, and the activation of pro-arrhythmic ionic volume regulatory processes (e.g., K+ efflux via opening of KATP channels) (Yan et al. 1996; Priebe & Beuckelmann 1998) that modify the passive electrical properties of the myocardium and lead to injury currents (ST/TQ segment changes). Moreover, the net result of these changes is a decreased space constant and, thereby, a reduction in the electrotonic

32 influence that neighboring cells exert on each other (Cascio et al. 2005). This loss of homogenizing passive electrical (i.e., electrotonic) coupling between adjacent regions of myocardium exacerbates the intrinsic electrical heterogeneities within the ventricle, especially during repolarization (Antzelevitch 2005; Laurita et al. 1997; Rudy 2005).

Increased dispersion of repolarization is a common finding in patients at risk of sudden cardiac death (Antzelevitch & Oliva 2006). In addition, depressed electrotonic coupling may facilitate triggered activity (de Groot 2002) and repolarization alternans (Wilson &

Rosenbaum 2007) leading to arrhythmias.

Hence, it is possible that increased cardiac parasympathetic activation during ischemia may protect against arrhythmias by modulating the passive electrical properties governing electrotonic coupling in the myocardium. Notably, the vagal innervation of the ventricles is sparser and less well-defined when compared to that of the atria (Higgins et al. 1973; Zang et al. 2007), as reflected, for example, by the dominant chronotropic effects of vagal stimulation (e.g., Brack et al. 2004). Nonetheless, changes in cardiac parasympathetic drive can alter (via the heart rate modulation) metabolic demand during ischemia (Senges et al. 1983; Sammel et al. 1983; Sidi et al. 1995), and therefore, it may indirectly influence the ventricular passive electrical properties early during ischemia.

Thus, it was the purpose of the present study first to investigate the effects of increased or decreased cardiac parasympathetic nerve activity on the early electrotonic uncoupling elicited by a coronary artery occlusion and then determine the mechanisms responsible for these vagally mediated changes electrotonic coupling. Myocardial electrical impedance was used as a marker of passive electrotonic coupling of the ventricular tissue. In particular,

33 the hypothesis that cardiac vagal stimulation would attenuate the ischemically induced early increase in myocardial eIectrical impedance secondary to reductions in heart rate was tested.

Methods

The principles governing the care and treatment of animals, as expressed by the

Declaration of Helsinki and as adopted by the American Physiological Society, were followed at all times during this study. In addition, the Ohio State University Institutional

Lab Animal Care and Use Committee approved all the procedures used in this study.

Surgical preparation: Mongrel dogs (19.5 ± 0.4 kg, 15.4 to 23.6 kg, n = 36) were sedated with morphine sulfate (1mg/kg sc) and anesthetized to effect by intravenous administration of alpha-chloralose/urethane (50mg/kg and 500mg/kg, respectively) via a catheter percutaneusly placed into a cephalic vein. This anesthetic regime has been previously shown to preserve indices of parasympathetic tone (Halliwill et al. 1992). Artificial ventilation with room air was established and maintained by means of a cuffed endotrachreal tube connected to a respirator (Harvard Apparatus, model 613). A heparinized saline-filled catheter was inserted into a femoral artery and connected to a pre- calibrated pressure transducer (Biopac Systems, Inc., model TSD104A/DA-100C) to measure arterial pressure. Body temperature was monitored with a rectal probe (Yellow

Springs Instrument Co, Inc., model 701) and maintained within the physiological range with a circulating heated-water blanket (Gaymar Industries, Inc., model TP-500).

A midline incision in the neck was performed and the vago-sympathetic trunks were carefully isolated at the mid-cervical level. A silk suture was loosely placed around each nerve bundle in order to facilitate future localization and decentralization. The nerves were kept moist throughout the experiment and care was taken to avoid stretching them during manipulation. Subsequently, the chest was opened through a left thoracotomy in the fifth intercostal space and the heart was suspended in a pericardial cradle. A silk suture was loosely passed around the proximal left circumflex coronary artery. The epicardial inter- coronary collaterals in the area perfused by the left circumflex coronary artery were ligated to reduce inter-subject variability in the size of the ischemic area that would result from the coronary occlusion.

Myocardial Electrical Impedance: A commercially available bipolar temporary pacing lead (Medtronic, Inc., Streamline™ 6495) was sutured into the mid-myocardial free-wall supplied by the distal left circumflex distribution (i.e., downstream from the circumflex coronary artery snare site), approximately in-line with the local epicardial fiber alignment.

The myocardial electrical impedance (MEI) lead consisted of a 30 cm insulated coaxial wire (diameter: 0.7 mm) with two discreet distal in-line electrodes (length: 4 mm; surface area: 8 mm2) located 8 mm apart, and was secured in the myocardium (at-risk) by means of a distal (non-conductive) fixation coil. During coronary occlusion, the two electrodes were visually confirmed to be both embedded in the myocardial muscle, and at the center of the region rendered cyanotic.

From this pacing lead, the complex myocardial electrical impedance spectrum of the myocardium was measured as previously described (del Rio et al.2005; Dzwonczyk et al. 2004; Howie et al. 2001). In short, (as summarized in Figure 3.1), a PC-controlled circuit probed the myocardium with a sub-threshold zero-mean bipolar current, consisting of two rectangular pulses of alternating polarity (± 5 µA, 100 µs wide) generated 200 ms apart.

Measurements were made only with the first pulse of each stimulation pair; the subsequent pulse (of opposite polarity) was only used to prevent possible artifacts introduced by the electrode-tissue interface. The complex myocardial electrical impedance spectrum (Zo) was calculated in the frequency domain using Fast-Fourier Transformation (FFT); the ensemble averages of ten stimulus (current) pulses (io) and their respective (voltage) responses (vo) were calculated, transformed into the frequency domain (via FFT), and the ratio between their spectra obtained (at each frequency). The mean modulus (MEIo) of the resulting complex spectrum (i.e., Zo) over the studied frequency range (0.27 – 5.90 kHz) was calculated every 3s and averaged over 30s (i.e., 10 samples).

Experimental protocol: After heart rate and arterial pressure had reached steady state values, several experiments were performed in order to evaluate parasympathetic neural effects on the early reversible electrotonic derangements evoked by coronary artery occlusion (see Fig. 3.2 for summary).

The electrotonic consequences of decreased cardiac vagal activity during an acute ischemic episode were studied by comparing the early myocardial electrical impedance response to coronary artery occlusion between neurally intact (CTRL, n = 7) and previously

36 vagotomized animals (VAG, n = 16) (Figs. 3.2A and 3.2B). In the latter group (i.e., VAG), the cervical vago-sympathetic nerve trunks were tightly ligated and transected before the coronary occlusion. In the former group (i.e., CTRL) the cervical vagus nerves were cut during the coronary occlusion.

The vagal interventions (see Fig. 3.2) were performed during the ischemic episode

(i.e., ligation of the left circumflex coronary artery). In order to investigate the effects of reductions in cardiac parasympathetic efferent activity on ischemic changes in myocardial electrical impedance, a bilateral cervical vagotomy (CTRL group, Fig. 3.2A) was performed after both early electrotonic and electrocardiographic ischemic changes became apparent (3.4 ± 0.3 min after onset of the coronary ligation). Conversely, in order to determine the effects of an acute increase in cardiac parasympathetic efferent activity on ischemic electrotonic changes, vagal nerve stimulation (VAG group, Fig. 3.2B) was performed after ischemia onset. The heart rate response to vagal nerve stimulation was first evaluated prior to the coronary occlusion. After heart rate had returned to baseline (pre- stimulation) values, the left circumflex coronary artery was ligated and the vagal nerve stimulation was repeated as soon as impedance and ECG derangements were obvious (3.5

± 0.6 min after coronary ligation). The vagal nerve stimulation was performed as follows.

The distal ends of the nerves were gently separated from the surrounding tissue and electrically stimulated for 1 min with rectangular pulses (10V, 1ms duration) at 10Hz using an impulse generator (Grass Medical Instruments, model Grass S44, via impulse-isolation unit model SIU105-B). The left and right nerves were stimulated independently (i.e., one after the other) and in random order (recovery for 1 min was allowed in between

37 stimulations). Three animals developed ventricular fibrillation between stimulations (i.e., upon termination of the initial stimulation episode). Unless stated otherwise, the mean response to the vagal nerve stimulation (either left or right) during ischemia is reported 30s after the onset of nerve stimulation.

Two additional sets of animals, also vagotomized prior to coronary artery occlusion

(Fig. 3.2C), were used to investigate any contribution of bradycardia or simultaneous reflex sympathetic activation on the myocardial electrical impedance response to the vagal nerve stimulation. These two groups were as follows:

• In a group of dogs (VAG/P group, n = 8) heart rate was maintained by dual-chamber

(right atrium and right ventricle) pacing during ischemia and vagal nerve stimulation.

Subsequently, pacing was discontinued and the vagal nerve stimulation was repeated (still under ischemic conditions). Overdrive pacing (220 bpm) was established following vagotomy either before (n = 6) or during (n = 2) the coronary ligation, using a programmable stimulator (Medtronic, Inc., model 5325) connected to two additional sets of bipolar wires sutured to the right atrium and the right ventricle.

• In another series of animals (VAG/S group, n = 5), the left and right stellate ganglia were removed prior to coronary occlusion and vagal nerve stimulation. In order to test the efficacy of the bilateral stellectomy, cardiac sympathetic responsiveness was tested before and after the procedure by a bolus injection of sodium nitroprusside (500 µg/kg iv) to decrease arterial pressure and to activate the baroreceptor reflex.

Data analysis: Femoral artery blood pressure (BP) and a standard bipolar single-lead (Lead

II) surface electrocardiogram (ECG) were recorded throughout the experiment. The ECG was recorded via subcutaneous needle electrodes (Biopac, Inc.; model EL450). The signals were band-pass filtered, amplified (Biopac Systems, Inc., BP: model DA100C/ECG: model

ECG100C), and digitally stored (at 500Hz) using a digital data acquisition system (Biopac

Systems, Inc., model MP-100). ECG waveforms were analyzed offline, and fiducial points were determined with the aid of a digital signal processing software (The Mathworks, Inc.,

MATLAB). The R-R interval, QRS duration, SJ-amplitude, and ST-segment deflection

(60ms after the J-point, i.e., ST60) were calculated. The effects of the parasympathetic interventions on indices of ventricular repolarization (Tpeak-Tend interval, TPE [Yan &

Antzelevitch 1998; Opthof et al. 2007] and the QT-interval, QT) were also measured. Since repolarization duration is dependent on heart rate, both raw and rate-corrected values (e.g.,

QTc = QT/R-R0.5) are reported.

Myocardial electrical impedance, heart rate (HR), mean blood pressure (BP), and two hemodynamically-derived indices of myocardial metabolic state, the rate-pressure product (RPP = HR*BPsystolic) (Baller et al. 1981) and the pressure-rate quotient (PRQ =

BP/HR) (Buffington et al. 1989), were averaged over 30s time intervals. The ECG-derived indices were averaged over 5 consecutive beats. Values are reported before and after left circumflex coronary artery occlusion (CAO) at three time-points: 1) baseline: 1min before the onset of ischemia; 2) CAO: 30s before a given intervention during ischemia, i.e., before vagotomy (VAG) or vagal-nerve stimulation (VNS); and 3) CAO + intervention (e.g.,

CAO+VNS): 30s after the onset of these interventions.

All data are presented as mean ± standard error of the mean. Statistical analyses were performed with SigmaStat (Systat Software, Inc.). Within each group, mean differences between values recorded before and after coronary occlusion (i.e., CAO) and/or a selected intervention (e.g., VNS) were compared using a one-way analysis of variance

(ANOVA) with repeated measures. The sphericity assumption (i.e., homogeneity of the covariance matrix) was verified using the Mauchley’s test (NCSS, Inc., NCSS). If this assumption was not met, a non-parametric repeated measures ANOVA on Ranks

(Friedman’s test) was used. In either case, if significant differences were observed, post hoc pair-wise comparisons between time points (baseline, CAO before and after intervention) were made using the Student-Newman-Keuls method. Between groups comparisons (i.e., intact vs. vagotomized) of the changes observed during CAO were made using an unbalanced analysis of covariance (ANCOVA) with the appropriate baseline as the covariate. The effects of left and right vagal nerve stimulation during ischemia were compared using a two-way (nerve: left/right, and time: before/after stimulation) ANOVA with repeated measures on one factor (time). Multi-variable linear regression analyses were used to study the relationship between the effects of CAO and/or VNS on electrotonic coupling (i.e., MEI) with those on ECG-derived parameters (considering HR as a covariate). In all cases, P<0.05 was considered to be statistically significant.

Results

Twelve dogs died prematurely during the experiments. Seven vagotomized animals

(VAG group, n = 5; VAG/P group, n = 1; and VAG/S group, n = 1) developed ventricular

40 fibrillation during coronary artery ligation prior to the onset of the vagal stimulation, while five dogs had malignant arrhythmias following either acute vagotomy (CTRL group, n =

2) or the termination of vagal stimulation (VAG group, n = 3) during ischemia. No attempt was made to resuscitate animals that developed ventricular fibrillation during the experiments.

As expected, myocardial electrical impedance increased significantly after coronary artery occlusion in all animals studied (+69 ± 5.8Ω from a baseline of 626 ± 12.5

Ω, n = 36). These data are consistent with ischemia-induced electrotonic uncoupling that likely reflects early changes in extracellular resistance due to shrinkage of the interstitial space and osmotic cell-swelling (Kléber et al. 1987; Yan et al. 1996; Fleischhauer et al.

1995). However, a larger and faster myocardial electrical impedance increase (P<0.05) was observed in vagotomized animals (VAG: +85 ± 9.1Ω from a baseline of 611 ± 23.6 Ω, at the rate of +34 ± 6.5 Ω/min, n = 16), when compared to intact controls (CTRL: +43 ± 4.6

Ω from a baseline of 620 ± 19.5Ω, at the rate of +12 ± 1.1 Ω /min, n = 7). Vagotomized dogs had higher heart rate (VAG: pre-occlusion 190 ± 5.6 bpm, post-occlusion 207 ± 4.5 bpm vs. CTRL: pre-occlusion 147 ± 14.2 bpm, post-occlusion 155 ± 10.7 bpm, P<0.05) and rate-pressure product (VAG: pre-occlusion 25.7 ± 1.42, post-occlusion 26.9 ± 1.54 x103bpm*mmHg vs. CTRL pre-occlusion 15.3 ± 1.40 x103bpm*mmHg, post-occlusion

15.3 ± 1.46 x103bpm*mmHg, P<0.05) than the intact animals. Thus, myocardial electrical impedance differences following vagotomy likely resulted as a consequence of higher cardiac metabolic demand during ischemia (e.g., faster ATP-depletion, increased anaerobic byproduct accumulation, and greater osmotic cell-swelling). As previously noted, all the

41 intact animals survived the left circumflex occlusion, while five vagotomized dogs developed ventricular fibrillation during ischemia (VAG: 5/16 vs. CTRL: 0/7).

Similarly, vagotomy during coronary occlusion (in CTRL animals) abruptly increased heart rate (from 155 ± 10.7 to 193 ± 14.6 bpm, P<0.05) and negatively affected hemodynamic markers of myocardial oxygen consumption/balance (see Table 3.1).

Myocardial electrical impedance (MEI) also increased rapidly (ΔMEI: +12 ± 1.1Ω, from

663 ± 17.9 to 675 ± 17.2 Ω) following the vagotomy. Moreover, acute vagotomy in the setting of ischemia led to the onset of ventricular fibrillation in 2 animals.

In contrast, as summarized in Figures 3.3-4A and Table 3.2, vagal nerve stimulation during the coronary ligation markedly reduced heart rate and myocardial metabolism (i.e., decreased rate pressure product, RPP and increased pressure rate quotient, PRQ); these effects were accompanied by an abrupt myocardial electrical impedance decrease (VAG group, n = 11; ΔMEI: -16 ± 2.1 Ω, from a baseline of 700 ± 43.7 to 684 ± 44.1 Ω, P<0.05).

Comparable myocardial electrical impedance responses were recorded after either left

(ΔMEI: -16 ± 3.0 Ω; from 675 ± 51.1 to 659 ± 51.4 Ω, n = 9) or right (ΔMEI: -14 ± 1.6 Ω, from 704 ± 48.0 to 690 ± 48.2 Ω, n = 10) vagus nerve stimulation (VNS). Both interventions provoked large, physiologically comparable (albeit statistically different,

P<0.05), reductions in heart rate (Left VNS: from 189 ± 11.4 to 88 ± 12.3 bpm; Right VNS: from 192 ± 11.9 to 63 ± 8.9 bpm). Moreover, as shown in Figure 3.4B and Table 3.2, the electrotonic effects of vagal stimulation were abolished when heart rate was held constant by atrio-ventricular overdrive pacing (VAG/P group, ΔMEI: 0 ± 1.1 Ω; from 697 ± 16.6 to 697 ± 16.6 Ω), but were restored (ΔMEI: -11 ± 0.6 Ω; from 700 ± 15.8 to 689 ± 16.1 Ω)

42 when vagal stimulation was repeated after termination of pacing (HR decreased from 201

± 7.0 to 58 ± 6.4 bpm). Taken together, these data suggest that the myocardial electrical impedance changes induced by the vagal stimulation resulted secondarily from a reduction in heart rate.

In order to control for any confounding effects resulting from a simultaneous cardiac sympathetic activation via the baroreceptor and/or the cardio-cardiac (Malliani et al. 1969; Minisi 2005) reflexes during coronary occlusion or the vagal stimulation, studies were also performed after bilateral stellectomy. Sodium nitroprusside (SNP) injections were used to confirm that bilateral stellectomy eliminated reflex sympathetic regulation of heart rate (i.e., to lower arterial pressure and activate the baroreceptor reflex). Before stellate ganglionectomy, the SNP-induced decreases in blood pressure (-17 ± 3.2 mmHg) were associated with significant HR increases (+26 ± 4.8 bpm). However, this heart rate response was absent (ΔBP: -18 ± 2.5 mmHg, ΔHR: +4 ± 3.2 bpm) following bilateral stellectomy (i.e., blunted baro-receptor reflex, see Fig. 3.5A). After stellectomy, coronary ligation also provoked minimal heart rate changes (ΔHR: +1 ± 2.4 bpm, n = 5), despite a well-defined impedance increase (ΔMEI: +60 ± 6.1 Ω; from a baseline of 678 ± 21.6 to

737 ± 24.5 Ω, n = 5) indicative of ischemia. Although direct comparison should be taken with care, removal of the stellate ganglia may have attenuated the early ischemic myocardial electrical impedance response in vagotomized animals (ΔMEI; VAG/S vs.

VAG, P<0.05).

Vagal nerve stimulation during ischemia elicited similar responses post-bilateral stellectomy (VAG/S group). As illustrated in Figure 3.5B, both heart rate (ΔHR: -120 ±

18.4 bpm; from 155 ± 17.9 to 35 ± 6.9 bpm) and myocardial electrical impedance (ΔMEI:

-23 ± 1.6 Ω; from 749 ± 30.6 to 726 ± 30.9 Ω) decreased markedly with the onset of vagal stimulation in the presence of bilateral stellectomy (VAG/S group; n = 4, P<0.05). These data suggest that myocardial electrical impedance changes induced by vagal nerve stimulation did not result from a concomitant reflex sympathetic activation during ischemia and/or stimulation of the cervical vago-sympathetic trunks (see Limitations section).

Electrocardiographic Data: As expected, coronary artery occlusion elicited a significant

ST-segment elevation (ST60; 0.02 ± 0.01 to 0.19 ± 0.02 mV, P<0.05) and progressive QRS widening (QRS; 76.1 ± 1.2 to 85.5 ± 1.1 ms) in the animals studied (n = 17; VAG and

CTRL groups). However, consistent with the larger ischemic impedance increase described above, the coronary occlusion increased QRS duration to a greater extent in vagotomized dogs (VAG: 75.9 ± 2.0 to 87.5 ± 1.5 ms vs. CTRL: 75.4 ± 1.5 to 82.4 ± 1.4 ms, P<0.05).

In contrast, vagus nerve stimulation during the coronary occlusion attenuated these ischemic electrocardiographic changes (see Table 3.3). Interestingly, the vagal nerve stimulation mediated QRS changes (shortening) during ischemia were linearly correlated with corresponding changes (decrease) in myocardial electrical impedance (R2 = 0.707,

P<0.05). Moreover, the overall myocardial electrical impedance change (ΔMEI) elicited during ischemia and subsequent vagal stimulation provided an independent linear predictor

2 2 of both ST-segment (ΔST60: R = 0.406, P<0.05) and QRS (ΔQRS: R = 0.756, P<0.001) changes.

Coronary ligation prolonged both the QT-interval (QT) and the duration of T- wave’s terminal-portion (TPE), with overlapping vagal stimulation (i.e., during ischemia) producing further lengthening of these intervals (see Table 3.3). However, when corrected for heart rate, these indices of repolarization (i.e., QTc and TPEc) were 1) prolonged by ischemia, and then, 2) significantly shortened by vagal nerve stimulation. No significant linear relationships were found between the vagal nerve stimulation mediated changes in repolarization (whether HR-corrected or not) and the decrease in myocardial electrical impedance. However, when both ischemic and vagally induced effects were considered simultaneously, the prolongation or shortening of HR-corrected parameters could be predicted by changes in impedance (ΔQTc: R2 = 0.887 and ΔTPEc: R2 = 0.809, P<0.05).

The changes in QTc (ΔQTc) were also strongly dependent on heart rate (P<0.05), as previously reported (Chevalier et al. 1998). Throughout the experiment a significant and

HR-independent correlation was also found between absolute TPEc and myocardial electrical impedance values (R2 = 0.561, P<0.001). Although good linear correlation does not imply causality, when considered together, these data suggest that differences in the degree of ventricular electrotonic coupling can explain, at least partially, the variability in myocardial repolarization.

Discussion

The present study investigated the cardiac parasympathetic effects on the early electrotonic changes triggered by acute coronary occlusion, as measured by myocardial electrical impedance. The main findings of the study are: 1) myocardial electrical

45 impedance increased more rapidly following coronary occlusion in vagotomized animals when compared to neurally intact dogs; 2) Bilateral vagotomy during the coronary occlusion further increased myocardial electrical impedance; 3) Acute vagal nerve stimulation abruptly decreased myocardial electrical impedance during the coronary occlusion. This effect was promptly reversed upon termination of vagal nerve stimulation and was abolished when heart rate was maintained by cardiac pacing but was not affected by bilateral stellectomy; and 4) Acute vagal nerve stimulation during the coronary occlusion significantly attenuated electrocardiographic indices of ischemia-induced injury and dispersion of repolarization. Taken together, these results suggest that vagal nerve activity indirectly modulates the passive electrical properties of the myocardium, attenuating the degree of electrotonic uncoupling induced by myocardial ischemia, possibly via a bradycardia-dependent reduction in cardiac metabolic demand.

Sympathetic reflex activation during acute ischemia has been shown to play a fundamental role in reducing the electrical stability of the ventricle, facilitating the development of arrhythmias (Zipes & Rubart 2006). As such, interventions that favor cardiac parasympathetic control have potent cardio-protective actions during ischemia. For example, it is well established that vagus nerve stimulation can limit ischemia-induced arrhythmias (Ando et al. 2005; Corr & Gillis 1974; Vanoli et al., 1991). Conversely, pharmacological or surgical parasympathetic denervation will increase arrhythmia susceptibility (Corr & Gillis 1974; Huang and Yang 1985). Huang and Yang (1985), for instance, reported that bilateral vagotomy lowered the ventricular fibrillation threshold in both normal and ischemic myocardium. However, despite the well-recognized contribution

46 of electrotonic derangements to pro-arrhythmic substrates, to the best of our knowledge, no study to date has investigated parasympathetic influences on the passive electrical changes that accompany myocardial ischemia.

The present study examined, in particular, the initial myocardial electrical impedance increase observed shortly after the onset of ischemia, a period closely associated with the development of the type 1A arrhythmias linked to the activation of the autonomic nervous system (Zaza & Schwartz 1985; de Groot & Coronel 2004). This initial increase in myocardial electrical impedance was first described by Kléber et al. (1987), and has been subsequently found to be sensitive to osmotic cell-volume changes (swelling) due to the intracellular accumulation of anaerobic byproducts during early ischemia, reflecting the concomitant collapse of the extracellular space (Fleischhauer et al. 1995; Yan et al.

1996). In fact, these investigators (Fleischhauer et al. 1995; Yan et al. 1996) proposed that ischemic anaerobic glycolysis and ATP-hydrolysis modulates the passive electrical properties of the myocardium by altering the relationship between extra- and intracellular volumes (as reflected by changes in myocardial electrical impedance), thereby promoting electrotonic uncoupling. As such, early impedance changes after coronary occlusions are sensitive to factors that influence the extent of the ischemic metabolic impairment (e.g., beta-adrenergic blockade, collateral density, ischemic preconditioning) (Jain et al. 2003; del Rio et al. 2005).

Interestingly, and in agreement with the acute attenuation of the early ischemic myocardial electrical impedance increase observed in the present study, vagal nerve stimulation has been shown to reduce myocardial energy demand and metabolic

47 derangements during ischemia (Senges et al. 1983; Sammel et al. 1983; Sidi et al. 1995).

For example, Sammel et al. (1983) reported that, in addition to a potent anti-arrhythmic effect, vagal stimulation during coronary occlusion reduced myocardial creatine kinase depletion, roughly in proportion to a reduction in the heart rate-blood pressure product. In the present study, vagal nerve stimulation elicited marked reductions in both heart rate and hemodynamic indices of metabolic demand during the coronary occlusion (see Table 3.2).

In agreement with previous reports (Kjekshus et al. 1981; Vilaine et al. 1980) electrocardiographic indices of ischemia severity, such as QRS-prolongation and ST- segment elevation were also attenuated by the vagal stimulation. Indeed, vagally mediated

QRS shortening (during ischemia) strongly correlated with the impedance changes following nerve stimulation. These data are consistent with a direct electrotonic modulation of ventricular conduction while the reduced ST-segment changes probably reflect slowed

ATP-depletion and K+ efflux secondary to decreased metabolic demand during vagal nerve stimulation. In the present study, atrio-ventricular pacing abolished the metabolic actions of vagal nerve stimulation (see Table 3.2). As such, the potent parasympathetic modulation of myocardial electrical impedance most likely resulted secondary to vagally mediated changes in metabolic demand during acute coronary occlusion rather than as a consequence of direct modification of myocardial electrotonic coupling.

It should be acknowledged, however, that although undetected in the present study, the activation the ventricular parasympathetic efferent nerves (Zang et al. 2005) might also directly modulate the passive electrical properties of the ischemic myocardium. Ando et al. (2005), for instance, suggested that the anti-arrhythmic effects of vagal stimulation

48 during ischemia might be mediated by changes in the gap-junction proteins. However, while gap junction conductance can be neurally modulated in vitro (Burt & Spray 1988), the established time-course for the ischemia-mediated changes in gap junction conductance is much too slow to explain the rapid changes in impedance that accompanied vagal stimulation or vagotomy in the present study. For example, alterations in the gap junction protein phosphorylation (Beardslee et al. 2000) and intracellular (junctional) resistance

(Kléber et al. 1987) have not been reported during the early (reversible) ischemic period, the time studied in the present study. However, changes in gap junction proteins occur during the secondary, sustained and generally irreversible myocardial impedance increase associated with late 1B-arrhythmias (Smith et al. 1995; Cascio et al. 2005; de Groot &

Coronel 2004). In marked contrast to the myocardial electrical impedance reduction that was observed in the present study, vagal stimulation would tend to “close” gap junctions via increases in cGMP, decreasing intercellular conduction (Burt & Spray 1988) and would thereby increase myocardial electrical impedance.

Acetylcholine (ACh) released during vagal stimulation may also directly modulate early myocardial electrical impedance changes during ischemia by modifying the resistance of the sarcolemmal membrane, via the opening of ion channels under parasympathetic control (e.g., IKACh) (Carmeliet 1999) and/or the activation of cGMP- mediated volume-regulatory mechanisms (Clemo et al. 1992). Some evidence also suggests that vagal stimulation can mimic the effects of ischemic preconditioning (Ando et al. 2005), possibly via a recently reported nitric oxide pathway (Brack et al. 2007).

Ischemic preconditioning is known to delay passive electrical changes following coronary

49 occlusion (Cinca et al. 1997; Jain et al. 2003). Finally, it must be emphasized that the myocardial impedance response to vagal stimulation was abolished when atrio-ventricular pacing prevented the vagally mediated reductions in heart rate. This observation strongly argues against a significant direct action of vagal nerve stimulation on myocardial impedance during acute myocardial ischemia.

Implications: In the present study, acute vagal nerve stimulation attenuated early ischemic passive electrical derangements, even when initiated after the onset of ischemia. This observation may have direct applications in the clinic, where early impedance changes following coronary occlusion have also been reported (Dzwonczyk et al. 2004; del Rio et al. 2005), and may facilitate electrical heterogeneities (Taggart et al. 2000) leading to early arrhythmias (de Groot JR & Coronel 2004; Tice et al. 2007). Moreover, the present study can also provide further insights into the mechanisms mediating parasympathetic protection against arrhythmic death.

Ng et al. (2007) recently reported that stimulation of the vagus nerve reduced the maximum slope of the action potential duration (APD) restitution curve, increasing the ventricular fibrillation threshold. Both steeper (slope >1) and regionally inhomogeneous

(Nash et al. 2006) APD/diastolic-interval relationships can facilitate wave-breaks leading to ventricular fibrillation during ischemia (Chen et al. 2003). Interestingly, electrotonic coupling can prevent pro-arrhythmic alternans (in silico), even if the isolated cells present steep APD restitution properties (Cherry & Fenton, 2004). Thus, besides shifting and flattening the operational point of restitution (Ng et al. 2007), vagal stimulation also tends

50 to homogenize myocardial APDs via preserved electrotonic coupling during ischemia.

Although further examination is needed, this hypothesis is supported by the observation that vagal nerve stimulation attenuated the ischemic changes in ECG-derived indices of repolarization.

In addition, the lessening of the electrical impedance changes (uncoupling) during ischemia via vagal stimulation could improve the electrical stability of ischemic myocardium by suppressing pro-arrhythmic triggered activity and alternans. For example, it has been shown in silico (de Groot, 2002) that the generation and/or propagation of afterdepolarizations can be attenuated by preserved electrotonic coupling. In vivo, Kjølbye et al. (2008) recently reported that preserved electrotonic coupling can suppress arrhythmogenic discordant alternans during global ischemia. Vagal stimulation has also been shown to reduce the occurrence of both cesium-induced early after-depolarizations

(Takahashi et al. 1992), and alternans (Ng et al. 2007).

However, it should be noted that, despite remarkable cardio-protective properties, artificially enhanced parasympathetic drive might also have concomitant deleterious effects. For example, while in the setting of acute myocardial ischemia vagal stimulation may limit energy consumption and spare myocardial tissue from ischemic injury, it also could interfere with normal compensatory contractile recruitment via reflex autonomic activation, further compromising ventricular function. In patients, the risks and benefits of vagal nerve stimulation need to be evaluated. Similarly, therapeutic vagal stimulation protocols need to be designed carefully; for example, acute withdrawal of parasympathetic drive may cause sinus tachycardia and rebound sympathetic activation, increasing

51 metabolic demand as well as calcium entry (Carmeliet 1999), thus facilitating arrhythmias.

In fact, five dogs developed ventricular fibrillation during ischemia following either acute vagotomy (CTRL group, n = 2) or the termination of vagal stimulation (VAG group, n =

3) in the present study.

Limitations of the Study: In the present study, cardiac parasympathetic activity during ischemia was enhanced by electrically stimulating the cardiac ends of the decentralized cervical vagus nerves. However, in dogs, the vagal nerve trunks also contain sympathetic efferent fibers (Randall et al. 1967). Therefore, the simultaneous activation of sympathetic and parasympathetic efferent nerve fibers by electrical stimulation could elicit a mixed response. Indeed, Priola and Fulton (1969) demonstrated that sympathetic ganglionectomy, not only enhanced the cardio-depressant effects of cervical vagal stimulation in normal animals, but also, eliminated a positive inotropic response observed in atropinized dogs.

The stellate ganglia were shown to be the source of these sympathetic fibers that would be activated by the electrical stimulation of the cervical vagus nerves (e.g., see Higgins et al.

1973). In the present study, bilateral stellectomy was performed in a set of animals (VAG/S group), and (as mentioned above) the response to vagal stimulation during ischemia was preserved. Similarly, Rosenshtraukh et al. (1994) observed that complete β-adrenergic receptor blockade did not prevent the QTc response (shortening) during vagal stimulation.

Thus, it is unlikely that cardiac sympathetic activation can account for the acute passive electrical changes (impedance decrease) observed during vagal nerve stimulation in the ischemic myocardium.

On the other hand, cardiac pacing abolished the impedance response to vagal nerve stimulation in the present study. As previously mentioned, these data strongly suggest that vagally mediated reductions in myocardial electrical impedance occur secondary to heart rate-dependent reductions in cardiac metabolic demand. Although these results are in agreement with previous observations on the vagal influence during ischemia (Senges et al. 1983), it should be noted that dual-chamber pacing (right atrium and right ventricle) was used to maintain heart rate during vagal stimulation (VAG/P group). The resulting atrio-ventricular synchronization could alter ventricular filling (stroke volume) or sympatho-vagal balance (Chiladakis et al. 2004) thereby confounding the response to vagal nerve stimulation.

It also should be noted that mongrel dogs subjected to myocardial ischemia induced by coronary artery ligation were used in this study. However, canine myocardium possess a well-developed collateral system (Maxwell et al. 1987), and therefore, coronary occlusion (even after the ligation of visible epicardial collaterals) is unlikely to result in a fully ischemic area. The residual collateral flow, although negligible during normal conditions (as evidence by the rapid onset of ischemic changes), in the setting of a prolonged diastolic interval (e.g., during vagal stimulation) may facilitate oxygen delivery and the washout of metabolites to/from ischemic myocardium, reducing the cell- swelling/interstitial shrinkage that increases impedance. Notably, the prompt reversal of early ischemic myocardial electrical impedance changes during ischemia has also been reported following reperfusion (Cascio et al. 2001; Howie et al. 2001).

In summary, vagal nerve stimulation provoked rapid and significant reductions in the early myocardial electrical impedance increase that accompanied myocardial ischemia.

Conversely, vagotomy provoked further increases in the impedance during coronary artery occlusion. The response to vagal stimulation was not altered by bilateral stellectomy, demonstrating that reflex sympathetic activation did not contribute to the changes induced by the nerve stimulation. However, when heart rate was maintained by cardiac pacing, vagal stimulation failed to alter the ischemic impedance response. Thus, the reductions in the ischemic impedance response elicited by vagal nerve stimulation would appear to occur as a consequence of the reductions in cardiac metabolic demand (i.e., attenuation of the ischemic insult) that arise secondarily to the neurally mediated bradycardia.

Table 3.1: Hemodynamic and myocardial electrical impedance (MEI) response to acute LCX coronary artery occlusion (CAO) and subsequent (overlapping) acute bilateral cervical vagotomy (VAG) in intact animals (CTRL group, n = 7).

Parameter Baseline CAO CAO+VAG P<0.05

Heart Rate 193.1 ± 14.6 147.1 ± 14.2 155.1 ± 10.7 † (bpm) (+38.0 ± 8.5)

Blood Pressure 105.3 ± 5.3 99.1 ± 7.6 97.9 ± 8.9 - (mmHg) MEI 662.8 ± 17.9 675.0 ± 17.2 620.3 ± 19.5 *,† (Ω) (+42.5 ± 4.6) (+12.2 ± 1.1)

(CTRL, n = 7) = n (CTRL, RPP Intact Animals Intact 15.4 ± 1.4 15.3 ± 1.5 18.8 ± 2.2 - (x103·bpm·mmHg) PRQ 0.66 ± 0.07 0.53 ± 0.07 0.76 ± 0.09 *,† (mmHg/bpm) (-0.10 ± 0.03) (-0.13 ± 0.02)

*: P<0.05 Baseline vs. CAO; †: P<0.05 CAO+VAG vs. CAO

Table 3.2: Hemodynamic and myocardial electrical impedance (MEI) response to LCX coronary artery occlusion (CAO) and subsequent (overlapping) vagal nerve stimulation (VNS) in previously vagotomized animals (i.e., post bilateral vagotomy) with (VAG/P) and without (VAG) atrio-ventricular-pacing.

Parameter Baseline CAO CAO+VNS P<0.05

Heart Rate 205.8 ± 6.4 72.8 ± 8.5 189.4 ± 8.5 *,† (bpm) (+16.4 ± 4.8) (-133.0 ± 7.7)

Blood Pressure 97.8 ± 10.6 136.1 ± 6.4 129.2 ± 8.4 † (mmHg) (-31.5 ± 9.4) MEI 700.2 ± 43.7 684.1 ± 44.1 618.4 ± 32.8 *,† (Ω) (+81.8 ± 14.2) (-16.1 ± 2.1)

(VAG, n = 11) n = (VAG, RPP 7.7 ± 1.5 3 26.1 ± 2.2 26.9 ± 2.3 † Bilateral Vagotomy Bilateral (x10 ·bpm·mmHg) (-19.2 ± 2.0) PRQ 1.42 ± 0.12 0.72 ± 0.03 0.63 ± 0.03 † (mmHg/bpm) (+0.79 ± 0.12)

Heart Rate 220.0 ± 2.2 222.0 ± 0.8 221.9 ± 0.6 - (bpm)

Blood Pressure 74.7 ± 11.7 106.4 ± 7.9 95.9 ± 10.6 † (mmHg) (-21.2 ± 6.4) MEI 696.9 ± 16.6 620.2 ± 11.9 696.9 ± 16.6 * (Ω) (+75.2 ± 10.5) RPP 16.6 ± 2.6 (VAG/P, n = 7) = n (VAG/P, 22.3 ± 1.7 21.3 ± 2.3 † (x103·bpm·mmHg) (-4.7 ± 1.4) PRQ 0.34 ± 0.05 Bilateral Vagotomy & Pacing Vagotomy Bilateral 0.51 ± 0.06 0.43 ± 0.05 † (mmHg/bpm) (-0.09 ± 0.03)

*: P<0.05 Control vs. CAO; †: P<0.05 CAO+VNS vs. CAO. RPP = Rate Pressure Product, PRQ = Pressure Rate quotient

ST60 ΔSJ QRS TPE RR TPEc QTc Time (mV) (mV) (ms) (ms) (ms) (ms) (ms) Baseline 0.04 ± 0.01 0.15 ± 0.04 75.9 ± 2.0 29.8 ± 1.3 313 ± 16 51.9 ± 2.5 314 ± 9 CAO* 0.22 ± 0.03 0.08 ± 0.05 87.5 ± 1.5 46.9 ± 2.6 289 ± 7 84.5 ± 4.2 340 ± 9 CAO+VNS 0.15 ± 0.03 0.18 ± 0.07 81.5 ± 1.9 60.1 ± 5.3 935 ± 144 65.8 ± 5.9 254 ± 16

P <0.05†       

*: P<0.05 Baseline vs. CAO (all parameters). †, Arrows (,): P<0.05 CAO vs. CAO+VNS.

ST60 = ST changes 60 ms after the J-point; SJ = change in the SJ amplitude; TPE = Tpeak-Tend; TPEc = Tpeak-Tend corrected for heart rate; QTc = QT interval corrected for heart rate.

Stimulus Complex MEI Spectrum MEI values over time, MEI (t) (current, i) 740 Vo(f) Zo (f) = = Ro(f) + j·Xo(f) MEI samples (every 3s) * i1 Io(f) impedance resistance reactance 720 30s (10 sample) averages * (complex) (real) (imaginary) i2 LCX tied

average 1.5 0 700 io (t) 1 -0.5 i10 resistance reactance

0.5 -1 IN 680 FFT Ro(f) Xo(f) 0 * -1.5 * 0 2 4 6 0 2 4 6 Io (f) Frequency (f, kHz) Frequency (f, kHz) Zo (f) 2 2 0.5 660 σ = 1.3 Ω (STD) / modulus (R + X ) 1.8 Vo (f) 1.6 |Zo(f)| Impedance modulus 640 OUT FFT 1.4 (magnitude) 1.2 v1 620 1 MEIo * v2 0.8 mean(|Zo(f)|) 600 average 0.6 623 Ω vo (t) Baseline CAO 0.4 0 1 2 3 4 5 6 v10 every 3s Frequency (f, kHz) -2 Response -10 -8 -6 -4 0 2 4 (voltage, v) Time (t, min) *: Values in Ohms (Ω)

Figure 3.1: Myocardial electrical impedance (MEI) technique (left) and recordings from a representative experiment (right). LEFT (see text for details): 1) the myocardium is probed with ten bipolar current pulses (i1-10), and the response recorded (v1-10); 2) using FFTs, the spectra of the averaged current (io) and voltage (vo) signals are calculated (Io and Vo, respectively); 3) the MEI spectrum (Zo), consisting of real (resistance, Ro) and imaginary (reactance, Xo) components, is given by the Vo/Io ratio; and 4) a single value (MEIo), representing the mean MEI modulus (|Zo|) over frequency, is reported. RIGHT: MEI samples were taken every 3s (circles), and 30s averages were studied (squares). Over time, MEI values remain stable (i.e., low variance, σ), until the onset of ischemia (coronary artery occlusion, CAO; at t = 0 min). NOTE: The spectra shown correspond to one of the MEI measurements taken during baseline conditions. For all animals (n = 36), the MEI variability (σ) was 1.9 ± 0.14Ω.

Intervention LCX tied N=7 VF: 0/7 A Baseline CAO CTRL

Vagotomy

VNS N=16 LCX tied VF: 5/16 B Baseline CAO VAG

Vagotomy

VNS LCX tied N=8 VF: 1/8 Baseline CAO VAG/P (*)

Vagotomy + Pacing (220bpm)

C VNS LCX tied N=5 VF: 1/5 Baseline CAO VAG/S

Vagotomy + Stellectomy

0 3 Time (min)

Figure 3.2: Experimental protocol and outline of the study (see text for details). Left circumflex (LCX) coronary artery occlusions (CAO) were performed in both neurally intact (A, CTRL group) and vagotomized animals (i.e., post bilateral-vagotomy; B, VAG group). CAO was followed by parasympathetic interventions: acute vagotomy in CTRL dogs or acute vagal nerve stimulation (VNS, 10 Hz, 10V) in VAG dogs. VNS was also studied in two additional sets of animals (C). In one set, heart rate was maintained by atrio- ventricular pacing (VAG/P group), while in the other set, bilateral stellectomy preceded CAO (VAG/S group). The response to VNS after CAO was only studied in animals vagotomized prior to ischemia onset (i.e., VAG, VAG/P and VAG/S groups). Seven vagotomized dogs (VAG n = 5; VAG/P n = 1; and VAG/S n = 1) developed ventricular fibrillation (VF) during CAO prior to VNS. (*) In VAG/P dogs: after VNS (shown), pacing was discontinued and stimulation was repeated (not shown). Pacing was established after the CAO onset in two dogs in the VAG/P group.

2s 1s 2s 2s

2.0 )

1.0

mV (

0 ECG ECG

-1.0

250 228 bpm

) 210 bpm

bpm 150 ( LCX tied 103 bpm

HR HR 50 VNS (10Hz)

Baseline CAO

0 3 4 Time (min)

Figure 3.3: Representative electrocardiogram (ECG) and heart rate (HR) tracings in a vagotomized animal (i.e., post bilateral-vagotomy, from VAG group) showing the response to the left circumflex (LCX) coronary artery occlusion (CAO, at t = 0min) and subsequent (overlapping) vagal nerve stimulation (VNS). Note early ST-segment elevation and progressive QRS widening induced by myocardial ischemia that was attenuated during overlapping VNS.

750 A. 193 bpm 54 bpm LCX tied

700

) VNS (10Hz)

Ω (

MEI MEI 137 bpm N=11, P<0.05 650 ΔHR = -133±7.4 bpm

ΔMEI = -16±2.1 Ω

600 750 B. Pacing @ 220bpm

700 LCX tied

) VNS (10Hz)

Ω (

MEI MEI N=7, N.S. 650 ΔHR = - 0.1±0.4 bpm

ΔMEI = - 0.1±1.1 Ω

600 Baseline CAO

-1 0 1 2 Time (min)

Figure 3.4: Representative myocardial electrical impedance (MEI) recordings in two previously vagotomized animals (i.e., post bilateral-vagotomy) showing the response to the left circumflex (LCX) coronary artery occlusion (CAO, at t = 0 min) and subsequent (overlapping) vagal nerve stimulation (VNS) when A) heart-rate was allowed to change freely (top, VAG group) or B) held-constant by atrio-ventricular pacing (bottom, VAG/P group) during stimulation. Inserts show mean responses during VNS for the respective groups (i.e., VAG and VAG/P).

A. Baseline Post-Stellectomy 2s SNP 2s SNP

150 ])

100

mmHg (

BP BP N=5, P<0.05 N=5, N.S.

ΔBP = -17±3.2 ΔBP = -18±2.5 50

160 ΔHR = 26±4.8 ΔHR = 4±3.3

)

bpm

( HR HR 100

0 2 0 2 Time (min) Time (min) 750 B. VNS (10Hz)

Post-Stellectomy 154 bpm

700

) LCX tied Ω ( 35 bpm

MEI MEI N=4, P<0.05 650 ΔHR = -120±18 bpm 151 bpm ΔMEI = -23±1.6 Ω

600 Baseline CAO

0 2 4 6 Time (min)

Chapter 4: Electrotonic Remodeling following Myocardial Infarction

Passive electrical remodeling following myocardial infarction (MI) is well established. These changes can alter electrotonic loading and trigger the remodeling of repolarization currents, a potential mechanism for ventricular fibrillation (VF). However, little is known about the role of passive electrical markers as tools to identify VF- susceptibility post-MI. This study, published (in part) in the Journal of Applied Physiology

(del Rio et al., 2008b), investigated electrotonic remodeling in the post-MI ventricle, as measured by myocardial electrical impedance (MEI), in animals prone to and resistant to

VF. In short, MI was induced in dogs by a two-stage left-anterior descending (LAD) coronary artery ligation. Before infarction, MEI electrodes were placed in remote (left- circumflex, LCX) and infarcted (LAD) myocardium. MEI was measured in awake animals at 1, 2, 7, and 21 days post-MI. Subsequently, VF-susceptibility was tested by a 2-min LCX occlusion during exercise; 12 animals developed VF (susceptible, S) and 12 did not

(resistant, R). The healing infarct had lower MEI than the normal myocardium. This difference was stable by day 2 post-MI (287 ± 32Ω vs. 425 ± 62Ω, P<0.05). Significant differences were observed between resistant and susceptible animals 7 days post-MI: susceptible dogs had a wider electrotonic gradient between remote and infarcted myocardium (R: 89 ± 60Ω vs. S: 180 ± 37Ω). This difference increased over time in susceptible animals (252 ± 53Ω at 21-days), due to post-MI impedance changes on the remote myocardium. These data suggest that early electrotonic changes post-MI could be 63 used to assess later arrhythmia susceptibility. In addition, passive-electrical changes could be a mechanism driving active-electrical remodeling post-MI, thereby, facilitating the induction of arrhythmias.

Introduction

Healed myocardial infarction is one of the most important structural substrates of sudden cardiac death (SCD) (Sheppard and Davies, 1998). Fatal arrhythmias (ventricular fibrillation) arising from the boundary of scarred myocardium are the cause of sudden death in a significant percentage of deceased patients with a known cardiac lesion. Despite substantial stratification and therapeutical advances, the risk of sudden death remains the highest in the first 30 days after myocardial infarction (Bunch et al., 2007; Solomon et al.,

2005). Moreover, common arrhythmia risk-stratification variables have limited predictive power identifying patients at risk of SCD after MI (Huikuri et al., 2003; Al-Khatib et al.,

2007). Hence, research on both mechanisms and new markers of arrhythmia susceptibility, especially in the setting of healing myocardial infarction, is needed.

A potential mechanism for ventricular fibrillation (VF) and sudden cardiac death following myocardial infarction (MI) is remodeling of repolarization currents, leading to increased action potential duration (APD) and dispersion of repolarization (Pinto and

Boyden, 1999; Al-Khatib et al., 2007). Recently, it has been shown in vitro that changes in electrotonic loading, which may occur from passive electrical heterogeneities in the ventricle, can trigger the remodeling of these sarcolemmal currents (Libbus et al., 2004).

Notably, the remodeling of myocardial structures (e.g., gap junctions) mediating passive

64 electrical coupling/loading in ventricles prone to arrhythmias is well established after MI

(e.g., for review see Veenstra, 2006). Several investigations suggest that discontinuities in coupling resistance between neighboring myocardial regions can lead, by itself, to unidirectional conduction block, a critical factor for re-entrant arrhythmias (de Groot and

Coronel, 2004; Cascio et al., 2005). Moreover, Sahakian et al. (1992) showed (in silico) that conduction block is more likely to occur with steeper electrotonic gradients

(differences) and/or when propagation encounters an abrupt decrease in coupling resistance. Interestingly, healing/healed myocardial infarction has lower electrical impedance than normal non-infarcted myocardium (Fallert et al., 1993; Cinca et al., 1998;

Schwartzman et al., 1999; Warren et al., 2000; Wolf et al., 2001; del Rio et al., 2004;

Salazar et al., 2004) as a consequence of ischemic sarcolemmal rupture (Rodriguez-

Sinovas et al., 2005). For example, Fallert et al. (1993) reported that following infarction

(in sheep), scar impedance was approximately 60% of the non-infarcted tissue.

Nonetheless, the prognostic value of this electrotonic disparity as a tool to assess the susceptibility to ischemia-induced arrhythmias following myocardial infarction has not been established.

Thus, it was the purpose of this study to investigate electrotonic coupling in the post-MI heart, as measured by myocardial electrical impedance (MEI), and its relationship to arrhythmia susceptibility. In particular, the hypothesis that post-MI hearts with the highest degree of ventricular electrotonic (MEI) heterogeneity also exhibited the greatest susceptibility to VF was tested.

Methods

The principles governing the care and treatment of animals, as expressed by the

American Physiological Society, were followed at all times during this study. In addition, the animal protocols and experimental procedures were approved by the Institutional Lab

Animal Care and Use Committee (ILACUC) at this institution, and adhered to the statutes of the Animal Welfare Act and the guidelines of the Public Health Service.

Chronic Model of Ischemia-Induced VF: The studies were performed on a well- characterized canine model of sudden cardiac death, known to mimic the main clinical features associated with the disease: healed myocardial ischemic injury, acute myocardial ischemia, and cardiac autonomic activation. The surgical preparation for this model was initially presented by Billman, Schwartz and Stone (Billman et al., 1982; Schwartz et al.,

1984), and has been subsequently reviewed in detail (Billman, 2005 and 2006).

In short, forty-three (n = 43) heartworm-free, purpose bred, mixed-breed dogs weighing 16.0 to 25.7 kg (20.1 ± 2.7 kg) were sedated (morphine sulfate 15mg IM, and thiopental sodium 20mg/kg IV), and connected to a respirator via an endotracheal-cuffed tube. Anesthesia was maintained with inhaled isoflurane (1-1.5%) mixed with oxygen

(100%). Under sterile conditions, the chest was opened via a left thoracotomy (fifth intercostal space), the heart was exposed, and was suspended with a pericardial cradle.

Subsequently, a large antero-lateral myocardial infarction (MI) was created by ligature of the left anterior descending (LAD) coronary artery, proximal to its first diagonal branch; in order to limit acute ischemic arrhythmias and increase survival a modified (Billman et

66 al., 1982; Schwartz et al., 1984) two-stage LAD occlusion was used. Briefly, as described by Harris (1950), two silk snares were loosely placed around the LAD coronary artery; these sutures were used first to constrict the vessel by tying a ligature against a 20-gauge hypodermic needle that was then removed (stage I), and 20 minutes later, to occlude this vessel completely (stage II). In addition, the left circumflex (LCX) coronary artery was dissected free of the surrounding tissue near its origin (under the edge of the left auricular appendage) and was chronically instrumented with a 20 MHz Doppler-flow probe and an hydraulic coronary artery occluder. Inflation of this balloon would later render a portion of the LCX distribution acutely ischemic (see Arrhythmia Susceptibility).

Before infarction, as required for MEI measurements (see below), two bipolar pacing electrodes (Medtronic Inc., model Streamline™ 6495) were placed (one each) in the distal LAD and LCX coronary artery distributions. These two regions are referred to as the ischemic/infarcted and the remote/non-infarcted myocardium, respectively. Each lead was placed (parallel to the local fiber alignment) into the mid-myocardial wall, downstream of the respective ligation/occlusion site (i.e., LAD ligatures/LCX hydraulic occluder), and was firmly secured in place with non-absorbable sutures (prolene 2-0). After acute LAD coronary ligation (infarction), the ischemic/infarcted electrodes were visually confirmed to be in the center of the region rendered ischemic (i.e., cyanotic/dyskinetic) during coronary occlusion. The pericardial cradle was released, the chest closed in layers and evacuated of air, restoring the negative intra-thoracic pressure. All leads were tunneled under the skin, exited at the neck, and were carefully bandaged.

In order to reduce mortality during the acute infarction phase, the procedures were performed under prophylactic anti-arrhythmic therapy instituted both before surgery

(procainamide 500mg IM) and during each stage of the coronary occlusion (lidocaine HCl

60mg IV, bolus). In addition, the dogs were given a broad-spectrum antibiotic post- operatively (amoxicillin, 500 mg B.I.D. PO for 7 days) to reduce the risk of infection.

Nonetheless, out of the forty-three animals that were surgically prepared, seventeen did not complete the experimental procedures (and were excluded from analysis) due to either premature death (n = 11) or to failure of the MEI electrodes (n = 6, see below).

Arrhythmia Susceptibility: The surviving animals were allowed to recover for 3-4 weeks and were trained to run on a motor-driven treadmill. Subsequently, the susceptibility to ischemia-induced ventricular fibrillation was assessed using a standardized protocol, generally referred as the “exercise-plus-ischemia” test (Billman et al., 1982; Schwartz et al., 1984; Billman, 2005). Briefly, after a 3 min warm-up period (4.8 km/h, 0% grade), the dogs ran (6.4 km/h) for 15 min with the grade (incline) increased every 3 min (i.e., 0, 4, 8,

12, and 16%) in order to activate the autonomic nervous system and to achieve a submaximal (~70%) heart rate of 210 beats/min. During the last minute of exercise (i.e., while running at 6.4 km/h, 16%), the animals were subjected to a brief (2 min) LCX occlusion.

This combination of exercise plus ischemia yields two stable and well-differentiated populations of animals: one susceptible to ischemia-induced ventricular fibrillation and the other resistant (for review see Billman, 2006). In this study, 12 animals developed VF

(susceptible, S) and 12 did not (resistant, R) during the exercise-plus-ischemia test. Two

68 animals (n = 2) could not be classified due to equipment failure. A single-lead electrocardiogram (ECG) was recorded at the time of classification (30.8 ± 8.0 days).

Myocardial Electrical Impedance (MEI): As has previously been described (del Rio et al.,

2005; del Rio et al., 2008a), a computer-controlled circuit developed in this laboratory

(Howie et al., 2001) was used to measure the complex electrical impedance of the myocardium. In short, using a bipolar pacing lead (see above), the myocardium was probed with a sub-threshold zero-mean bipolar current, consisting of two rectangular pulses of alternating polarity (± 5 µA, 100 µs wide) generated 200 ms apart. Measurements were made only with the first pulse of each stimulation pair; the subsequent pulse (of opposite- polarity) was used to mitigate possible artifacts introduced by the electrode-tissue interface

(e.g., polarization; see Limitations). The complex MEI spectrum was calculated in the frequency domain as the ratio (at each frequency) of the current and voltage spectra resulting from the combined averages of ten stimulus pulses and their respective (voltage) responses. The mean modulus of the complex MEI spectrum in the 0.27 – 5.90 kHz frequency range was examined.

Experimental Protocol: As described above, animals were instrumented with MEI electrodes in remote (non-infarcted) and ischemic/infarcted myocardium. Using these leads, MEI was recorded from each myocardial region (distribution) either intra- operatively (under anesthesia, open-chest), at the end-of-surgery (under anesthesia, closed-

69 chest) and/or during recovery (awake unsedated at rest). As mentioned before, six animals

(n = 6) were excluded from the chronic study due to lead malfunction (i.e., dislodgement).

Thus, the studies were performed on twenty-six dogs (n = 26). In order to study the early time-course of electrotonic (MEI) remodeling after MI, a group of animals (n = 14), regardless of arrhythmias susceptibility (5 resistant, 7 susceptible, and 2 unable to be classified), had MEI measurements collected both acutely (i.e., at the end-of-surgery), and chronically (i.e., on days 1, 2, 7, and 21) after myocardial infarction (LAD ligation). The remaining animals (n = 12), consisting of both VF-resistant (n = 7) and VF-susceptible (n

= 5) dogs, had MEI data recorded only on post-MI day 7 and day 21. Subsequently, in order to study the differences between animals susceptible (S, n = 12) and resistant (R, n =

12) to ischemia-induced arrhythmias, data collected at post-MI day 7 and day 21 time- points were pooled among all dogs successfully classified with the exercise-plus-ischemia test (n = 24). The susceptibility to ischemia-induced arrhythmias was evaluated at the end of the study, i.e., 4 weeks (30.8 ± 8.0 days) after MI. In all cases, chronic post-MI data were collected from unrestrained, awake, unsedated animals at rest (in a dim lighted/quiet room) and were averaged over at least 1 min (for each myocardial region).

As mentioned before, a modified Harris (1950) two-stage occlusion was used to create an anterior myocardial infarction (Billman et al., 1982; Schwartz et al., 1984).

Notably, although the electrotonic derangements triggered by a single-stage acute coronary occlusion have been well described (Kléber et al., 1987; Howie et al., 2001; de Groot and

Coronel, 2004; Cascio et al., 2005; del Rio et al., 2005), no study to date has evaluated the passive electrical changes that follow a two-stage procedure. Thus, in a subset of animals

(n =10), MEI was continuously recorded (every 3 s) intra-operatively during the 2-stage

LAD ligation. MEI data were collected distal to the LAD snares (ischemic region), and are reported (averaged over 30 s) at eight time-points before/during the ischemic insult: pre- ischemic (baseline) values were taken 2 minutes before coronary constriction (at t = 0 min).

Following the onset of occlusion (post-CAO), ischemic minutes 2, 5, 10, 20 (onset of LAD ligature), 22, 25 and 30 were studied. It should be noted that given the unknown electrotonic effects of anesthesia, acute (i.e., collected during or at the end of surgery) and chronic MEI measurements were not compared.

Data analysis: Electrocardiographic signals (ECG) were band-pass filtered and digitally recorded (at 500Hz) using a data acquisition system (Biopac Systems, Inc., model MP-

100). Stored waveforms were analyzed offline, and fiducial points were determined with the aid of personal computer software (Biopac Systems, Inc., Acknowledge). As such, heart rate (HR), R-R and QT interval durations were measured. Since repolarization duration

(QT) is rate-dependent, QT was corrected for heart-rate values (QTc = QT/R-R0.5). Data were averaged over 5 consecutive beats at rest.

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed with SigmaStat (Systat Software, Inc.) and NCSS (NCSS, Inc.). Mean impedance differences between values recorded before/after acute two-stage coronary occlusion (i.e., during surgery), were tested using a one-way analysis of variance

(ANOVA) with repeated measures. The time-course of electrotonic (MEI) remodeling after

MI was evaluated using a two-way (time: days 1/ 2/ 7/21 post-MI, and distribution:

71 infarct/remote) ANOVA with repeated measures on both factors. Similarly, intergroup comparisons (i.e., resistant vs. susceptible) were made using a three-way (time: day 7/day

21 post-MI, distribution: infarct/remote, and group: susceptible/resistant) ANOVA with repeated measures on two factors (time/distribution). A significant second-order interaction (time x distribution x group) was found (F1/15 = 24.69, P<0.001), and four two- way ANOVA were subsequently performed to investigate this effect: two group x distribution (at each time-point) and two distribution x time (within each group) tests were done. In addition, the group-wise difference (contrast) between remote and infarct impedance (ΔMEI), reflecting the degree of electrotonic heterogeneity within the myocardium, was studied over time using a two-way (time/group) ANOVA with repeated measures on one factor (time). If significant F-values were observed, post hoc pair-wise comparisons were made using the Tukey test; P<0.05 was considered to be statistically significant (a priori). The repeated measures sphericity assumption (i.e., homogeneity of the covariance matrix) was verified using the Mauchley’s test (NCSS, Inc.). Receiver- operator characteristic (ROC) curves were generated for the ΔMEI values measured 7 and

21 days post-MI. The area under these ROC curves (AUC) was calculated using a bi- normal model (NCSS, Inc.), and is reported (with its standard error, SEAUC) as an overall measure of diagnostic accuracy (Zou et al., 2007).

Results

Acute ischemia (two-stage CAO): During the two-stage ischemic insult MEI increased rapidly (2-5 min) following the initial vessel constriction (stage I) but subsequently

72 recovered (10-20 min). Subsequently, the complete ligation of the LAD coronary artery

(stage II) led to a sustained impedance rise (>20 min). These data are displayed in Figure

4.1.

Chronic ischemia (infarction): As expected, healing myocardial infarct tissue had significantly lower MEI than normal myocardium (see Figure 4.2 and Table 4.1). This difference was stable by day 2 post-MI (2.3 ± 0.6 days), when the infarct impedance had reached 70 ± 12% of that recorded in non-infarcted tissue (MI: 287 ± 32Ω vs. LCX: 425 ±

62Ω, n = 14, P<0.05), coinciding (temporally) with the peak number of premature deaths; five animals (5/8) died 48-72 hours after coronary ligation. One week after MI (7.2 ± 0.6 days), significant differences were observed between resistant (R) and susceptible (S) animals (see Fig. 4.3 and Table 4.2): susceptible dogs had a wider electrotonic disparity

(ΔMEI) between remote (non-ischemic) myocardium and the infarcted tissue (R: 89 ± 60Ω vs. S: 180 ± 37Ω, P<0.05). Three weeks after MI (21.5 ± 1.7 days), this difference had increased significantly, but only in VF-susceptible animals (R: 62 ± 101Ω vs. S: 252 ±

53Ω, P<0.001) (see Table 4.2). It is likely that remodeling of remote (LCX) myocardium

(R: 435 ± 78Ω vs. S: 585 ± 91Ω, P<0.001) was responsible for this increased electrotonic dispersion, as no intergroup differences were found among impedance measurements recorded in infarcted tissue (R: 373 ± 69Ω vs. S: 333 ± 77Ω, P=0.3, N.S.).

The diagnostic accuracy of ΔMEI for predicting arrhythmia susceptibility was studied from ROC curves for measurements made on days 7 and 21 post-MI; the areas under the ROC curves (AUC) were 90.3% (SEAUC = 6.2%, see Fig. 4.4) and 95.3% (SEAUC

= 4.9%), respectively. The optimal impedance difference for classification one week after

MI (i.e., ΔMEIo~145 Ω) was selected at the point of intersection of the sensitivity and specificity curves (~83%), providing positive (PPV)/negative (NPV) predictive values of

82.8% (see Fig. 4.4, inset).

Furthermore, at the time of classification (29.3 ± 1.4 days post-MI), animals prone to ischemia-induced arrhythmias had longer QTc intervals (R: 323 ± 4.2 ms vs. S: 341 ±

6.3 ms, P<0.001) despite comparable heart rates (R: 119 ± 18.8 bpm vs. S: 130 ± 20.0 bpm,

P=0.16, N.S.). These data are consistent with prolonged repolarization in the VF- susceptible dogs.

Discussion

The present study investigated chronic electrotonic remodeling in the left ventricle following an anterior myocardial infarction, as described by the electrical impedance of both ischemic and remote (non-infarcted) myocardium. Unlike prior studies (Fallert et al.,

1993; Cinca et al., 1998; Schwartzman et al., 1999; Warren et al., 2000; Wolf et al., 2001;

Salazar et al., 2004), these passive electrical changes were studied in awake animals and, importantly, in dogs that were subsequently shown to be susceptible/resistant to ischemia- induced ventricular fibrillation (VF). In agreement these previous reports, the healing infarction had a lower impedance than did the remote, non-infarcted, myocardium. In addition, dogs prone to VF had a wider electrotonic gradient (impedance difference, ΔMEI) between these myocardial regions. This broader “impedance mismatch” between infarcted and non-infarcted myocardium was observed as early as one week following infarction,

74 and more importantly, provided a reliable marker for subsequent susceptibility to VF.

Furthermore, in animals prone to arrhythmias this “mismatch” increased over time due to

MEI changes (increase) in the non-infarcted myocardium, facilitating the impedance-based classification of arrhythmic susceptibility.

Acute MEI Changes (two-stage CAO): The present study also evaluated (for the first time) the passive electrical changes that follow an acute Harris two-stage LAD coronary artery occlusion. Following the initial constrictive ischemic insult (stage I), MEI increased rapidly but subsequently recovered. A larger and sustained rise was observed after the complete ligation of the LAD (stage II). In short, after 20 minutes of constriction and 10 minutes of coronary occlusion (30 minutes of combined ischemia), MEI rose +81 ± 41.3Ω from the pre-ischemic value of 812 ± 106.6Ω (i.e., +10.0 ± 5.2%, P<0.05). In a previous study, performed also in isoflurane-anesthetized dogs, MEI was shown to increase +24.1 ± 5.4%

(from 809 ± 133Ω) after a 30 min (single-stage) acute LAD occlusion (Howie et al., 2001).

Taken together, these observations suggest that the two-stage occlusion procedure attenuated the MEI progression (electrotonic uncoupling) during ischemia. Interestingly, interventions that delay the passive electrical changes triggered by acute coronary occlusion are known to limit ischemia-induced arrhythmias (e.g., see Cinca et al., 1997).

Thus, although further research is needed, these results suggest that the attenuation of ischemia-induced electrotonic changes (MEI increase) may be a mechanism that mediates the anti-arrhythmic protection of the two-stage Harris occlusion.

Chronic MEI Changes (infarction remodeling): Following infarction, the non-ischemic myocardium is known to undergo adaptive processes resulting in detrimental mechanical and electrical changes (Pinto and Boyden, 1999; Al-Khatib et al., 2007). In agreement with the present observations, clinical and experimental data suggest that the extent of this maladaptation, or remodeling, is well correlated with the risk of ventricular arrhythmias

(Sutton et al., 2003; Solomon et al., 2005). During post-MI cardiac remodeling, impulse conduction in the surviving myocardium is altered by changes in both passive electrical coupling and tissue architecture, leading to highly heterogeneous tissue which may serve as an arrhythmogenic trigger/substrate (Saffitz et al., 1999; Severs et al., 2004).

For example, in surviving post-infarct myocardium, disarray and downregulation of the gap junctions mediating cell-to-cell electrotonic communication have been shown to correlate spatially with pathways of reentry (Peters et al., 1997). In a canine model of healed myocardial infarction, Luke and Saffitz (1991) reported that myocytes at the scar border presented not only less interconnections but also smaller gap junctions, limiting intercellular current transfer. Notably, intercellular resistance and therefore, whole-tissue impedance, is determined partially by the conductance properties of the gap junctions

(Kléber et al., 1987; de Groot and Coronel, 2004; Cascio et al., 2005).

Similarly, post-MI changes in cellular and interstitial space morphology also can alter electrotonic coupling and action potential propagation properties. Cooklin et al.

(1997), for instance, showed that preparations from hypertrophied ventricles, characterized by larger myocytes, had higher longitudinal impedance than those from sham-operated controls. Interestingly, Swann et al. (2003), using the same animal model as the present

76 study, reported that susceptible but not resistant animals exhibited a significant hypertrophy (by echocardiography) of non-infarcted myocardial regions. Thus, it is plausible that more severe remodeling, involving both abnormal (decreased) gap-junction coupling and increased cell-size, could underlie the higher impedance recorded from regions remote to a myocardial infarction in animals prone to ischemia-induced VF. In agreement with this hypothesis, post-MI patients with severe left ventricular remodeling

(hypertrophy) have the highest risk for sudden cardiac death due to lethal arrhythmias

(Sutton et al., 2003; Solomon et al., 2005).

Regardless of the underlying mechanism(s), passive-electrical changes following

MI, as assessed by the myocardial electrical impedance, were able to differentiate between animals later shown to be either prone to or resistant to malignant arrhythmias. In particular, the degree of electrotonic dispersion, or impedance disparity (ΔMEI) between the remote (non-ischemic) myocardium and the healing infarct, identified VF-susceptible dogs with high sensitivity and specificity, even when measured just 7 days post-MI (see

Fig. 4.4). Notably, the risk of sudden death remains the highest in the first 30 days after myocardial infarction (Solomon et al., 2005; Bunch et al., 2007), and the prognostic significance of early intervention/therapy is well documented. Thus, although a direct comparison with indices of post-MI arrhythmic-risk currently used in the clinic is difficult

(and perhaps unwarranted at this time given the relatively small sample), the results of the present study suggest that markers of electrotonic remodeling (especially in the surviving myocardium) could be valuable identifying patients vulnerable to arrhythmic-events earlier. Furthermore, several groups have presented clinically-applicable catheter-based

77 impedance mapping techniques (e.g., Warren et al., 2000; Salazar et al., 2004; Wolf et al.,

2001), that could be used concurrently with existing methods investigating active-electrical remodeling post-MI (e.g., indices dispersion of repolarization, and/or programmed electrical stimulation protocols), thereby providing a more complete evaluation/understanding of the electrical substrate for arrhythmias.

Interestingly, electrotonic remodeling, occurring as a consequence of myocardial infarction, could also play a role triggering and/or modulating pro-arrhythmic adaptive processes affecting active electrical properties (i.e., ionic currents). For example, Libbus et al. (2004) found that changes in electrotonic loading acutely reduced early (phase 1) action potential (AP) repolarization in the epicardium, consistent with remodeling (attenuation) of the transient outward potassium current (Ito). Downregulation of Ito, as well as other repolarizing currents, provides the cell with the capacity to deliver greater electrotonic current to partially uncoupled cells downstream of the conduction (Spitzer et al., 2006), thereby acting as an intrinsic “impedance matching” (or load balancing) mechanism allowing “optimal” source-load coupling.

Thus, a wider electrotonic gradient (impedance mismatch) following infarction, as observed in animals that either died early (data not shown) or survived but were susceptible to VF, could mediate a more severe attenuation/remodeling of these sarcolemmal ionic currents. Notably, Yao et al. (1999) reported that changes in repolarizing potassium currents (such as Ito) were detectable just three days after infarction, coinciding with the onset of impedance heterogeneities as reported in the present study. Furthermore, it is generally accepted that post-MI slowing of repolarization prolongs action potential

78 duration (APD) and increases heterogeneities in the time course of repolarization (Pinto and Boyden, 1999). Swann et al. (2003) reported that when compared to VF-resistant animals, VF-susceptible dogs presented marked ventricular repolarization heterogeneities.

In the present study, QTc was longer in VF-susceptible animals, an observation consistent with slowed/heteregoneous repolarization. Furthermore, at the cellular level, animals prone to ischemia-induced VF were found to have longer (and more disperse) APDs when compared to either control and/or VF-resistant animals (Sridhar et al., 2006). Taken together, these observations further strengthen the potential role of electrotonic remodeling of the non-ischemic myocardium as a substrate for VF in post-infarct hearts.

Study Limitations: Mongrel dogs subjected to coronary artery occlusion-induced myocardial ischemia/infarction were used in this study. It is well established that dogs possess a well-developed coronary collateral system (Maxwell et al., 1987), and therefore are more resistant to supply ischemia than other species lacking such innate protection

(e.g., pigs). As a result, the effects of infarction on the chronic passive electrical properties of the myocardium may have been under-estimated in the present study. For example,

Legato (1993) reported that in this canine model “infarcts were almost never completely homogeneous, but consisted of patches of fibrosis separated by grossly normal tissue”.

Schwartzman et al. (1999) found that the electrical impedance of inhomogeneous infarct, where myocytes and collagen bundles coexist, has a higher value (380 ± 60 Ω•cm) than that of densely infarcted myocardium (160 ± 30 Ω•cm). As such, although the impedance values obtained in this study for infarcted myocardium (e.g., 320 ± 46Ω at day 7 post-MI)

79 are consistently higher than those previously reported in the literature for other species

(e.g., 110 ± 30 Ω•cm by Cinca et al. [1998], 90 ± 29 Ω•cm by Fallert et al. [1993], 122 ±

26 Ω•cm by Salazar et al.[2004]), the higher infarct impedances are consistent with the observations of Legato and the presence of a prominent coronary collateral density in dogs.

The chronic measurement of the myocardial passive electrical properties in vivo also has limitations. In order to track electrotonic changes following myocardial infarction,

MEI electrodes were implanted chronically, and measurements were taken over several weeks. However, as pointed out by Grill and Mortimer (1994) the electrical properties of the tissue surrounding implanted electrodes changes over time. Notably, myocardial electrical impedance, as measured in this study, results from the combination of the true impedance of the myocardium and that of the electrode-tissue interface. As such, the results reported in the present study could be confounded not only by changes occurring over time at the electrode-tissue interface (e.g., electrode-encapsulation), but also, by polarization artifacts characteristic of this metal-electrolyte boundary. Similarly, (although each animal served as its own control), any confounding electrotonic remodeling triggered by the electrode implantation/surgical procedure alone (i.e., besides the MI) could not be resolved neither, as no sham-operated (i.e., non-infarcted) controls were studied.

However, several studies demonstrate that chronic MEI measurements are stable and remain sensitive to pathologies known to alter electrotonic coupling in the myocardium

(ischemia [del Rio et al., 2004], rejection [Grauhan et al., 1996; Pfitzmann et al., 2000]).

For example, both Grauhan et al. (1996) and Pfitzmann et al. (2000), measured MEI chronically in non-infarcted (sham) dogs using a two-pole technique similar to that

80 employed in this study, reporting that after an initial decrease following implantation (2 days, as observed in the present study), impedance values were not only “completely stable” for up to 40 days, but also, that polarization artifacts were negligible. Furthermore, the experiments in the present study were designed to mitigate the possible bias introduced by concomitant changes/artifacts at the electrode-tissue interface: e.g., relative comparisons were made between MEI measurements taken from different regions of the heart using identical stimuli (current/frequency) and electrodes (size, surface area), either over time for each animal (remote vs. infarct), and/or at a given time-point between different animals (susceptible vs. resistant). Moreover, as described above, the bipolar current stimuli used to measure myocardial impedance were charge-balanced; this technique reduces residual electrical charge at the tissue-metal interface, improving the signal-to-noise ratio and reducing polarization at the interface resistances of chronically implanted electrodes (Johnson et al., 2005; Merrill et al., 2005). Therefore, it seems unlikely that the reported MEI differences between animals susceptible and resistant to VF reflected group-specific changes mediated by the electrode tissue-interface, the surgical procedure, and/or by polarization artifacts, rather than by the remodeling of electrotonic coupling (in remote myocardium) following infarction.

In summary, animals prone to ischemia-induced VF and characterized by prolonged QTc intervals following myocardial infarction were found to have a wider electrotonic gradient (impedance difference) between infarcted and non-infarcted myocardium than was noted in those animals resistant to malignant arrhythmias. These

81 differences were obvious as early as 7 days following myocardial infarction (becoming progressively greater over time) and allowed the stratification of later arrhythmic susceptibility. Furthermore, this electrotonic (impedance) mismatch could provoke remodeling of ionic currents and, could thereby, lead to repolarization heterogeneities.

Thus, the results of the present study suggest that early passive electrical changes following

MI could be used to assess later arrhythmia susceptibility. In addition, such electrotonic changes could be a mechanism contributing to the active-electrical remodeling in the ventricles following MI, creating a substrate that favors arrhythmia formation.

Table 4.1: Myocardial electrical impedance (MEI) of ischemic/infarcted and remote (non- infarcted) myocardium following left-anterior descending (LAD) coronary artery ligature (myocardial infarction, MI). Acute MI measurements (shaded) were obtained in anesthetized animals (at the end of the surgical preparation/LAD ligation). Chronic post- MI measurements (infarct healing; day 1 – day 21) were taken in awake-unsedated animals (n = 14, except for Day 21 where n = 12 due to equipment failure in 2 dogs).

Acute MI Infarct Healing

Distribution Day 0 Day 1 Day 2 Day 7 Day 211 P<0.05 Remote (LCX) (Ω) 765 ± 88 447 ± 72 425 ± 62 442 ± 92 525 ± 117 21 vs 1,2,7 Infarct (LAD) (Ω) 917 ± 139 340 ± 48 287 ± 32 294 ± 39 340 ± 77 N.S.

Ratio LAD/LCX (%) 121 ± 19* 79 ± 15* 70 ± 12* 70 ± 19* 70 ± 24*

MEI values in Ohms (Ω). LCX: left-circumflex coronary artery 1n= 12 on Day 21. *: P<0.05 Remote vs. Infarct.

Table 4.2: Myocardial electrical impedance (MEI) data recorded from infarcted and remote (non-infarcted) myocardium after left-anterior descending (LAD) coronary artery ligation. Data are grouped according to subsequent susceptibility to ischemia-induced ventricular fibrillation.

Group Remote Infarct ΔMEI

Susceptible (n = 12) (Ω) 483 ± 49 303 ± 45 180 ± 37 * Resistant (n = 12) (Ω) 427 ± 68 338 ± 41 89 ± 60 *

Day 7 Day P-value (S vs. R) 0.012 0.106 0.001

Susceptible (n = 9) (Ω) 585 ± 91† 333 ± 77 252 ± 53† * Resistant (n = 8) (Ω) 435 ± 78 373 ± 69 62 ± 101 *

Day 21 Day P-value (S vs. R) <0.001 0.300 <0.001

MEI values in Ohms (Ω). ΔMEI: MEI Remote – MEI Infarcted. *: P<0.05 Remote vs. Infarct; †: P<0.05 Day 7 vs. Day 21.

920 A. LAD tied

880

)

Ω (

840 MEI

800 Stage I Stage II

Constriction Ligature 760

140

120 B. LAD tied )

100 N=10

Ω ( *: P<0.05 vs. pre-CAO (t=0min) 80 * * 60 * 40

Change Change * * 20

Stage I Stage II MEI 0 Constriction Ligature -20 2-stage CAO

-5 0 5 10 15 20 25 30 35 Time (min)

1000 N=14 Infarct (LAD)

*: P<0.05, Infarct vs. Remote Remote (LCX) ) 800 Ω P<0.05

, (Infarct)

600 * Ohms

( * * MI * 400

MEI MEI ΔMEI

Surgery AWAKE 200 0 1 2 7 Time (days)

Infarct (LAD) 7 ΔMEI 600 Remote (LCX) R vs. S, P<0.05

day day *: P<0.05, Infarct vs. Remote

500 @

) 400 Ω

, * * 300

200

Ohms (

100 MEI 0 RESISTANT SUSCEPTIBLE n=12 n=12 B.

7 R vs. S, P<0.05 250

day day R: 89 ± 60 Ω

200

@ )

Ω 150 ,

100 S: 180 ± 37 Ω

Ohms 50 ( Resistant (R) 0

Susceptible (S) MEI MEI

Δ -50 RESISTANT SUSCEPTIBLE n=12 n=12

100 %) , ΔMEI @ day 7 80

AUC: 90.3% (SEAUC=6.2%)

60 120 Sensitivity Specificity 100 ~145

true positivestrue 80

( 40 60 @ΔMEIo~145 Ω: 40

20 PPV: 82.8 % 20 0 NPV: 82.8 % -50 50 150 250 350

ΔMEI (Ohms, Ω) Sensitivity Sensitivity 0 0 20 40 60 80 100 100 – Specificity (false positives, %)

Figure 4.4: Empirical (gray) and bi-normal (fitted, black) receiver-operator characteristic (ROC) curves generated for the myocardial electrical impedance (MEI) differences between infarcted and remote (non-infarcted) myocardium (i.e., ΔMEI) 7 days after left- anterior descending (LAD) coronary artery ligation; the area under the ROC curve (AUC) provides a measure of diagnostic accuracy (48). Inset: Percent correct classification of susceptible (sensitivity, %) and resistant (specificity, %) animals as a function of ΔMEI; the optimal classification threshold (i.e., ΔMEIo~145 Ω) was selected at the point of intersection of the two curves (~83%), providing positive (PPV) and negative (NPV) predictive values of 82.8%. Data were collected in awake-unsedated animals at rest.

Chapter 5: β-Adrenoceptor Mediated Enhancement of Electrotonic Coupling

Autonomic neural activation during cardiac stress testing is an established risk- stratification tool in post-myocardial infarction (MI) patients. However, autonomic activation can also modulate myocardial electrotonic coupling, a known factor to contribute to the genesis of arrhythmias. The present study, published (in part) in Frontiers of Physiology (del Rio et al., 2015), tested the hypothesis that exercise-induced autonomic neural activation modulates electrotonic coupling (as measured by myocardial electrical impedance, MEI) in post-MI animals shown to be susceptible or resistant to ventricular fibrillation (VF). In short, dogs (n = 25) with healed MI instrumented for MEI measurements were trained to run on a treadmill and classified based on their susceptibility to VF (12 susceptible, 9 resistant). MEI and ECGs were recorded during 6-stage exercise tests (18 min/test; peak: 6.4 km/h @ 16%) performed under control conditions, and following complete β-adrenoceptor (β-AR) blockade (propranolol); MEI was also measured at rest during escalating β-AR stimulation (isoproterenol) or overdrive-pacing.

Exercise progressively increased heart rate (HR) and reduced heart rate variability (HRV).

In parallel, MEI decreased gradually (enhanced electrotonic coupling) with exercise; at peak exercise, MEI was reduced by 5.3 ± 0.4% (or -23 ± 1.8Ω, P<0.001). Notably, exercise- mediated electrotonic changes were linearly predicted by the degree of autonomic activation, as indicated by changes in either HR or in HRV (P<0.001). Indeed, β-AR blockade attenuated the MEI response to exercise while direct β-AR stimulation (at rest) 89 triggered MEI decreases comparable to those observed during exercise; ventricular pacing had no significant effects on MEI. Finally, animals prone to VF had a significantly larger

MEI response to exercise. These data suggest that β-AR activation during exercise can acutely enhance electrotonic coupling in the myocardium, particularly in dogs susceptible to ischemia-induced VF.

Introduction

Myocardial infarction is a well-established risk factor for sudden cardiac death

(SCD) due to malignant arrhythmias (Adabag et al., 2010; Zaman and Kovoor, 2014).

However, despite significant advances in the understanding of the physiological substrate(s) mediating/facilitating the onset of arrhythmias, risk stratification for SCD in post-MI patients remains difficult and insufficient (Goldberger et al., 2014; Wellens et al.,

2014; Zaman and Kovoor, 2014). Indeed, the majority of SCD episodes occur in patients with either low-/intermediate- or without known risk factors (e.g., Wellens et al., 2014).

In these patients, underlying ionic current abnormalities, particularly those mediating repolarization, either co-exist with and/or are exacerbated by autonomic imbalances favoring enhanced sympathetic drive (e.g., Chen et al., 2007; Pokorný et al.,

2011; Wellens et al., 2014). As such, multi-modality stratifications techniques, encompassing electrocardiographic evaluation of repolarization abnormalities during states of autonomic activation, such as exercise, are favored (Goldberger et al., 2014;

Wellens et al., 2014). For instance, the assessment of microvolt T-wave alternans (TWA or MTWA) during low-intensity exercise has been shown to predict not only arrhythmic

90 events in post-MI patients but also arrhythmia-free survival in patients with LV dysfunction (Cantillon et al., 2007; Amit et al., 2010; Verrier et al., 2011; Merchant et al.,

2012; Shizuta et al., 2012).

Interestingly, activation of the autonomic nervous system (e.g., during exercise), and its concomitant catecholamine release, may in turn also modulate the passive electrical properties that govern electrotonic interactions in the myocardium. For example, several studies have shown that catecholamines (and increased cAMP levels) enhance junctional coupling in myocytes (e.g., see de Mello, 1996; Dhein, 2004; Salameh and Dhein, 2011).

Notably, electrotonic coupling is a well-established factor modulating both repolarization disturbances and arrhythmic risk, as poorly coupled cells are more likely to exhibit pro- arrhythmic behaviors (e.g., Pastore and Rosenbaum, 2000; de Groot and Coronel, 2004;

Wit and Peters, 2012; Saffitz and Kléber, 2012). For instance, enhanced electrotonic coupling has been shown to suppress early after-depolarizations (EADs) (Huelsing et al.,

2000; Himel et al., 2013), and reduce transmural dispersion of repolarization (Quan et al.,

2007). Similarly, preserved electrotonic interaction has been shown to modulate TWA in silico, and more recently, also in vivo (Pastore and Rosenbaum, 2000; Watanabe et al.,

2001; Cherry and Fenton, 2004; Sato et al., 2006; Kjølbye et al., 2008; Jia et al., 2012).

Remarkably, no study to date has investigated concomitant passive electrical (electrotonic) changes during autonomic neural activation in vivo.

It was, therefore, the purpose of this study to investigate myocardial electrotonic coupling changes induced by submaximal exercise in the left-ventricle of post-MI animals, as measured by myocardial electrical impedance (MEI). Specifically, the hypothesis that

91 exercise-induced autonomic activation can modulate myocardial electrotonic coupling

(i.e., MEI) was tested in animals with healed myocardial infarctions later demonstrated to be either susceptible or resistant to ischemia-induced VF. Briefly, β-adrenoceptor (β-AR) activation during submaximal exercise acutely decreased the electrical impedance of the surviving myocardium, particularly in animals susceptible to VF, consistent with an increased electrotonic coupling.

Methods

The principles governing the care and treatment of animals, as expressed by the

American Physiological Society, were followed at all times during this study. In addition, the animal protocols and experimental procedures were approved by The Ohio State

University’s Institutional Lab Animal Care and Use Committee (ILACUC) at this institution, and adhered to the statutes of the Animal Welfare Act and the guidelines of the

Public Health Service.

Surgical Preparation: The studies were performed using a well-characterized canine model of sudden cardiac death, known to mimic/combine the most prevalent features associated with this disease in the clinic: healed myocardial ischemic injury, acute myocardial ischemia, and cardiac autonomic activation (see Billman, 2006).

Briefly, thirty-five (n = 35) heartworm-free purpose bred mixed-breed dogs

(weight: 16.1 to 24.1 kg, 19.0 ± 0.4 kg) were sedated (morphine sulfate 15 mg IM, and thiopental sodium 20 mg/kg IV), and connected to a respirator via an endotracheal cuffed

92 tube. Anesthesia was maintained with inhaled isoflurane (1-1.5 %) mixed with oxygen

(100%). Under sterile conditions, the chest was opened via a left thoracotomy (fifth intercostal space); the heart was exposed, and suspended with a pericardial cradle.

Subsequently, an antero-lateral myocardial infarction (MI) was created by a two-stage ligature of the left anterior descending (LAD) coronary artery. The left circumflex (LCX) coronary artery was dissected free of the surrounding tissue near its origin (under the edge of the left atrial appendage) and was instrumented with a 20 MHz Doppler-flow transducer, and a hydraulic coronary artery occluder; inflation of this balloon would later render a portion of the LCX distribution acutely ischemic (see Arrhythmia Susceptibility).

As required for MEI measurements (see below), a bipolar pacing electrode

(Medtronic Inc., model Streamline™ 6495) was placed remote to the infarct, in the distal

(non-ischemic) distribution of the LCX coronary artery. In a subset of animals (n = 10), a second MEI electrode was placed in the healthy (non-infarcted) anterior myocardium for pacing purposes (see Experimental Protocol). The leads were inserted into the mid- myocardial wall (parallel to the local fiber alignment), and were firmly secured in place with non-absorbable sutures (prolene 2-0). The pericardial cradle was released, the chest closed in layers and evacuated of air restoring the negative intra-thoracic pressure. All leads were tunneled under the skin, exited at the neck, and were carefully bandaged.

Exercise Test Protocol: The animals were allowed to recover for 3-4 weeks, and subsequently, were trained to run on a motor-driven treadmill. A 6-stage submaximal exercise stress-test (SMT), as initially described by Stone (1977), was used to activate the

93 autonomic nervous system. This protocol is summarized in Figure 5.1, and consisted of a

3 min warm-up walking period (4.8 km/h, 0% grade; level L1), followed by running (6.4 km/h) for 15 min with the grade (incline) increased every 3 min (i.e., 0, 4, 8, 12, and 16%; levels L2-L6).

Arrhythmia Susceptibility: The susceptibility to ischemia-induced ventricular fibrillation was assessed at the end of the study using a standardized protocol, generally referred as the

“exercise-plus-ischemia” test (see Billman, 2006). In short, a submaximal exercise bout was performed (as described above) and during the last minute of exercise, the animals were subjected to a brief (2 min) LCX occlusion (i.e., while running at 6.4 km/h, 16%).

This combination of exercise plus ischemia, when applied post-MI, yields two stable and well-differentiated populations of animals: one susceptible and the other resistant to ischemia-induced malignant arrhythmias, such as ventricular fibrillation (see Billman

[2006] for review). In this study, 12 animals developed VF (susceptible, S) and 9 did not

(resistant, R) during the exercise-plus-ischemia test. Four animals (n = 4) could not be classified due to equipment failure (e.g., occluder rupture).

Myocardial Electrical Impedance (MEI): As has previously been described, a computer controlled circuit developed in this laboratory was used to measure the complex electrical impedance of the myocardium (Howie et al., 2001; Dzwonczyk et al., 2004; del Rio et al.,

2005 and 2008). In short, using a bipolar pacing lead (see above) the myocardium was probed with a sub-threshold zero-mean bipolar current, consisting of two rectangular pulse

94 of alternating polarity (± 5 µA, 100 µs wide) generated 200 ms apart. The complex MEI spectrum was calculated in the frequency domain, as the ratio (at each frequency) of the current and voltage spectra resulting from the ensemble averages of ten stimulus pulses and their respective (voltage) responses. The mean modulus of the complex MEI spectrum in the 0.27 – 5.90 kHz frequency range was examined (del Rio et al., 2008).

Experimental Protocol: As described above, thirty-five animals (n = 35) were instrumented with MEI electrodes in the remote, non-infarcted myocardium. However, five animals (n

= 5) experienced lead malfunctions (e.g., dislodgement) either before or at the time of experimentation, and therefore, were excluded from the analysis, while another five animals (n = 5) failed to acclimatize to the treadmill exercise protocol. Thus, the studies were performed in thirty animals (n = 30), with exercise-data successfully collected and analyzed in twenty-five dogs (n = 25).

First, in order to investigate the time-course of the electrotonic coupling (i.e., MEI) during submaximal exercise, all animals, regardless of arrhythmias susceptibility (9 resistant, 12 susceptible, and 4 unable to be classified), had MEI measurements collected during a submaximal exercise test (SMT) performed approximately 1-month after the LAD ligature (28 ± 1.7 days post-MI).

On a different day (26 ± 1.7 days post-MI), a subset of animals (5 resistant, 7 susceptible, and 4 unable to be classified; n = 16) performed the submaximal exercise test, but after pretreatment with the β-adrenoceptor antagonist propranolol HCl (1.0 mg/kg IV,

Sigma Chemical, St. Louis, MO). Previous studies demonstrated that this dose of

95 propranolol 1) completely abolished the cardiac response to the β-adrenoceptor agonist isoproterenol HCl (1 µg/kg IV) (Collins and Billman, 1989), and 2) did not compromise the exercise capacity during the submaximal exercise test in the presence of a 1-month-old anterior wall myocardial infarction (Brice and Stone, 1986). Propranolol was given intravenously (cephalic vein) as a bolus injection 3 min before the onset of exercise. A partially counter-balanced design was used: some dogs (6/16) were first exercise-tested under the influence of this β-adrenoceptor antagonist, and on a later day, had a control test

(i.e., with no drug) performed; whereas in the remaining animals (10/16) the response to the submaximal exercise test (SMT) was first studied under control conditions, and on a subsequent day, following β-adrenoceptor blockade. In all cases, MEI measurements during the exercise tests were taken (continuously) from the distal LCX distribution

(remote non-ischemic region) in awake, unsedated, and otherwise unstressed, post-MI animals (in a quiet and dimly lit room).

In order to investigate further the role of exercise-induced autonomic neural activation on myocardial electrotonic coupling (MEI), the total β-adrenoceptor response was quantified at rest in some animals (n = 10). Briefly, the dogs were lightly sedated with acepromazine (0.5 mg/kg IM; Ft. Dodge Animal Health, Ft. Dodge, IA), and a (cephalic vein) catheter was percutaneously placed for the administration of isoproterenol HCl

(Sigma Chemical, St. Louis, MO); five increasing doses of this β-adrenoceptor agonist were given: 0.005, 0.015, 0.05, 0.15, and 0.5 µg/min/kg. MEI measurements were obtained continuously during isoproterenol infusion and washout. Data are reported (averaged over

30s) when a steady-state response was achieved at each dose, and 2 minutes after dosing discontinuation.

Finally, the possible confounding effects of exercise-mediated heart rate changes were evaluated in another subset of dogs (n = 10) via overdrive left-ventricular pacing at rest. Briefly, with the animal standing on the treadmill (awake and unsedated), an impulse generator (Grass Medical Instruments, model Grass S44, via impulse-isolation unit model

SIU105-B) was used to maintain ventricular rates of 180 and 210 beats/min, mimicking those observed during moderate (L2; 6.4 km/h, 0%) and peak exercise (L6; 6.4 km/h, 16%).

In these animals, a second bipolar MEI/pacing electrode was placed at the time of instrumentation (see Surgical Preparation above); the pacing protocol was repeated from each lead (while MEI was simultaneously recorded from the other, i.e., the non-stimulating electrode), and, as similar impedance responses were obtained, the results for the two sites

(leads) were combined. MEI data collected before pacing onset, and after stabilization at each pacing rate, are reported (averaged over at least a 30s interval).

Data analysis: A single-lead bipolar electrocardiogram (ECG) was recorded during each presentation of the submaximal exercise test. The ECG signals were band-pass filtered and digitally sampled (1 kHz)/analyzed (on-line) using a heart rate variability (HRV) monitor

(Delta-Biometrics, Inc.; Urbana-Champaign, IL). Briefly, using a previously well- described (Billman and Hoskins, 1989; Billman and Dujardin, 1990) R-R interval time- series analysis technique, the heart rate (HR) mean and its variability (i.e., HRV) were determined continuously from non-overlapping 30s-segments of the ECG. Two kinds of

HRV indices were studied simultaneously: 1) two measures of statistical dispersion, namely the standard deviation (RRSD) and the range (RRRNG; longest – shortest R-R interval) of the R-R intervals within each 30s analysis-window; and 2) an index estimating the amplitude of the respiratory sinus arrhythmia (R-R interval variability in the 0.24 - 1.04

Hz frequency range), or vagal tone index (VT). MEI, HR and HRV data are reported

(averaged over 30s) at eight time-points sampled before (one), during (six), and after (one) the bouts of submaximal exercise. Pre- (baseline) and post-exercise (recovery) values were taken 2 minutes before/after exercise onset/offset (i.e., at t = -2 min, and t = 20 min, see

Fig. 5.1) with the animals standing on the treadmill. Meanwhile, the six exercise data- points were recorded during the last 30s of each stage in the submaximal stress protocol

(i.e., L1 - L6).

In addition, exercise-induced changes in the ECG morphology as well as on ECG- derived indices of the duration and heterogeneity of ventricular repolarization were evaluated in a subset of animals (n = 19). In short, with the aid of pattern-recognition software (ECG Auto; EMKA Technologies, France), fiducial points/intervals were determined and measured offline from two sets of thirty consecutive ECG complexes

(beats), one recorded before (i.e., at rest) and the other immediately following a control submaximal exercise test (i.e., within 5 beats of stopping the treadmill). The effects of exercise on the duration of the T-wave’s terminal portion (i.e., peak-to-end interval, TPE

(Yan and Antzelevitch, 1998; Opthof et al., 2007) and on the QT-interval’s length, as well as on the relationship between cardiac electrical systole and diastole (i.e., ratio of QT- and

TQ-intervals, QT/TQ) (Fossa et al., 2007; Kijtawornrat et al, 2010) were evaluated; both

98 absolute (QT) and rate-corrected (QTc, via van de Water’s formula; van de Water, 1989)

QT-intervals are reported. In addition, the standard deviation of the T-wave amplitude within each 30-beat epoch (TSD) was calculated and used as a surrogate-marker of temporal repolarization variability (e.g., T-wave alternans; Nearing and Verrier, 2002).

All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed with SigmaStat (Systat Software, Inc., San Jose, CA) and NCSS

(NCSS, Inc., Kaysville, UT). The mean time-course of electrotonic coupling (i.e., MEI), and ECG-derived variables during the submaximal exercise tests (SMT) was evaluated using a one-way (exercise level: baseline, L1 to L6, and recovery) analysis of variance

(ANOVA) with repeated measures. Intergroup comparisons (i.e., resistant vs. susceptible) were made using a two-way (exercise level, and group: susceptible/resistant) ANOVA with repeated measures on one factor (exercise level). Similarly, the responses to exercise, recorded under control conditions (control) and after β-adrenoceptor blockade (beta), were compared using a two-way (exercise level, and control/beta tests) ANOVA with repeated measures on both factors. Finally, the statistical significance of any impedance changes induced by either pacing (3 levels: baseline/two rates) and/or by isoproterenol infusion/washout (7 levels: baseline/five doses/recovery) was evaluated using one-way

ANOVA with repeated measures. The sphericity assumption (i.e., homogeneity of the covariance matrix) was verified using the Mauchley’s test (NCSS, Inc.). If this assumption was not met, then a non-parametric repeated measurements ANOVA on Ranks (Friedman) test was used. In all cases, if significant F-values (or Q-values in the non-parametric case) were observed, post hoc pair-wise comparisons were made using the Tukey test.

Linear regression analyses were performed as well in order to study the relationship

(interaction) between exercise-induced changes in electrotonic coupling (i.e., ΔMEI) and in two indices of autonomic activation, the heart rate (ΔHR) and the vagal-tone index

(ΔVT); the regression data were “centered”, i.e., deviations from each animal’s mean values (over the whole exercise bout) were studied. The equality of the ΔMEI/ΔHR (and

ΔMEI/ΔVT) linear models fitted to the different groups and conditions studied (susceptible vs. resistant, and control vs. β-AR blockade) was tested by multiple linear regression analysis, considering both qualitative (group) and interaction terms (i.e., simultaneously testing the differences in slope and intersect of the regression functions). For all analyses,

P<0.05 was considered, a priori, to be statistically significant.

Results

Effects of Exercise: As expected and consistent with previous studies (Billman and

Hoskins, 1989; Billman and Dujardin, 1990; Billman, 2006), submaximal treadmill exercise resulted in a progressive acceleration of heart rate and a concomitant decrease in heart rate variability (see Fig. 5.2, and Table 5.1). At peak exercise (L6; 6.4km/h, 16%) heart rate increased on average 78 ± 4.3% (HR: from 119 ± 4 at rest to 208 ± 4 bpm at L6,

P<0.05), while the cardiac vagal-tone index, for instance, decreased 86 ± 2.3% (VT: from

7.9 ± 0.3 at rest to 1.2 ± 0.2 ln(ms2) at L6, P<0.05). In parallel with these changes indicative of strong cardiac autonomic neural activation, MEI decreased progressively during exercise in all animals studied (see Fig. 5.2, and Table 5.1), suggesting enhanced electrotonic coupling. For example, at the highest exercise level (i.e., L6) MEI decreased -

100

23 ± 1.8Ω (or 5.3 ± 0.4%) from the pre-exercise (at rest) values (MEI: from 446 ± 16 to

423 ± 16Ω at L6, P<0.05). Moreover, following discontinuation of exercise offset (i.e., recovery), all parameters returned towards the pre-exercise (baseline) values.

Notably, exercise-mediated impedance changes (ΔMEI) were linearly predicted by

(i.e., correlated with) the degree of autonomic neural activation, as indicated by either changes in heart rate (ΔHR; slope ΔMEI vs. ΔHR = -0.249 Ω/bpm; R2 = 0.83, P<0.05), or in vagal tone index (ΔVT; slope ΔMEI vs. ΔVT = 3.134 Ω/ln(ms2); R2 = 0.77, P<0.05) (see

Fig. 5.3 and Table 5.1).

Indeed, pretreatment with the (non-selective) β-AR antagonist propranolol significantly attenuated the MEI response to exercise (e.g., at L6, CTRL: -23 ± 2.5Ω vs.

BB: -11 ± 2.0Ω; P<0.05, n = 15) (see Fig. 5.4, Table 5.2), markedly reducing the slope of the ΔMEI vs. ΔHR (-0.250 vs. -0.139 Ω/bpm; R2 = 0.78, P<0.05) and the ΔMEI vs. ΔVT relationships (Ω/ln(ms2); R2 = 0.77, P<0.05). Similarly, as expected, β-AR blockade blunted the exercise-induced heart rate increase (e.g., at L6, CTRL: +47 ± 6 vs. BB: +30 ±

5 bpm; see Fig. 5.4), but accentuated cardiac parasympathetic withdrawal (e.g., lower vagal tone index values were recorded). Moreover, direct β-adrenoceptor stimulation at rest (with isoproterenol infusions) triggered a dose-dependent MEI response (decrease) comparable to that observed during submaximal exercise (see Fig. 5.5). On average, at the highest dose- level assayed (i.e., at 0.5 µg/min•kg), isoproterenol decreased MEI by -14 ± 1.7Ω (from

453 ± 40 to 440 ± 45Ω, P<0.05). Thus, when considered together, these data suggest that the acute electrotonic (impedance) changes induced by exercise are predominantly mediated by sympathetic β-adrenoceptor activation.

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On the other hand, while both submaximal exercise and direct β-adrenoceptor stimulation (at rest) led to acute MEI reductions, suggestive of favored increased electrotonic coupling, elevations in heart-rate via overdrive left-ventricular pacing had no significant effects on the passive electrical properties of the myocardium (see Fig. 5.6). For instance, when resting animals were paced at 210 beats/min (from a basal rate of 123 ± 7 bpm) MEI changed only +2 ± 0.8Ω (from 426 ± 28Ω at baseline to 428 ± 29Ω, n = 10;

N.S.); meanwhile, when the same subset of animals performed a submaximal exercise test,

MEI decreased -20 ± 3.3Ω (from 435 ± 38Ω at rest to 414 ± 38.3Ω at L6, n = 8; P<0.05) although heart rate increased similarly (from 124 ± 7 at rest to 213 ± 9 bpm at L6, P <

0.05). These data support the conclusion that the effects of exercise on electrotonic coupling (enhancement) are not mediated by its concomitant chronotropic effects (i.e., rate acceleration).

Arrhythmia Susceptibility: Although (as mentioned above), MEI decreased with exercise onset in all animals studied, the degree of reduction was modulated by the underlying arrhythmic susceptibility of each animal: Animals prone to ischemia-induced VF had a significantly larger MEI response to submaximal exercise (see Fig. 5.7, Table 5.2). At the peak exercise level (i.e., at L6), for instance, MEI decreased -30 ± 1.6Ω in dogs susceptible to VF (S, n = 12) and only -17 ± 2.1Ω in those resistant (R, n = 9) (P<0.05), albeit comparable heart rates (e.g., S: 211 ± 5 vs. R: 210 ± 6 bpm at L6, N.S.) and heart rate variability indices (e.g., VT; S: 1.3 ± 0.3 vs. R: 0.7 ± 0.2 ln(ms2) at L6, N.S.) were reached

102 by both groups; these observations suggest an increased impedance responsiveness to exercise-induced autonomic neural activation in dogs prone to arrhythmias.

In fact, susceptible animals had significantly (P<0.001, see Table 5.2) steeper relationships between the exercise-mediated changes in impedance (ΔMEI) and those recorded for the heart rate (slope of ΔMEI vs. ΔHR; S: -0.295 vs. R: -0.180 Ω/bpm, R2 =

0.87) and/or the vagal tone index (slope of ΔMEI vs. ΔVT; S: 4.11 vs. R: 2.09 Ω/ln (ms2),

R2 = 0.85). Notably, such marked intergroup differences were evident even when the slopes of the ΔMEI vs. ΔHR (S: -0.31 ± 0.2 vs. R: -0.18 ± 0.2 Ω/bpm, P=0.001) and/or the ΔMEI vs. ΔVT (S: 4.3 ± 0.27 vs. R: 2.4 ± 0.46 Ω/ln ms2, P=0.002) relationships were calculated individually for each animal, rather than from the study groups. Moreover, complete β-AR blockade (with propranolol) blunted the exercise-induced impedance differences between animals susceptible (S, n = 7) and resistant (R, n = 5) to ischemia-induced arrhythmias

(during control test; S: -30 ± 2.4 vs. R: -17 ± 2.7 Ω, P<0.05, but after β-AR blockade; S: -

16 ± 1.5 vs. R: -10 ± 4.1Ω, N.S.).

Electrocardiographic Data (response to exercise): Concomitantly with the above mentioned changes on indices of autonomic neural activation (cardio-acceleration, decreased heart-rate variability), exercise shortened the PR-interval and flattened the T- wave (data not shown) while decreasing indices of ventricular repolarization temporal duration (QTc, TPE) (see Table 5.3). For instance, on average, exercise shortened the rate- corrected QT-interval from 251 ± 4 msc at rest to 229 ± 4 msc (P<0.05), suggesting a faster and/or more homogeneous repolarization. On the other hand, exercise increased the QT/TQ

103 ratio (an index of the steepness of ventricular restitution, Fossa et al., 2007; Kijtawornrat et al, 2010]) as well as the standard deviation of the T-wave amplitude (a marker of repolarization variability and/or T-wave “alternans”). Notably, exercise-induced changes on the T-wave amplitude variability were larger in animals susceptible to ischemia-induced

VF (from 20.5 ± 3.1 μV at rest to 50.5 ± 9.0 μV post-exercise, P < 0.05) than in those resistant such arrhythmias (from 25.8 ± 4.7 μV at rest to 39.2 ± 5.6 μV post-exercise, N.S.); no other significant electrocardiographic differences, either at rest or following exercise, were noted between animals susceptible and resistant to VF.

Discussion

The present study investigated the acute effects of submaximal exercise and its resulting autonomic neural activation on the myocardial electrotonic coupling of dogs with healed myocardial infarctions, as described by changes in the electrical impedance of surviving remote (i.e., non-infarcted) myocardium. This study demonstrated that acute β- adrenoceptor activation (either during bouts of exercise or via a direct pharmacological challenge) acutely increased passive (electrotonic) coupling in the myocardium, with the largest changes noted in those animals that were subsequently shown to be susceptible to malignant ventricular arrhythmias. In short, myocardial impedance was shown to decrease gradually as the level of exercise and autonomic neural activation increased. In contrast, ventricular overdrive pacing (at heart rates matched to those seen with exercise) had no significant effects on MEI.

104

As noted by Kléber et al. (1987), the gross myocardial electrical impedance measurements used in this study as a surrogate of electrotonic coupling represent the combined passive electrical properties of the intra-, extra- and inter- (i.e., junctional) cellular pathways. Thus, the observed changes (i.e., decreases) in impedance can be a reflection of both direct changes (increases) in cell-to-cell coupling at the gap-junctions, and/or geometrical effects affecting the myocyte/interstitial space ratios during exercise

(Fleischhauer et al., 1995; de Mello, 2010) (see below). Notably, pharmacological interventions that result in stimulation of the β-AR/adenylyl-cyclase/PKA pathway and increase cAMP concentration have been shown to enhance gap junctional coupling in many preparations (e.g., see Dhein, 2004; Manoach et al., 1995; deMello, 1996). In agreement with these in vitro reports, complete β-AR blockade attenuated the MEI response to exercise while direct β-AR stimulation (at rest with isoproterenol) triggered MEI decreases comparable to those observed during exercise.

Moreover, in the present study, β-AR meditated exercise-induced electrotonic changes were not only demonstrated in vivo (conscious animals), but more importantly, the magnitude of these changes were shown to differ between post-MI dogs subsequently shown to be susceptible or resistant to ischemia-induced ventricular fibrillation (VF). In this clinically-relevant scenario, a significantly larger MEI response to exercise was noted in animals prone to malignant arrhythmias (i.e., VF), suggesting increased electrotonic responsiveness to autonomic (β-AR) activation. The VF-susceptible animals have been extensively studied, presenting marked derangements in ionic current (Sridhar et al., 2008;

Bonilla et al., 2012), electrotonic coupling (del Rio et al., 2008), intracellular calcium

105 homeostasis (Belevych et al., 2009 and 2012), and autonomic control (see Billman, 2006).

For instance, susceptible dogs have been shown to have a larger degree of electrotonic remodeling post-MI (del Rio et al., 2008), presenting moderately (albeit not statistically significant) higher remote MEI values at rest which could favor the observed increased electrotonic responsiveness. Indeed, in this study, animals prone to arrhythmias tended to have slightly higher baseline (i.e., pre-exercise) impedances but reached similar (absolute) values during exercise, suggesting, perhaps, a role of basal electrotonic derangements to their increased passive electrical responses to β-AR activation. Interestingly, the post-MI remodeling (e.g., down-regulation) of junctional proteins mediating electrotonic coupling is a well-established risk factor for malignant arrhythmias (e.g., see Saffitz and Kléber,

2012). On the other hand, susceptible animals also exhibit enhanced cardiac β-AR responsiveness, presenting a dominant functional contribution of the β2-adrenergic receptors following MI, both in vivo and in vitro (Billman et al., 1997; Houle, 2001).

Notably, acute β2-AR stimulation can increase junctional conductance and protein expression in cardiac myocytes (Xia et al., 2009), likely via the exchange protein directly activated by cAMP (Epac)/Rap1 signaling pathway (Somekawa et al., 2005; Duquesnes et al., 2010; Mostafavi et al., 2014).

It also should be noted that much like patients prone to SCD (Rubart and Zipes,

2005), post-MI VF-susceptible dogs have well-documented abnormalities in myocardial calcium handling, being characterized by leaky and oxydized ryanodine receptors

(Belevych et al., 2009) as well as increased calcium (Ca2+) entry and Ca2+ transients, particularly during β-AR stimulation (Billman et al., 1997; Altschuld and Billman, 2000;

106

Belevych et al., 2012). Moreover, in these animals, β2-AR mediated increases in Ca2+- transient amplitudes and Ca2+-entry have been reported (Billman et al., 1997), rendering them exceptionally responsive (i.e., anti-arrhythmic protection) to the pharmacological blockade of L-type current (Billman, 1989). Interestingly, intracellular Ca2+ and junctional/electrotonic conductance are tightly coupled. For instance, inhibition of junctional communication can attenuate Ca2+ transients and sparks (Li et al., 2012).

Meanwhile, pathologically-elevated intracellular Ca2+ levels (e.g., during a sustained ischemic insult) have been shown to decrease junctional conductance leading to electrotonic uncoupling (Cascio et al., 1990; Kléber, 1992; Smith et al., 1995; Owens et al., 1996; García-Dorado et al., 2004; de Groot and Coronel, 2004; Cascio et al., 2005), likely as a protective mechanism against the spread of calcium overload (i.e., myocytes live and work together but die alone; quote from Engelmann [1875] in Janse et al., 2002).

In contrast, moderate (i.e., within physiological levels) intracellular Ca2+ changes can enhance junctional coupling (perhaps via Ca2+-activated kinases) (e.g., see Delage and

Délèze, 1999]. Indeed, Joyner at al. (1997) using a hybrid (both in vitro and in silico) paired-myocyte model showed that both β-AR stimulation (with isoproterenol) and direct opening of the L-type Ca2+ channels (with Bay K8644), facilitated cell-to-cell coupling and impulse propagation (an effect prevented by the L-type Ca2+ channel antagonist nifedipine). Thus, β-AR mediated electrotonic changes can provide mechanistic explanation(s) for the enhanced electrotonic responsiveness observed in the present study, particularly when the fact that complete β-AR blockade abolished the exercise-induced

(electrotonic) differences with their VF-resistant counterparts is considered.

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Clinical Implications: Regardless of the potential mechanism(s), the observed increase in electrotonic coupling during exercise and/or β-adrenergic autonomic activation may have important clinical implications. Notably, enhancements in electrotonic coupling can reduce repolarization heterogeneities thereby blunting or masking intrinsic pro-arrhythmic ionic substrates. Indeed, increased electrotonic coupling has been shown to suppress early afterdepolarizations (EADs) and reduce pro-arrhythmic dispersion of repolarization (e.g., Quan et al., 2007; Huelsing et al., 2000; Himel et al., 2013). Interestingly, Vanoli et al. (1995) showed that adrenergic activation via either left stellate ganglion stimulation (in vivo) or isoproterenol administration (in vitro) blunted the d-sotalol-mediated prolongation of the action potential duration. Similarly, Jarvenpaa et al. (2007) reported that post-MI patients susceptible to VF have an impaired capacity of the autonomic nervous system to alter (i.e., prolong) electrocardiographic indices of repolarization. These observations are in agreement with the results of the present study; namely, the observed shortening of both the rate-corrected QT interval and the terminal portion of the T-wave (reflecting spatial repolarization heterogeneities); changes that are consistent with the exercise-mediated increases in myocardial electrotonic coupling.

Finally, substantial evidence supports the role of abnormal repolarization and calcium mishandling during neural autonomic activation in the onset and maintenance of lethal arrhythmias, particularly in the setting of both congenital and acquired (e.g., post-

MI) electro-mechanical remodeling. For instance, exercise testing has been shown to amplify the arrhythmic genotype-phenotype relationship in patients with both long-QT

108 syndrome (Takenaka et al., 2003) and catecholaminergic polymorphic ventricular tachycardia (Obeyesekere et al., 2011). However, in a stark contrast, exercise-driven risk stratification of post-MI patients (particularly those with preserved ejection fraction) remains difficult and, at times, counter-intuitive. For instance, in a recent meta-analysis,

Chan et al. (2010) showed that abnormal microvolt TWA results were more likely to reflect an increased risk for arrhythmic events only when β-adrenoceptor blocker therapy was not withheld prior to testing. In a similar manner, cardiac pacing elicited not only positive

TWA responses in susceptible patients (Hohnloser et al., 1997, Raatikainen et al., 2005), but led to a lower incidence of indeterminate test results when compared to exercise testing

(Kraaier et al., 2009). In this study, exercise not only shortened the Tpeak to Tend interval

(TPE), an index that has been reported to prolong during pro-arrhythmic stimulation

(Johnson et al., 2013), but failed to induce significant TWA differences between susceptible and resistant animals (i.e., S: 50.5 ± 9.0 vs. R: 39.2 ± 5.6 μV, N.S.). Thus, enhanced myocardial electrotonic coupling mediated by exercise-induced β-adrenoceptor activation could mask (e.g., via reductions in repolarization heterogeneities) changes in indices of risk for arrhythmia and could thereby explain the false negative results often obtained by exercise stress testing.

On the other hand, it is also important to note that improved electrotonic coupling has been shown to blunt myocardial ionic heterogeneities (e.g., Quan et al., 2007; Huelsing et al., 2000; Himel et al., 2013), masking and even reducing pro-arrhythmic risk (e.g., via pharmacological gap-junction modulation,; Hennan et al., 2006; Kjølbye et al., 2008).

However any improvement in electrotonic coupling that was induced by acute exercise was

109 of insufficient magnitude to prevent the onset of ischemic (coronary artery occlusion) arrhythmias in susceptible animals. Indeed, myocardial ischemia has been shown to depress electrotonic coupling acutely (e.g., Kléber et al., 1987; Cascio et al., 1990; Smith et al., 1997; del Rio et al., 2005 and 2008). Thus, in the setting of a healed infarction/remodeling (del Rio et al., 2008) and exercise-mediated electrotonic enhancements, concomitant regional ischemia likely resulted in marked local passive electrical heterogeneities, which are pro-arrhythmic (e.g., Bishop et al., 2014). Similarly, as noted above, impedance measurements represent the “average” electrotonic properties

(Kléber et al. (1987) of a specific myocardial region. Therefore, the observed MEI decreases are unlikely to reflect a homogenous enhancement of electrotonic coupling.

Indeed, the heterogeneous distribution of the myocardial autonomic innervation, would favor the onset of “focal” arrhythmias during β-AR stimulation (Myles et al., 2012).

Study Limitations: This study demonstrated that β-adrenoceptor activation mediates exercise-driven changes (increased) in myocardial electrotonic coupling. However, it also should be noted that despite complete β-adrenoceptor blockade, a moderate decrease in

MEI was observed during exercise. Several factors, that were not assessed in the present study, may have contributed to the residual (non- β-AR mediated) increases in myocardial electrotonic coupling during exercise.

For instance, during exercise, circulating cathecholamines activate both β- and α- adrenoceptors. Interestingly, sub-chronic α-adrenergic stimulation has been reported to exert PKC-mediated enhancements in connexin43 expression (Salameh et al., 2006.

110

Furthermore, Rojas-Gomez et al. (2008) found that phenylephrine (an α-adrenergic receptor agonist) enhanced Cx43 expression in neonatal rat cardiac myocytes, resulting in enhanced gap-junction conductance. However, it also should be noted that opposite electrotonic (i.e., reductions in junctional conductance) have been reported during acute α- adrenoceptor stimulation (deMello, 1997; deBoer et al., 2007), albeit these effects may vary in the setting of concomitant β-adrenoceptor stimulation (Salameh et al., 2010).

Similarly, the renin-angiotensin system is also acutely activated during exercise, increasing the circulating levels of angiotensin-II, which can modulate the passive electrical properties of the myocardium (de Mello, 1996 and 2014; Sovari et al. 2013).

Also, as noted above, changes in both spatial/geometrical and/or ionic composition of the myocardium can alter its electrotonic properties. For example, Veeraraghavan et al.

(2012) recently reported that changes in interstitial volume can modulate both conduction velocity and its dependence on gap-junction conductance. Changes in interstitial and/or myocyte volumes during exercise are likely, particularly given the reported ionic, metabolic, and plasma-volume changes during exercise (Paterson, 1996); interestingly, β-

AR activation has been implicated in the volume-regulation (i.e., decrease) of cardiac myocytes (Wang et al., 1997). Thus, β-adrenoceptor mediated changes in myocyte volume could also contribute to the exercise-induced changes MEI reported in the present study.

Similarly, changes in both cell-to-cell coupling (e.g., Burt and Spray, 1988; Saffitz and Yamada, 1998; Salameh and Dhein, 2013) and global indices of myocardial passive electrical properties (e.g., Sasaki et al., 1994; Dekker et al., 1996; Howie et al., 2001) have been linked/associated with alterations in the mechanical properties of the myocardium.

111

Indeed, in isolated myocytes, positive/negative inotropic agents have been shown to enhance/depress (respectively) junctional coupling in parallel with their functional effects

(Burt and Spray, 1988; Dhein, 2004). In the present study, both exercise and direct pharmacological β-AR stimulation (with isoproterenol), two well-defined inotropic interventions, decreased MEI, consistent with an enhanced electrotonic coupling. Although neither systemic/cardiac hemodynamics nor mechanics were assessed directly in the present study, previous studies have extensively documented the hemodynamic/functional responses of susceptible/resistant post-MI dogs, both at rest and during exercise (e.g.,

Avendano and Billman, 1994; Billman et al., 1985 and 1997; de Ferrari et al., 1993). For instance, Billman et al., (1985) reported comparable increases in the peak rate of left- ventricular pressure change (i.e., dP/dtmax – an inotropic index) during exercise in both groups of post-MI dogs, with animals prone to arrhythmias showing larger elevations in ventricular filling (end-diastolic) pressures (consistent with the exercise-driven preload changes [Miyazaki et al., 1990]) and blunted increases in systolic pressures. Meanwhile,

Avendano and Billman (1994) described similar inotropic/hemodynamic changes (i.e., dP/dtmax and systolic pressure increases) following isoproterenol administration (and other interventions that increased cAMP levels) in post-MI dogs and, in contrast to exercise, moderate isoproterenol-mediated end-diastolic pressure reductions were also noted (consistent with pre-load reductions [Barnes et al., 1979]). Hence, as mechanical stretch can alter intercellular coupling (Salameh and Dhein, 2013), it is possible that exercise-mediated changes in myocardial wall-tension could also play a role in the passive electrical changes that were observed in the present study. Finally, neither myocardial nor

112 core body temperature were measured during exercise in this study. Notably, the resistivity of a medium can decrease as temperature increases (e.g., 2%/°C; Tsai et al., 2002).

However, previously unpublished data from our laboratory (Billman GE, and del Rio CL) found that core body temperature not only increased moderately (~1°C) during exercise, but also, recovered slowly post-exercise (consistent with the limited heat-dissipation of dogs); an observation that contrasts with the rapid restoration of electrotonic coupling following the termination of exercise. Thus, changes in myocardial temperature induced by exercise probably did not contribute to the MEI changes noted in the present study.

In conclusion, the results of the present study demonstrate that β-AR activation during exercise can acutely enhance passive electrical properties (i.e., electrotonic coupling) of the myocardium, particularly in post-MI dogs susceptible to ischemia-induced

VF. Increased coupling during β-AR stimulation may have important clinical implications, as it could mask intrinsic (and/or acquired) pro-arrhythmic repolarization abnormalities during states of autonomic activation (e.g., exercise) in vivo.

113

Baseline Submaximal Exercise Test (SMT) Correlation

Parameter 0 km/h, 0% 6.4 km/h, 0% 6.4 km/h, 8% 6.4 km/h, 6% vs. ΔMEI MEI (Ohms) 446 ± 16 431 ± 16* 427 ± 16* 423 ± 16* - Heart rate (bpm) 119 ± 3 176 ± 4* 190 ± 4* 208 ± 4* (-) R2 = 0.83 Vagal Tone (ln ms2) 7.9 ± 0.3 3.1 ± 0.3* 2.2 ± 0.3* 1.2 ± 0.2* (+) R2 = 0.77

RRSD (ms) 71 ± 6 22 ± 2* 14 ± 1* 8 ± 1* n/s

RRRNG (ms) 329 ± 30 101 ± 8* 63 ± 7* 38 ± 4* n/s

*: P<0.05 vs. Baseline. n/s: not studied

114

Table 5.2: Exercise-induced changes in both the myocardial electrical impedance (MEI) and the heart-rate (HR) of awake-unsedated dogs with healed left-anterior descending (LAD) myocardial infarct; comparative effects of the underlying susceptibility to malignant arrhythmias (S vs. R), and of β-adrenoceptor blockade (+BB).

Baseline SMT (Peak) Slope (Correlation)

Parameter / Sub-Group 0 km/h, 0% 6.4 km/h, 16% vs. ΔMEI Susceptible (S, n = 12) 454 ± 24 424 ± 24* (-30 ± 1.6)

n/a Resistant (R, n = 9) 439 ± 28 422 ± 26* (-17 ± 2.1)†

MEI

(Ohms) Control (n =16) 433 ± 22 409 ± 22* (-23 ± 2.5) n/a + BB (n = 16) 470 ± 26 459 ± 27* (-11 ± 2.0)ǂ Susceptible (n = 12) 114 ± 4 211 ± 6* (+97 ± 5) -0.30 ± 0.02

Resistant (n = 9) 124 ± 8 210 ± 6* (+86 ± 4) -0.19 ± 0.03

(bpm) Control (n =16) 121 ± 5 211 ± 6* (+91 ± 5) -0.25 ± 0.03

Heart Rate Heart Rate + BB (n = 16) 111 ± 5 ǂ 180 ± 4* (+70 ± 5) ǂ -0.16 ± 0.03

*: P<0.05 vs. Baseline; †: P<0.05 vs. Susceptible; ǂ: P<0.05 vs. Control

115

Table 5.3: Electrocardiographic response(s) to submaximal exercise. Data collected immediately after the discontinuation of exercise.

RR PR QRS QT QT TPE QT/TQ T Time c SD (ms) (ms) (ms) (ms) (msc) (ms) (n/u) (μV)

Baseline 479 ± 19 92 ± 3 66 ± 2 206 ± 5 251 ± 4 51 ± 4 0.79 ± 0.04 25 ± 3 Exercise 329 ± 8 79 ± 3 65 ± 3 170 ± 4 229 ± 4 39 ± 2 1.11 ± 0.06 45 ± 5

P <0.05†   -     

†, Arrows (,): P<0.05 Exercise vs. Baseline (rest). QTc = van der Water’s rate-corrected QT interval; TPE = Tpeak-Tend; QT/TQ= index reflecting the steepness of restitution; TSD = Standard deviation of the T-Wave amplitude.

116

L1 L2 L3 L4 L5 L6

16 12 8

Incline 4 0 %

0 4.8 6.4 km/h Speed

PRE Submaximal Exercise Test (SMT) POST

0 3 6 9 12 15 18 Time (min)

Figure 5.1: Schematic representation of the six-level submaximal exercise test (SMT).

117

220 505 offset

) 200

) 495

Ω ,

bpm 180 ( 485

160 Ohms

( 475 140

MEI MEI 465 Heart Rate Rate Heart 120 P < 0.05 onset

100 455

) 9 5 2 8

0 P < 0.05 )

ln ms ln (

P < 0.05 , -5 6

5 -10 Ohms

4 ( -15 3 -20

2 MEI Δ -25 1

Vagal Tone Index Index Tone Vagal 0 -30 SMT SMT

PRE L1 L2 L3 L4 L5 L6 POST PRE L1 L2 L3 L4 L5 L6 POST Exercise Level Exercise Level

118

30 25 ΔMEI/ΔHR ) 20

Ω -0.249 Ω/bpm

, 15 R2 = 0.83, P < 0.001 (95% C.I.) 10

Ohms

(

o 0 -5

MEI -10 Δ -15

-20 -80 -20 0 40 80

ΔHRo (beats/min, bpm)

30 25

) 20 Ω

, 15 10

Ohms

(

o 0 ΔMEI/ΔVT -5 2

MEI +3.134 Ω/ln ms -10 2

Δ R = 0.77, P < 0.001 -15 (95% C.I.)

-20 -4 -2 0 2 4 6 8 2 ΔVTo (ln ms )

119

220 5

) 200 P < 0.05 )

Ω -5 ,

bpm 180 ( -10

160 Ohms

( -15 140 -20

CTRL MEI Δ Heart Rate Rate Heart 120 -25 BB P < 0.05 100 -30 SMT SMT

PRE L1 L2 L3 L4 L5 L6 POST PRE L1 L2 L3 L4 L5 L6 POST Exercise Level Exercise Level

Figure 5.4: Effects of (non-selective) β-adrenoceptor blockade (+BB, propranolol) in the myocardial electrical impedance (MEI, right), and heart-rate (HR, left) response(s) to exercise; β-adrenoceptor blockade blunted the MEI response to exercise. SMT = submaximal exercise test.

120

520

) 510

Ω , ,

500

Ohms (

490 MEI MEI

start end 480 5

) 0 P < 0.05

Ω , ,

-5 Ohms

( -10 MEI MEI

Δ -15

-20 Isoproterenol

PRE 0.005 0.015 0.05 0.15 0.5 POST Dose (µg/min·kg)

121

) Ω

, -5

-10 Ohms

( P < 0.05

-15 MEI

Δ P < 0.05 -20 Pacing Exercise -25 180 210 Heart Rate (beats/min)

Figure 5.6: Comparative myocardial electrical impedance (MEI) response to either submaximal exercise (white) or acute rate-matched ventricular pacing (black); only exercise decreased MEI.

122

0 P < 0.05

) -5

Ω , -10

-15

Ohms ( -20

MEI -25 Δ S (VF) -30 R

-35 SMT

PRE L1 L2 L3 L4 L5 L6 POST Exercise Level

Figure 5.7: Effects of underlying arrhythmic susceptibility of each animal in the myocardial electrical impedance (MEI) response to exercise; animals prone to ischemia-induced VF (i.e., S; n = 12) had a significantly larger MEI response to submaximal exercise, when compared those that were resistant (i.e., R; n = 9).

123

Chapter 6: Ischemia-Induced Electrotonic Uncoupling in the Surviving post-MI Myocardium and SCD

Sudden cardiac death (SCD) due to ventricular fibrillation (VF) remains a leading cause of death in most industrially developed countries (Zipes and Wellens. 1998; Janse,

2003; Mozaffarian et al., 2015). The temporary electrotonic depression of intrinsically viable tissue has been proposed as potential mechanism underlying these arrhythmias, as passive electrical decoupling can set/facilitate regional conduction slowing and blockade, a known “substrate” for re-entrant arrhythmias (Janse and Wit, 1989; de Groot and

Coronel, 2004; Coronel et al., 2010).

Indeed, electrotonic coupling is a well-established factor modulating both repolarization disturbances and heterogeneities, a known risk-factor for malignant arrhythmias in the clinic; in short, poorly-coupled cells have limited passive-electrical influence(s) over their neighbors (i.e., lower space constant facilitating heterogeneities) and therefore, are more likely to exhibit source-sink mismatches and pro-arrhythmic behaviors (e.g., Joyner and Sugiura, 1991; Shaw and Rudy, 1997; Pastore and Rosenbaum,

2000; de Groot and Coronel, 2004; Wit and Peters, 2012; Saffitz and Kléber, 2012).

Moreover, pro-arrhythmic temporal/spatial repolarization heterogeneities, such as those reflected by T-wave alternans (TWA), can be both determined and modulated by the loss of electrotonic interactions between cells (Pastore and Rosenbaum, 2000; Watanabe et al.,

2001; Cherry and Fenton, 2004; Sato et al., 2006; Kjølbye et al., 2008; Myles et al., 2010;

124

Storm et al., 2010; Jia et al., 2012), as the reduction of passive electrical coupling/loading can lead to localized heterogeneities (i.e., a substrate for reentry) (Bishop et al., 2014).

In particular, acutely depressed electrotonic coupling during myocardial ischemia

(e.g., Kléber et al., 1987; Cascio et al., 1990; Smith et al., 1997), such as triggered by acute coronary events (an established risk factor for SCD, e.g., see Mehta et al., 1997; Myerburg and Junttila, 2012; Tikkanen et al., 2012), has been shown to both facilitate and coincide with the onset of arrhythmias (e.g., Smith et al., 1997; de Groot and Coronel, 2004).

Thus, this study, partially published (conference proceeding) in Computers in

Cardiology (del Rio et al., 2004) and presented (in part) at the scientific sessions of both the American Heart Association (del Rio et al., Circulation 2005; 112(SII):11-16) and the

European Congress of Cardiology (del Rio et al., Eur Heart J 2007; 28(S): 29-30), investigated myocardial electrotonic coupling changes induced by acute ischemia

(coronary artery occlusion) in the non-infarcted left-ventricle of post-MI animals, as measured in vivo by myocardial electrical impedance (MEI). Specifically, the hypotheses that intrinsic arrhythmia susceptibility and autonomic activation can modulate ischemic myocardial electrotonic uncoupling (i.e., ME changes) were tested in animals demonstrated to be either prone or resistant to ischemia-induced VF. Briefly, acute ischemia at rest induced comparable electrotonic depression regardless of the underlying

125 arrhythmic susceptibility, however, such uncoupling uncovered pro-arrhythmic ventricular electrical oscillation (such as TWA) only in of post-MI animals prone to malignant arrhythmias and SCD. The data also demonstrates that acute electrotonic uncoupling during ischemia can be modulated by interventions affecting β-adrenoceptor (β-AR) signaling, as passive electrical derangements were blunted by complete/partial β-AR blockade but enhanced by exercise, particularly in SCD-susceptible animals.

Methods

The principles governing the care and treatment of animals, as expressed by the

American Physiological Society, were followed at all times during these studies. In addition, the animal protocols and experimental procedures were approved by The Ohio

State University’s Institutional Lab Animal Care and Use Committee (ILACUC) at this institution, and adhered to the statutes of the Animal Welfare Act and the guidelines of the

Public Health Service.

Surgical preparation: The surgical preparation of this model has been well described in the literature (e.g., see Billman, 2006), and is summarized in the previous chapters. Briefly, isoflurane-anesthetized heartworm-free mongrel dogs (n = 28) had a large antero-lateral infarction created by a two-stage permanent ligature of the distal left anterior descending coronary artery (LAD). At the time of infarction the animals were chronically instrumented with both a 20 MHz Doppler-flow probe (Crystal Biotech; Northborough, MA) and a pneumatic occluder (In Vivo Metrics; Ukiah, CA) placed/secured around the proximal left

126 circumflex coronary artery (LCX). In addition temporary pacing electrodes (Medtronic

Streamline™, 8mm2), were sutured (~1cm apart) into the mid-myocardial wall of the distal

LCX distribution, i.e., the remote (non-infarcted) myocardium for MEI measurements.

Myocardial Electrical Impedance: MEI was measured in a fashion previously well described (del Rio et al., 2005 and 2008). In short, using the pacing electrode(s), a computer controlled circuit stimulated the myocardium with a sub-threshold zero mean bipolar current, consisting of two alternating rectangular pulses (±5µA, 100µs wide) generated

10ms apart. The (positive) current stimulus and the respective voltage response of the myocardium were band-pass filtered (0.27 – 5.90 kHz), digitized (@ 22.0 kHz) and transformed into the frequency domain by a radix-2 Fast Fourier Transformation (FFT).

The complex MEI spectrum was calculated as the voltage to current ratio at each frequency component. Here, MEI is reported every 3s as the ensemble average (10 measurements) of the mean MEI modulus in the 0.5 kHz to 5.0 kHz range.

Arrhythmia susceptibility: In all animals, the susceptibility to ischemia-induced ventricular fibrillation was established using a standardized protocol, generally referred as the

“exercise-plus-ischemia” test (see Billman, 2006). In short, a six-stage 18min submaximal exercise test was performed (as described before; e.g., see del Rio et al., 2015) and during the last minute of exercise, the animals were subjected to a brief (2 min) LCX occlusion

(i.e., while running at 6.4 km/h and 16%;). This combination of exercise plus ischemia, when applied post-MI, yields two stable and well-differentiated populations of animals:

127 one susceptible and the other resistant to ischemia-induced malignant arrhythmias, such as ventricular fibrillation (for review see Billman, 2006). For this study, 19 animals that developed VF during the exercise-plus-ischemia test (i.e., susceptible; S) and 9 that did not

(i.e., resistant; R) were used.

Study Protocol: Following surgical instrumentation, the animals were allowed to recover and were subjected to brief (2min) LCX occlusions at rest (conscious) either under control conditions (CTRL, 23.5 ± 0.8 days post-MI; n = 28, 19 susceptible and 9 resistant) and/or following complete β-AR blockade (+BB, 23.6 ± 0.9 days post-MI; n = 19, 14 susceptible and 5 resistant) with propranolol HCL (1.0 mg/kg IV; Sigma Chemical Co., St. Louis,

MO). In addition, a subset of animals (n = 9, 6 susceptible and 3 resistant) also underwent brief (2min) LCX occlusions following either β1- (bisoprolol 0.6 mg/kg IV; Merck,

Darmstadt, Germany) or β2-AR selective antagonism (ICI 118,551 0.2 mg/kg IV; RBI,

Natick, MA) in order to establish the independent contribution of each receptor sub-type to passive electrical changes during ischemia. Finally, MEI changes during the exercise plus ischemia test were also recorded in a subset of animals (n = 10, 5 susceptible and 5 resistant), in order to establish the role of exercise-induced autonomic/metabolic activation in the electrotonic derangements triggered by acute ischemia.

Since repolarization heterogeneities (such as those reflected by T-wave alternans,

TWA) have been linked to increased VF vulnerability, temporal repolarization variability during ischemia (i.e., 60-90s following LCX occlusion) was assessed in a subset of animals

(n = 19, 10 susceptible and 9 resistant). Both temporal and power spectral analyses of a

128 single-lead bipolar electrocardiogram (ECG, 15s-epochs) were performed (see Fig. 6.1).

The standard deviation of the T-wave amplitude within each analysis epoch was calculated with the aid of pattern-recognition analysis software (ECG Auto; EMKA Technologies,

France) and used as a surrogate-marker of T-wave alternans (e.g., Nearing and Verrier,

2002). In addition, the power spectral density (PSD) of the ECG within each analysis window was estimated using a modified Welch periodogram (MATLAB; Mathworks, Inc), and the power ratio (PSDratio) between the spectral-densities at the heart beat frequency

(fbeat) and its second harmonic (fHIGH = 2*fbeat) was used to quantify higher-order (i.e., non- alternating) variability; when applied to cardiac action potentials isolated from resistant/susceptible animals (Sridhar et al., 2009), a PSDratio elevation can be observed in

(susceptible) cells presenting of premature depolarization and variable action-potential durations (see Fig. 6.2).

In Vivo Data Analysis: All in vivo data are presented as mean ± standard error of the mean

(SEM). Statistical analyses were performed with SigmaStat (Systat Software, Inc., San

Jose, CA). Changes in electrotonic coupling (i.e., ΔMEI) induced by coronary artery occlusion either under control (CTRL) and/or following β-AR stimulation (i.e., exercise,

+SMT)/blockade (+BB) were evaluated using a both one- (intervention, for ΔMEI) or two- way (time-point and intervention) analysis of variance (ANOVA) with repeated measures.

Intergroup comparisons (i.e., resistant vs. susceptible) were made using a two-way

(intervention, and group: susceptible/resistant) ANOVA with repeated measures on one factor (intervention). Finally, the statistical significance of any impedance changes induced

129 by either pacing (3 levels: baseline/two rates) and/or by isoproterenol infusion/washout (7 levels: baseline/five doses/recovery) was evaluated using one-way ANOVA with repeated measures. The normality and equal variance assumptions were verified; if these assumptions were not met, then a non-parametric repeated measurements ANOVA on

Ranks (e.g., Friedman) test was used. In all cases, if significant F-values (or Q-values in the non-parametric case) were observed, post hoc pair-wise comparisons were made using the Tukey test.

Linear regression analyses were performed in order to study the relationship

(interaction) between ischemia-induced electrotonic uncoupling (i.e., ΔMEI) and the estimated TWA amplitude, a clinical index of repolarization heterogeneities. The equality of the TWA/ΔMEI linear models fitted to the two different arrhythmic phenotypes studied

(i.e., susceptible vs. resistant) was tested by multiple linear regression analysis, considering both qualitative (group) and interaction terms (i.e., simultaneously testing the differences in slope and intersect of the regression functions). For all analyses, P<0.05 was considered, a priori, to be statistically significant.

In Silico Experiments: In order to model the heterogeneous electrotonic properties of the interface(s) between non-infarcted, ischemic, and infarcted (i.e., scar) myocardium, cable theory was used to simulate propagating cardiac action potentials (PAPs) in a one dimensional (1D) structure, as described by the following partial differential equation

(PDE):

130

 a   2    V  C  V  I V ,t (1)   2 m m m ionic   2  Ri  x t where Ri is the cell-to-cell coupling (or axial) resistivity (Ω·cm), Vm is the transmembrane potential (mV), and Iionic is the non-linear membrane current density (µA/cm2), which was modeled following the Luo-Rudy equations (Luo and Rudy, 1991 and 1994). The cable equation (1) was solved using the implicit Crank-Nicholson method described by Joyner et al. (1978), while the differential equations describing the ionic current kinetics were solved using the hybrid integration method. Additionally, symmetric propagation

(stimulus-end) and sealed-end (far-end) boundary conditions were used (Maglaveras et al.,

1988). The length (∆x = 100µm) and time (∆t = 10µs) increments were set as in Sahakian et al. (1992), for a cable of N = 40 segments and T = 40ms (i.e., 4000 iterations). The rest of the parameters were 2.5µm for the cable radius (a), and 1 µF/cm2 for the membrane capacitance (Cm).

The passive electrical properties of the boundaries between normal, ischemic myocardium and scar were modeled by means of a spatially heterogeneous coupling resistance Ri (e.g., Sahakian et al., 1992). Three regions (normal, ischemic, and infarcted) were defined along the cable, and previously reported MEI values were used as estimates of Ri for each region (Fig. 6.5). For this model, the axial resistivities, reflecting the electrotonic properties of normal, ischemic and scarred myocardial regions were set to 550,

630 and 160 Ω·cm (respectively), consistently with previous observations in both humans and experimental models (Howie et al., 2001; Dzwonzayk et al., 2004; del Rio et al., 2005,

2008a, and 2008b). Propagation was studied in three scenarios: baseline (i.e., pre-

131 ischemic), acute LCX occlusion both under control conduction (CTRL), and following β-

AR blockade (+BB). The β-AR blocked ischemic region was modeled by a 30-50% reduction of the ischemic MEI increase observed/established for the control occlusion, in accordance with the reported beneficial effects of β-blockers on the energy metabolism of ischemic myocardium (Nanki et al., 1987).

Results

In all cases, MEI increased, as expected (del Rio 2005 and 2008), rapidly and significantly following an acute coronary insult (occlusion) (see Fig. 6.2). However, the degree of these ischemia-induced electrotonic changes was modulated by interventions blunting/increasing β-AR signaling (see Table 6.1 and Fig. 6.3). Complete β-AR blockade blunted the ischemic MEI increase (ΔMEI, +BB: -30 ± 5%, 14.0 ± 1.3 vs. 21.6 ± 2.2 Ω in

CTRL, P< 0.05), while exercise enhanced it (+SMT: +60 ± 17%, 29.4 ± 3.5 Ω, P<0.05).

Small differences in baseline (i.e., pre-ischemic) impedance values were observed between control and β-AR blocked myocardium, but, consistently with previous observations (del

Rio et al., 2015), exercise appeared to enhance electrotonic coupling (i.e., decrease MEI) in non-ischemic myocardium (see Table 6.1).

Notably, both β1- and β2-ARs appeared to mediate the early electrotonic decoupling during ischemia (see Fig. 6.2), as the independent antagonism of each receptor sub-type successfully blunted the MEI response (ΔMEI, CTRL: 23.7 ± 2.7 Ω vs. β1: -32 ±

5%, 15.5 ± 1.7Ω and β2: -21 ± 7%, 17.8 ± 1.5Ω, both P< 0.05).

132

Arrhythmia susceptibility: Surprisingly, acute ischemia resulted in comparable MEI changes in animals prone and resistant to arrhythmias (S: 23.4 ± 2.9Ω vs. R: 17.4 ± 2.5Ω,

N.S.), with β-AR blockade providing similar (significant) electrotonic protection to both groups (S+BB: -32 ± 6%, to 14.6 ± 1.6Ω vs. R+BB: -26 ± 7%, to 12.5 ± 2.8Ω; N.S.). On the other hand, exercise enhanced ischemic electrotonic changes preferentially in susceptible dogs (S: 36.2 ± 3.9 Ω vs. R: 22.7 ± 4.2 Ω, P < 0.05), likely via an exercise- mediated reduction in the pre-ischemic MEI values (S: -24.9 ± 9.5 Ω vs. R: 16.3 ± 3.6 Ω,

P = 0.2) (see Table 6.2). At rest, the remote (non-infarcted) myocardium had higher pre- ischemic MEI values in animals prone to arrhythmias (S: 533 ± 18 Ω vs. R: 446 ± 29 Ω, P

< 0.05); acute β-AR blockade blunted these baseline differences (S: 481 ± 24 Ω vs. R: 451

± 38 Ω, N.S.)

No TWA amplitude differences were observed between susceptible and resistant animals at rest (S: 6.6±1.9mV vs. R: 6.9±1.4mV, N.S.). However, after 90s of ischemia, alternans increased significantly more in susceptible animals (S: 24.4±5.9mV vs. R:

11.1±2.1mV, P<0.05), despite both groups presenting similar degrees of electrotonic uncoupling (see Table 6.2). Moreover, (only) in susceptible animals, the TWA amplitude during ischemia was well correlated (R2 = 0.82, P<0.05) with the degree of induced electrotonic decoupling (i.e., MEI changes) (see Fig. 6.4).

Finally, acute ischemia led to a marked increase in the relative power-density of inter-beat electrical phenomena (see Table 6.2) in animals prone to VF (S: 1.23±0.17 vs.

1.66±0.22, P<0.05) but not in those resistant (R: 0.97±0.13 to 1.00±0.22, N.S.), suggesting

133 the presence of higher-order (i.e., non-alternating) oscillations such as afterdepolarizations.

In Silico Model: Simulated action potential propagation in an electrotonically heterogeneous myocardium consisting of a non-infarcted, ischemic, and scar boundary is shown in Fig. 6.4. Simulated electrotonic changes (i.e., MEI rise) in the ischemic myocardium adjacent to scar, resulted in unidirectional block. However, moderate (30-

50%) attenuation of such uncoupling, as measured under β-AR blockade, allowed bidirectional conduction.

Conclusion

Taken together, these data show that ischemia-induced electrotonic uncoupling facilitates and uncovers pro-arrhythmic ventricular electrical oscillation (such as TWA) in post-MI animals susceptible to malignant arrhythmias and sudden death. The data also demonstrates that acute electrotonic uncoupling during ischemia can be modulated by interventions affecting β-AR signaling, as passive electrical derangements were blunted by complete/partial β-AR blockade but enhanced by exercise. Notably, β-adrenergic receptor blockade remains as the only antiarrhythmic drugs that offers significant protection against sudden death (Janse, 2003), and has been shown to significantly reduce TWAs (Rashba et al., 2002). The findings of this study suggest that β-AR blockade protection is achieved, at least partially, by attenuation of cell-to-cell electrical uncoupling.

134

Parameter / Sub-Group Baseline CAO ΔMEI

Control (n = 28) 505 ± 17 526 ± 17* +21.6 ± 2.2 + BB (n = 19) 473 ± 20 487 ± 21* +14.0 ± 1.3↓ (-30 ± 5%) + SMT (n = 10) 451 ± 26↓ 480 ± 27* +29.4 ± 3.5↑ (+60 ± 17%) Susceptible (S, n = 19) 533 ± 18† 556 ± 18*,† +23.4 ± 2.9

MEI (Ohms) MEI Resistant (R, n = 9) 446 ± 29 463 ± 31* +17.4 ± 2.5

*: P<0.05 vs. Baseline; †: P<0.05 vs. Resistant (R); ↑,↓: P<0.05 vs. Control (CTRL)

135

Table 6.2: Pro-arrhythmic effects of acute ischemia (LCX coronary artery occlusion, CAO) at rest in conscious post-MI dogs known to be either susceptible (S) or resistant (R) to malignant arrhythmias, as reflected by changes in electrotonic coupling (i.e., myocardial electrical impedance, ΔMEI) as well as by both the TWA amplitude and the relative power- density (PSDratio) of inter-beat electrical events; comparative effects of either β- adrenoceptor blockade (+BB) or autonomic activation (via submaximal exercise, +SMT) on MEI are also shown.

Parameter / Sub-Group Susceptible (S) ǂ Resistant (R) ǂ

ΔMEI (Ohms, CTRL) +23.4 ± 2.9 (n = 19) +17.4 ± 2.5 (n = 9) + BB (β-AR blockade) +14.6 ± 1.6 (n = 14) +12.5 ± 2.8 (n = 5) + SMT (Exercise) +36.2 ± 3.9† (n = 5) +22.7 ± 4.2 (n = 5) TWA (mV, CTRL) 6.6 ± 1.9 to 24.4 ± 5.9*,† 6.9 ± 1.4 to 11.1 ± 2.1* ,† PSDratio (n/u, CTRL) 1.2 ± 0.2 to 1.7 ± 0.2* 1.0 ± 0.1 to 1.0 ± 0.2*

†: P<0.05 vs. Resistant (R); *: P<0.05 vs. Baseline; ǂ: n = 10 unless indicated otherwise.

136

1 2s 2s 2s

0.5

) mV

( 0

ECG -0.5

-1 LCX CAO

250 ) 200

bpm 150 (

100 HR HR

50 VF

CAO

0 30 60 Time (s)

PSDratio = PSD(fbeat)/PSD(fHIGH)

) 0.5

2 fbeat TWA = [PSD(fTWA) - PSD(fnoise)]

mV ( fTWA= 0.5*fbeat fHIGH fnoise

fTWA PSD

Frequency (Hz) 15s-epoch Welch s Periodogram

Figure 6.1: Representative electrocardiogram (ECG) and respective Power-Spectral- Density (PSD) taken before/during an acute left-circumflex (LCX) coronary artery occlusion (CAO) in a conscious post-MI dog shown to be susceptible to ischemia-induced ventricular fibrillation. Both the standard deviation of the T-wave amplitude within each analysis epoch (a surrogate-marker of T-wave alternans, TWA) and the power ratio (PSDratio) between the spectral-density at the heart beat frequency (fbeat) and its first harmonic (fHIGH = 2*fbeat) were used in order to quantify temporal repolarization variability before and during ischemia.

137

80 6000

60 Resistant (R) R 5000 fbeat

40 )

) PSD(fbeat)/PSD(fHIGH)

4000 mV

20 Hz

(

/ 2

0 3000 mV

( PSDratio -20

2000 PSD PSD

Amplitude -40

1000 -60 fHIGH

-80 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.5 1 1.5 2 2.5 3 Time (s) f (Hz)

150 3500

Susceptible (S) 3000 S 100 fbeat

) 2500

)

Hz ( 50 / PSDratio

2 2000 mV

( 1500 0

PSD PSD 1000 fHIGH Amplitude -50 500

-100 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.5 1 1.5 2 2.5 3 Time (s) f (Hz)

138

30 CT RL 25 +BB

) 20 LCX CAO

Ω ( 15

10 β-AR (P<0.05) 5 -32.4 ± 5.4%

0 MEI ChangeMEI

-5

-10 -1 0 1 2

30 CT RL 25 +BB

) 20

Ω ( 15

10 β1-AR (P<0.05) 5 -32.4 ± 5.1%

0 MEI ChangeMEI

-5

-10 -1 0 1 2

30 CT RL 25 +BB

) 20

Ω ( 15

10 β2-AR (P<0.05) 5 -20.5 ± 6.6%

MEI ChangeMEI 0

-5

-10 Baseline CAO

-1 0 1 2 Time (min)

139

60 0.828*ΔMEI + 4.638 R² = 0.816 (P<0.05)

) 50 V

m 40 (

30 TWA

TWAs 20 SUSCEPTIBLE 10 @ CAO (90s)

0 0 10 20 30 40 50 60 70 80 70

) 50 -0.095*ΔMEI + 12.67 V R² = 0.008 (N.S.)

m 40 (

TWAs 20 RESISTANT 10 @ CAO (90s)

0 0 10 20 30 40 50 60 70 80 ΔMEI (Ohms)

Figure 6.4: Relationship(s) between the ischemia-induced electrotonic uncoupling, as reflected by myocardial electrical impedance changes (i.e., ΔMEI), and the ECG-derived TWA amplitude (an index of repolarization heterogeneity), in conscious post-MI dogs known to be either susceptible (S, top) or resistant (R, bottom) to malignant arrhythmias.

140

Axial Resistivity Baseline 700 20 A. B.

600 0 )

500 -20 10 20

)

mV ( normal ischemic scar ·cm 400 -40

Ω 26 (

Ri Ri 300 -60

Baseline VOLTAGE 32 200 CTRL -80 +BB 100 -100 0 10 20 30 40 0 10 20 30 40 CELL # TIME (ms)

CAO (CTRL) CAO (+BB) 20 20 C. D.

0 0

) )

-20 10 20 -20 10 20

( ( -40 -40 26 26

-60 -60

VOLTAGE VOLTAGE VOLTAGE VOLTAGE 32 32 -80 -80

-100 -100 0 10 20 30 40 0 10 20 30 40 TIME (ms) TIME (ms)

Figure 6.5: In an in silico model, a non-uniform cell-to-cell coupling resistivity (Ri) reflecting the heterogeneous electrotonic properties of the interface(s) between remote (non-infarcted), ischemic, and infarcted (scar) myocardium (A, top left) results in altered propagation: electrotonic uncoupling during modeled ischemia (i.e., coronary artery occlusion, CAO) results in unidirectional block (C, bottom left), while its attenuation by β-AR blockade prevents it (D, bottom right). Panels B-D show propagating action potentials in four cable segments (cells # 10, 20, 26 and 32).

141

Chapter 7: Summary

Despite significant advances in the understanding of the physiological substrate(s) mediating/facilitating malignant arrhythmias, arrhythmic sudden cardiac death remains a leading cause of death. Previous reports have suggested a role of electrotonic/passive electrical changes in both the onset and maintenance of arrhythmias, however, no study to date has assessed the ability of indices reflective of electrotonic changes to stratify intrinsic arrhythmic susceptibility in vivo. The experiments presented in this document leveraged both a well-established model of lethal arrhythmias post-MI and myocardial electrical impedance (MEI) spectral measurements in vivo, to establish the relevance and prognostic value of electrotonic changes in SCD. In particular, the studies described above evaluated electrotonic changes/heterogeneities in the setting of three well-established risk factors for arrhythmias/SCD (acute ischemia, myocardial infarction, and/or autonomic imbalances) in order to conclude the following:

• Early electrotonic changes (i.e., MEI increases) following an acute ischemic insult

(coronary artery occlusion) were shown not only to precisely indicate the onset of

ischemia in both pre-clinical models (dogs/pigs) and humans, but also, to be sensitive

to interspecies differences in collaterization modulating the severity of the ischemic

insult (Chapter 2).

• Changes in autonomic balance favoring parasympathetic control (e.g., vagal nerve

stimulation) were shown to modulate the passive electrical properties of the 142 myocardium, attenuating the degree of early electrotonic uncoupling induced by myocardial ischemia, possibly via a reduction in cardiac metabolic demand (i.e., attenuation of the ischemic insult) due to neutrally-induced bradycardia. In particular, it was shown that 1) myocardial electrical impedance increased more rapidly following coronary occlusion in vagotomized animals when compared to neurally intact dogs; 2)

Bilateral vagotomy during the coronary occlusion further increased myocardial electrical impedance; 3) Acute vagal nerve stimulation abruptly decreased myocardial electrical impedance during the coronary occlusion. This effect was promptly reversed upon termination of vagal nerve stimulation and was abolished when heart rate was maintained by cardiac pacing but was not affected by bilateral stellectomy; and 4)

Acute vagal nerve stimulation during the coronary occlusion significantly attenuated electrocardiographic indices of ischemia-induced injury and pro-arrhythmic dispersion of repolarization (Chapter 3).

• Both infarcted and remote (i.e., non-infarcted) myocardium undergo chronic electrotonic remodeling following a sustained ischemic insult (coronary artery ligation). Overall, the healing infarction was shown to have lower impedance than remote, non-infarcted, myocardium, with animals prone to malignant arrhythmias showing a wider electrotonic gradient (i.e., impedance difference) between these two myocardial regions. This broader “impedance mismatch” between infarcted and non- infarcted myocardium provided a reliable marker for subsequent susceptibility to VF.

Indeed, in SCD-susceptible animals this electrotonic “mismatch” increased over time

143

due to the remodeling in non-infarcted myocardium, facilitating the impedance-based

classification of arrhythmic susceptibility (Chapter 4).

• In the electrotonically-remodeled post-MI hearts, acute β-adrenoceptor activation

(either during bouts of exercise or via a direct pharmacological challenge) was shown

to acutely increase passive (electrotonic) coupling in the remote (non-infarcted)

myocardium, with animals susceptible to SCD showing the greatest electrotonic

responsiveness (Chapter 5).

• Finally, electrotonic uncoupling induced by an acute ischemic insult (coronary

artery occlusion) to the non-infarcted myocardium of post-MI animals susceptible to

malignant arrhythmias, was shown to facilitate/uncover (i.e., predict) pro-arrhythmic

ventricular electrical oscillations and repolarizarion abnormalities (such as TWA).

Moreover, acute electrotonic uncoupling during ischemia was shown to be modulated

by interventions affecting β-AR signaling, as passive electrical derangements were

blunted by complete/partial β-AR blockade but enhanced by exercise (Chapter 6).

Taken together, these experiments successfully establish passive electrical heterogeneities in vivo, and their modulation by autonomic changes, as a risk/prognostic factor for SCD/arrhythmic susceptibility.

Future directions: For instance, both cell-to-cell coupling (e.g., Burt and Spray, 1988;

Saffitz and Yamada, 1998; Salameh and Dhein, 2013) and global indices of myocardial passive electrical properties (e.g., Sasaki et al., 1994; Dekker et al., 1996; Howie et al.,

144

2001) have been linked/associated with alterations in the mechanical properties of the myocardium. Indeed, ex vivo, mechanical stretch can alter intercellular coupling (Salameh and Dhein, 2013), and positive/negative inotropic agents have been shown to enhance/depress (respectively) junctional coupling in parallel with their functional effects

(Burt and Spray, 1988; Dhein, 2004). However, neither systemic/cardiac hemodynamics nor mechanics were assessed in the studies presented within this document. Since functional depression is an important prognostic index in SCD, the potential pro- arrhythmic inter-relationship(s) between electrotoninc and mechanical dysfunction remain to be elucidated in vivo.

145

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