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UIVLI800-521-0600
ENHANCED IN VIVO AND IN VITRO RESPONSE TO BETA-2 ADRENERGIC RECEPTOR STIMULATION IN ANIMALS SUSCEPTIBLE TO VENTRICULAR FIBRILLATION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Melanie Taghon Houle, M.S.
*****
The Ohio State University 1999
Dissertation Committee: Approved By Professor George E. Billman, Adviser Professor Ruth A. Altschuld Professor Patrick Ward Professor Jack Rail Adviser Graduate Program in Physiology UMI Number: 9951667
UTVLI 9951667 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
Bell & Howell Biformation and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT
Sudden, cardiac death is an unexpected natural death from a cardiac cause within a short period of time from the onset of symptoms. A canine model of sudden cardiac death has been developed such that dogs with a healed myocardial infarct (MI) develop ventricular fibrillation (VF) induced by a 2 min coronary occlusion during the last min of exercise and maintained one min after exercise. An enhanced (3%-adrenergic receptor (P 2-
AR) response was noted in dogs that were susceptible to VF. It was previously demonstrated that the pz-AR agonist zinterol elicited larger increases in the transient amplitude in myocytes obtained from susceptible (S) as compared to resistant (R) dogs.
The contractile response to Pz-AR stimulation had not been determined in the intact animal. Therefore, the contractile response to P-adrenergic receptor (p-AR) stimulation was evaluated using echocardiography (velocity o f circumferential fiber shortening (Vcf) both before (n=35, S=19 & R=16) and after anterior wall infarction (MI, n=31, S=12 &
R=19). Before MI, increasing doses of isoproterenol (ISO) provoked similar responses in both groups of dogs. The Pi-adrenergic receptor (Pi-AR) or Pz-AR antagonists
(bisoprolol or ICI 118,551 respectively) had similar effects on the ISO response in both groups with a larger reduction noted after Pi-AR blockade. In contrast, after Ml, the susceptible dogs displayed a significantly (ANOVA p<0.01) larger Vcf response to ISO which was eliminated by both Pa-AR and Pi-AR blockade. The single cell unloaded
shortening response to ISO was also larger in cells obtained from susceptible animals as compared to resistant dogs. As was noted in the intact animal, the Pa-AR antagonist ICI
118,551 elicited a greater reduction in the susceptible dog myocytes
(S, -48%, n = 6 and R, -15%, n=9). The number o f Pa-ARs, as determined by radioligand binding, as well as the Kd value, was similar in both groups. As such, these data suggest that the enhanced Pa-AR response provoked by infarction in the susceptible dogs may result from altered Pa-AR coupling to G proteins resulting in cytosolic Ca“^ changes. The p44/42 mitogen activated protein (MAP) kinase cascade is coupled to a Gi protein and has been implicated as a possible alternative non-arrhythmogenic signaling pathway for the Pa-AR. Therefore, the hypothesis that the resistant animals activate the p44/42 MAP kinase pathway while the susceptible animals activate the Gs/cAMP pathway was explored. With Pa-AR as well as a i-adrenergic receptor (ai-AR) activation, the susceptible animals (n= 6 ) had a significantly greater upregulation of the p44/42 MAP kinase pathway as compared to the resistant animals (n= 6 ). Therefore, preferential activation of the p44/42 MAP kinase system cannot explain the observed differences in susceptibility to ventricular fibrillation. However, the possibility still exists for stronger coupling to the Gs protein of the cardiac pz-AR in susceptible animals thereby contributing to abnormalities in calcium handling in these animals.
Ill DEDICATION
“There are no secrets to success: Don’t waste time looking for them. Success is the result of perfection, hardwork, learning from failure, loyalty to those with whom you work and persistence.” General Colin Powell
I would like to dedicate this thesis to my husband, Tom, who never let me give up, and to my son, Sam, who never let me forget what is truly important.
IV ACKNOWLEDGMENTS
I wish to thank my adviser. Dr. George Billman, for many years of support and understanding as well as his patience in guiding me through the formation of this dissertation.
I would also like to thank Dr. Ruth Altschuld for the insightfid conversations, guidance and encouragement through the (at times) exasperating single cell and MAP kinase experiments.
1 am grateful for the technical help and encouragement of Lou Castillo who helped with the myocyte isolation procedure. Bob Kelley for help with the edge detection studies. Dr. Tomohiro Nakayama for his expert echocardiography skills, and Dr. Beth
Holycross for her insightful conversations.
I would also like to thank my husband, Tom Houle, for his expert computer assistance.
Finally, I must acknowledge the generous contributions of Merck Parmaceutical for supplying the bisoprolol as well as Proctor and Gamble for supplying some of the susceptible animals used for the single cell and MAP kinase studies. VITA
September 8 , 1968 ...... Bom - South Bend, Indiana
May 1990 ...... B.S. Biology, Saint Mary’s College, South Bend, Indiana
1990-1992 ...... AT.S. Biology, University o f Denver. Denver, Colorado
1992-1994 ...... Instructor, Chemistry University of Nebraska, Omaha Omaha, Nebraska
1995-present ...... Graduate Research Associate The Ohio State University Columbus, Ohio
PUBLICATIONS
1. Taghon, M. S. and S.E. Sadler, Insulin-like growth factor 1 receptor mediated endocytosis in Xenopus laevis oocytes: A role for receptor tyrosine kinase activity. Developmental Biology 163(l):66-74, 1994.
2. Houle, M. S. and G. E. Billman. Low frequency component of the heart rate variability spectrum: a poor marker of sympathetic activity. Am J Physiol. 276(Heart Circ. Physiol. 45)H215-H223, 1999
3. Lynch, J. J., M. S. Houle, G. L. Stump, A. A. Wallace, D. B. Gilberto, H. Jahansouz, G. R. Smith, A. J. Tebbens, N. J. Liverton, H. G. Seinick, D. A. Claremon, and G. E. Billman. Antiarrhythmic efficacy of selective blockade o f the cardiac slowly activating delayed rectifier current Iks in canine models of malignant ischemic ventricular arrhythmias. Circulation 1999;100:1917-1923.
VI ABSTRACTS
1 . Houle, M. S. and G. E. Billman. The autonomic response to exercise in animals susceptible to ventricular fibrillation. Physiologist 39: A17, 1996.
2. Houle, M. S., G. E. Billman, and P. Ward. In Vivo biological activities of angiotensin and kinin metabolites in dog vasculature. FASEB /II: A501, 1997.
3. Billman, G. E. and M. S. Houle. Heart rate variability in animals susceptible to ventricular fibrillation: low firequency power is a poor marker of sympathetic activity. JAuton Nerv Sys 65: 87, 1997.
4. Houle, M. S., G. E. Billman, J. Hensley, and R. A. Altschuld. Effects of calcium channel antagonists on Ca^^ transients in canine atrial cardiomyocytes. FASEB J 12: A74, 1998.
5. Billman, G. E., M. S. Houle, and J. J. Lynch. Selective Iks blockade protects against ventricular fibrillation induced by myocardial ischemia. Enr Heart J 19(Abstract Suppl): 17, 1998.
6 . Houle, M. S., L. Castillo, J. Hensley, C. M. Hohl, P. F. Binkley, R. A. Altschuld, and G. E. Billman. Enhanced in vivo response to Pi-adrenergic receptor activation in post- infarcted canines susceptible to ventricular fibrillation. Circulation 98:1-553, 1998.
7. Billman, G. E., M. S. Houle, and J. J. Lynch. Selective Iks, but not Ikt blockade protects against ventricular fibrillation induced by myocardial ischemia. Circulation 98: 1-52, 1998.
8 . Houle, M. S., T. Nakayama, R. A. Altschuld, and G. E. Billman. Enhanced in vivo and in vitro contractile responses to Pa-adrenergic receptor stimulation in animals susceptible to ventricular fibrillation. Circulation 100(Suppl. I)d-274,1999.
9. Billman, G. E., M. S. Houle, H. C. Englert, and Goegelein. Ischemically-induced changes in the T-wave and susceptibility to sudden death: evidence that activation of the ATP-sensitive potassium channel may contribute to ventricular fibrillation. Circulation 100 (Suppl. I) :I51-152, 1999.
FIELDS OF STUDY
Major Field: Physiology
VII TABLE OF CONTENTS
Page Abstract ...... ü
Dedication ...... iv
Acknowledgments ...... v
Vita...... vi
List of Figures ...... xi
Abbreviations ...... xvi
1. Introduction ...... I
1.1 Sudden Death and Arrhythmias ...... 2
1.1.1 Abnormal Automaticity ...... 4
1.1.2 Abnormal Impulse Conduction ...... 5
1.2 Overview of Cardiac P-Adrenergic Receptors ...... 6
1.3 Specifics of the Cardiac Pi-Adrenergic Receptor ...... 12
1.4 Mitogen Activated Protein Kinase ...... 16
1.5 Role of Mitogen Activated Protein Kinases in Cardiac Muscle...... 22
1.6 Proposed Studies ...... 25
2. Materials and Methods ...... 29
2.1 Surgical Preparation of the Canine Model of Sudden Death ...... 29
vin 2.2 Exercise plus Ischemia Test: Classification of the Dogs ...... 30
2.3 Echocardiography studies ...... 32
2.4 Isolation of Ventricular Myocytes ...... 33
2.5 Edge Detection Protocol ...... 35
2.6 Western blot Detection of p44/42 Mitogen Activated Protein Kinase or p38 Mitogen Activated Protein Kinase ...... 36
2.7 Mitogen Activated Protein Kinase Activity Assay ...... 3 8
2.8 Statistical and Data Analysis ...... 39
3. Results...... 41
3.1 Classification of Dogs ...... 41
3.2 Analysis of Pre-Infarct Echocardiograms ...... 41
3.3 Analysis of the Post-Infarct Echocardiograms ...... 42
3.4 Analysis of Single Cell Unloaded Shortening ...... 44
3.5 Analysis of the p44/42 and p38 Mitogen Activated Protein Kinase Western Blots ...... 46
3.6 Analysis of the p44/42 Mitogen Activated Protein Kinase Activity Assay...... 47
3.7 Summary o f Findings ...... 48
4. Discussion ...... 152
4.1 Analysis o f P2-Adrenergic Receptor Function in Susceptible and Resistant Dogs ...... 154
4.2 Pi-Adrenergic Receptor Activation wit Ischemia and the Development of Arrhythmias ...... 159
4.3 Intracellular Signaling of the Pz-Adrenergic Receptor ...... 163
4.4 Study Limitations...... 172
ix 4.5 Future Studies...... 172
References ...... 174
Appendix ...... 182 LIST OF FIGURES
Figure Page 1.1 P-Adrenergic Receptor Second Messenger System ...... 9
1.2 Desensitization and Downregulation of the P-Adrenergic Receptor ...... 11
1.3 Typical Three Kinase Pathway Observed for the Family of Mitogen Activated Protein Kinases ...... 18
1.4 Detailed Description of the Three Mitogen Activated Protein Kinase Signaling Pathways ...... 21
3.1 Representative M-mode Echocardiograms from a Resistant Dog Prior to Infarct ...... 51
3.2 Representative M-mode Echocardiograms from a Susceptible Dog Prior to Infarct ...... 53
3.3 Vcf Isoproterenol Dose Response for Susceptible and Resistant Dogs Prior to Infarct ...... 55
3.4 Pre-Infarct Vcf Isoprotereinol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Resistant Dogs...... 57
3.5 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and Without Bisoprolol for Resistant Dogs ...... 59
3.6 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and Without ICl 118,551 for Resistant Dogs ...... 61
3.7 Pre-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Blockade for Susceptible Dogs ...... 63
3.8 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs ...... 65
xi 3.9 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs ...... 67
3.10 Heart Rate Isoproterenol Dose Response for Susceptible and Resistant Dogs Prior to Infarct ...... 69
3.11 Pre-Infarct Heart Rate Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Resistant Dogs ...... 71
3.12 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Resistant Dogs ...... 73
3.13 Pre-Infarct Heart Rate Dose Response Curve for Isopoterenol With and Without ICI 118,551 for Resistant Dogs ...... 75
3.14 Pre-Infarct Heart Rate Isoproterenol Dose Response Curves in the Presence and Absence o f P-Adrenergic Receptor Blockade for Susceptible Dogs ...... 77
3.15 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs ...... 79
3.16 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs ...... 81
3.17 Representative M-mode Echocardiograms from a Resistant Dog Post Infarct ...... 83
3.18 Representative M-mode Echocardiograms from a Susceptible Dog Post Infarct ...... 85
3.19 Vcf Isoproterenol Dose Response for Susceptible and Resistant Dogs Post Infarct ...... 87
3.20 Post-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade For Resistant Dogs ...... 89
3.21 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and Without Bisoprolol for Resistant Dogs ...... 91
3.22 Post-Infarct Vcf Dose Response Curve for Isopoterenol With and Without ICI 118,551 for Resistant Dogs ...... 93
XU 3.23 Post-Infarct V cf Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Susceptible Dogs ...... 95
3.24 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs ...... 97
3.25 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs ...... 99
3.26 Percent Change of the Isoproterenol Response Due to ICI 118,551 in Susceptible and Resistant Animals ...... 101
3.27 Percent Change o f the Vcf Isoproterenol Dose Response from two Consecutive Infusions ...... 103
3.28 Heart Rate Isoproterenol Dose Response for Susceptible and Resistant Dogs Post Infarct ...... 105
3.29 Post-Infarct Heart Rate Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Resistant Dogs ...... 107
3.30 Post-Infarct Vcf Dose Response Curves for Isoproterenol With and Without Bisoprolol for Resistant Dogs ...... 109
3.31 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Resistant Dogs ...... 111
3.32 Post-Infarct Heart Rate Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Susceptible Dogs ...... 113
3.33 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs ...... 115
3.34 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs ...... 117
3.35 Representative Edge Detection Tracings from a Resistant Dog ...... 119
3.36 Representative Edge Detection Tracings from a Susceptible Dog 121
3.37 Percent Unloaded Cell Shortening for Resistant and Susceptible Dogs ...... 123 xiii 3.38 Percent Change from Control for Unloaded Single Cell Shortening ...... 125
3.39 Single cell +dL/dt max for Susceptible and Resistant Myocytes ...... 127
3.40 Time to 50% Relaxation for Stimulated Single Cardiomyocytes from Susceptible and Resistant Dogs ...... 129
3.41 Percent Unloaded Cell Shortening from Multiple Isoproterenol Treatments...... 131
3.42 Representative p44/42 MAP Kinase Western Blots from Susceptible and Resistant Dogs ...... 133
3.43 Presence of p44/42 MAP Kinase with Stimulation by Zinterol in Susceptible and Resistant Dogs ...... 135
3.44 Presence of p44/42 MAP Kinase with Stimulation by Phenylephrine in Susceptible and Resistant Dogs ...... 137
3.45 Presence of p44/42 MAP Kinase as Compared to Control with Stimulation by Zinterol in Susceptible and Resistant Dogs ...... 139
3.46 Presence of p44/42 MAP Kinase as Compared to Control with Stimulation by Phenylephrine in Susceptible and Resistant Dogs 141
3.47 Representative Radiographs from the p44/42 MAP Kinase Activity Assay for Susceptible and Resistant Dogs ...... 143
3.48 p44/42 MAP Kinase Activity for Susceptible and Resistant Dogs Following Stimulation by Zinterol ...... 145
3.49 p44/42 MAP Kinase Activity for Susceptible and Resistant Dogs Following Stimulation by Phenylephrine ...... 147
3.50 p44/42 MAP Kinase Activity Normalized to Control for Susceptible and Resistant Dogs Following Stimulation by Zinterol ...... 149
3.51 p44/42 MAP Kinase Activity Normalized to Control for Susceptible and Resistant Dogs Following Stimulation by Phenylephrine ...... 151 A. 1 Representative M-mode Echocardiogram of the Left Ventricle Demonstrating the LVIDd and LVTDs ...... 184
A.2 Representative M-mode Echocardiogram Demonstrating the Ejection Time as Measured from the Aortic Valve ...... 185
XV ABBREVIATIONS
ATP adenosine 5’-triphosphate
AY node atrial ventricular node
(3-ARK P-adrenergic receptor kinase
Pi-AR pi-adrenergic receptor
Pz-AR Pz-adrenergic receptor
BIS bisoprolol
BSA bovine serum albumin cAMP cyclic adenosine 5’-monophosphate
DADs delayed afterdepolarizations
EADs early afterdepolarizations
ERK extracellular signal-regulated protein kinase
ET ejection time
ISO isoproterenol
Ho null hypothesis
JNK c-jun kinase
LVIDd left ventricular internal diastolic diameter LVIDs left ventricular internal systolic diameter
MAP kinase mitogen activated protein kinase
MEKK MAP or ERK kinase kinase
MEK MAP or ERK kinase
PAK G-protein activated kinase
PKA protein kinase A
PTX pertussis toxin
R resistant
SA node sinoatrial node
SAPK stress activated protein kinase
S susceptible
TBS tris buffered saline
TEST tris buffered saline with tween
Vcf velocity of circumferential fiber shortening
VF ventricular fibrillation
xvu CHAPTER 1
INTRODUCTION
Ischemia is classically defined as a decrease in the blood flow to a tissue. In myocardial ischemia, the cells sufier firom a loss o f oxygen due to low blood flow as well as an accumulation of potentially harmful metabolites. It involves a decrease in the ATP supply thus causing a shift in the myocardial metabolism firom aerobic to anaerobic. This shift increases glycogenolysis resulting in an increase in lactate formation, a decrease in the intracellular pH, and a decrease in the tissue level of glycogen. It is well established that myocardial ischemia (35,55,62) and exercise (59,61) elicit an increase in eflerent sympathetic nerve activity to the myocardium thus causing increases in norepinephrine levels and activation of the Pi- and a- adrenergic receptors (Pi-AR and a-AR).
Accompanying the increase in sympathetic drive to the heart is an increase in the epinephrine release firom the adrenal glands thus further activating the Pi-AR as well as the Pi-adrenergic receptor (P%-AR) (57). It is well established that these G protein coupled receptors play an important role in the beat-to-beat regulation of cardiac function
(40,41). This alteration in the autonomic balance elicited by myocardial ischemia has been shown to reduce the cardiac electrical stability thereby increasing the propensity forventricular arrhythmias and the incidence of sudden death (21). Under normal as well as ischemic conditions, P-AR activation results in the phosphorylation of the L-type
calcium channels and the calcium release channels of the sarcoplasmic reticulum which
results in a large increase in the intracellular calcium levels. This increase in calcium is
normally quickly dissipated in part due to P-AR induced phosphorylation of
phospholamban. When this P-AR induced calcium release is coupled with myocardial
ischemia, the intracellular calcium levels can rise to even higher levels (9). The resulting
calcium overload could lead to the generation of potentially lethal arrhythmias that
frequently occurs during myocardial ischemia (9). Therefore, P-AR antagonists may be
beneficial in the management of these arrhythmias. In order to determine the therapeutic
potential of P-AR antagonists in the treatment of lethal arrhythmias, an in depth analysis
of sudden death, arrhythmias and p-AR signaling mechanisms must first be performed.
1.1 Sudden Death and Arrhythmias
Sudden death is characterized as an unexpected death from a cardiac cause within
a short period of time (<1 hour) from the onset of symptoms (78). Typically, patients
that experience sudden cardiac death have some underlying disease state including:
coronary artery disease (healed infarcts are present in >50% of all sudden death cases),
cardiomyopathy, left ventricular hypertrophy, valvular disease, congenital heart disease,
and electrophysiological abnormalities. Therefore, the mechanisms that precipitate
sudden cardiac death could be either ischemic, electrical or mechanical. Sudden death is usually associated with an external event or trigger such as an emotional, mental stress, or physical exertion (38). This leads to an activation of the autonomic nervous system causing an increase in the sympathetic drive accompanied with a decrease in
parasympathetic tone (38). This disruption in the autonomic balance causes an increased
blood pressure and heart rate while decreasing the heart rate variability thus decreasing
the ventricular fibrillation (VF) threshold (39). Since the patients usually die alone, the
exact cause of the sudden death is firequently unknown. However, cardiac arrhythmias
appear to be the primary culprit in most documented cases of sudden death
A brief description of normal impulse generation and conduction is needed in
order to understand the origin of abnormalities in cardiac rhythms. Typically, an impulse
originates in the sinoatrial (SA) node located in the right atrium. A group of pacemaker
cells fire spontaneously producing the electrical stimulus for the heart beat. This
electrical current then propagates throughout the right and left atria causing similar
depolarizations. Due to the low electrical resistance between the cells, the depolarizing
current spreads rapidly causing a coordinated contraction among the atrial myocytes.
Once depolarized the cell begins to repolarize in both a time and voltage dependent
manner. The cells will then stay refiactory to the generation of a new impulse for a
period of time such that a rapid second depolarizing current will not be able to stimulate
the cells. The impulse then travels through the atrial-ventricular (AV) node, bundle of
His, bundle branches and Purkinje fibers before spreading through the right and left
ventricles in an apex to base direction. Again, the cells of the ventricle will depolarize followed by a slower repolarization rate. They also remain refractory (unable to be stimulated) for a brief period of time. The ventricular cells contract in unison creating a forceful ejection of the blood. Alterations in these processes such as abnormal impulse generation or conductance can cause arrhythmias and thus irregular beating of the heart. 1.1.1 Abnormal Automaticity
As mentioned previously, the electrical stimulus for the heart beat originates in
the SA node. Abnormal automaticity occurs when the initiating stimulus is generated in
cells outside of the SA node. Abnormal automaticity can result when cells become
partially depolarized at rest and therefore their firing rate supercedes that of the SA node.
This reduction in the resting potential can be caused by a variety of conditions, such as stretch, calcium overload, and high [K]o (12). During myocardial ischemia, the resting potential of the ischemic area becomes less negative than the surrounding non-ischemic areas. As the ischemic membrane depolarizes, it is capable of spreading this depolarizing current, an injury current, to the surrounding non-ischemic tissue perhaps prior to when stimulation would normally occur. Thus, the abnormal automaticity results in premature systoles and tachycardias. A second cause of abnormal automaticity is known as triggered activity. Triggered activity results firom oscillations in the membrane potential and must be preceded by at least one action potential. These oscillations can be either early afterdepolarizations (EADs) or delayed afterdepolarization (DADs) (12).
The EADs occur during the onset of repolarization of the cardiac action potential (after the plateau) where as DADs occur after repolarization. If the amplitude of these oscillations is large enough to reach threshold, then sustained action potentials may be generated. Experimentally, DADs are commonly generated when there is an increase in intracellular calcium i.e., calcium overload. EADs can also result from an increase in inward calcium current as well as a decrease in outward potassium current ( 1 2 ). 1.1.2 Abnormal Impulse Conduction
Normal conduction of an action potential through, the cardiac myocardium is dependent on the rate of depolarization, the amplitude of the depolarization, and
intracellular and extracellular electrical resistance (36). If any one of these is impaired, then abnormal impulse conduction may result. As mentioned previously, proper conduction through the heart is necessary for a coordinated contraction.
Abnormal impulse conduction may result from a simple conduction block or it may involve a more complex model of reentry. During a reentrant arrhythmia, a certain area of the myocardium is reexcited by a circulating impulse. A reentrant arrhythmia is dependent on the presence of a unidirectional conduction block and slowed conduction in the opposite direction (12). The formation of a region of conduction block or conduction delay is common during ischemia. As mentioned previously, an ischemic area of the myocardium slowly depolarizes. With this rise in resting membrane potential comes the inactivation o f the fast sodium channels (9). The fast sodium channels dictate the rate of depolarization and thus contribute to how quickly an action potential will be conducted through a tissue. If an area has inactivated fast sodium channels, then the rate of depolarization will be decreased as well as the conduction velocity thus causing either a unidirectional block or an area of slowed conduction. Furthermore, as the ischemia progresses, intracellular levels of calcium and hydrogen increase (9). An increase in the intracellular concentrations of these ions increases the electrical resistance between the cells and further decreases the overall conductance of the electrical signal. If a signal is capable of propagating through the ischemic or impaired region, then depending on the refractoriness of the tissue surrounding the impaired region, a propagating impulse can 5 enter a recently excited region and trigger new action potentials in this tissue. Thus, the current will spread as long as it is capable of finding areas to excite, thereby causing abnormal beating o f the heart. If the reentrant circuits are characterized by random irregular pathways that continuously change the conduction pattern, then VF will rapidly ensue. The mechanisms that account for these arrhythmias, as well as the best possible treatment, is still being elucidated. As mentioned previously, activation of P-ARs have been implicated as contributing factors in the induction of malignant arrhythmias.
Therefore, the next section will focus on the P-AR signaling pathways and the intracellular events that may trigger arrhythmia formation.
1.2 Overview of Cardiac p-Adrenergic Receptors
In various mammalian species two classes of cardiac p-adrenergic receptors, the
Pi and p 2 subtypes with the possibility of a third class of receptors, the P 3 subtype have been described (17,37,72). The pi- and Pz-ARs although encoded by different genes are structurally and functionally similar (27). Like all G protein coupled receptors, they consist of seven membrane spanning domains which form a barrel like shape that is oriented perpendicular to the plasma membrane. In man and dog, activation o f both classes of p adrenergic receptors are known to increase the force of contraction, heart rate, and relaxation rate (37,42) while in rats, the relaxation rate is not altered by P 2-AR activity (2,43,72,73). The mechanisms that mediate these physiological changes are of particular interest. The classical view of P-ARs signaling involves the binding of an agonist to the receptor. The P-ARs are tightly coupled to the excitatory G protein, Gs. In fact, Pz-AR appears to have a tighter association with Gs than the pi-AR (16,29). Regardless, binding of an agonist is followed by the subsequent dissociation o f the Gs a subunit from the Gs Py subunit. The Gs a subunit goes on to activate adenylate cyclase which in turn catalyzes the conversion of ATP to cAMP. An increase in intracellular cAMP, in turn, increases the activity of protein kinase A (PKA) (Figure 1.1). PKA phosphorylates a multitude of myocardial proteins including: the L type Ca^^ channel, the Iks channel, phospholamban located on the sarcoplasmic reticulum, troponin I, and the metabolic enzyme glycogen phosphorylase (19,40,42). All of these actions work together to increase inotropy while also increasing lusitropy (the relaxation rate of the heart).
The P-ARs can also undergo desensitization and downregulation when faced with an increased amount of ligand (48). The downregulation process involves the phosporylation of the ligand bound receptor by a G-protein coupled receptor kinase. In the case of the P-ARs, this kinase is referred to as p-adrenergic receptor kinase (P-ARK).
P-ARK is located in the cytosol and translocates to the membrane in order to phosphorylate the occupied receptor. The mechanism for translocation includes the physical contact between p-ARK and the membrane bound GPy subunit. This action is then followed by the binding of a protein called p-arrestin. The P-arrestin sterically inhibits any further G-protein activation thus desensitizing the receptor (51). The desensitized receptor-arrestin complexes then undergo P-arrestin dependent internalization through clathirin coated pits (51). This process is commonly referred to as downregulation. Once internalized the sequestered receptor can either be dephosphorylated and recycled to the cell surface or degraded by various lysosomal enzymes (28) (Figure 1.2). p - Adrenergic
Plasma Membrane G„ adenylate cyclase GTP r \ ATP CAMP I *PKA
Figure 1.1 p-Adrenergic Receptor Second Messenger System This action of desensitizatioii followed by downregulation is an adaptive process
that protects the heart against over stimulation by catecholamines. However, there
appears to be some alterations in this process during various pathophysiological
conditions. In the healthy human heart, left ventricular Pi-ARs make up approximately
60% of all the P-ARs but mediate 80% of the P-ARs response (15,24). In heart failure,
the heart becomes weakened due to a variety of causes and is no longer capable of
beating strongly. As a consequence of the impaired cardiac function, the sympathetic
nervous system is reflexively activated resulting in an excess release of catecholamine in
an attempt to increase the pumping efGciency of the heart. However, with heart failure,
the Pi-AR number decreases. The increase in catecholamine release is also coupled to an
increase in the enzymatic activity of p-ARK which potentiates the loss of Pi-AR (15,40).
This decrease in receptor number is associated with a decrease in adenylate cyclase
activity and an increase in the amount of the adenylate cyclase inhibitory G protein, Gi
(39,74). In contrast, the pz-AR. number remains constant or even increased (15). As a
consequence, the Pi/Pz-AR ratio shifts thereby making the contribution of the pz-AR
more important (15,24). Interestingly, down regulation does not occur with ischemia
even though the catecholamine levels are sufficiently high. Mukheqee et al. (54)
reported an increase in the P-AR in the canine heart one hour after ischemia. This
increase in P-AR was associated with an increase in cAMP. In both pathophysiological cases, heart failure and myocardial ischemia, there is at the very least a maintenance of the pz-ARs which could play a critical role in the developed arrhythmias.
10 Desensitization
Activated p - Adrenergic Receptor
p-ARK'
PARK ATP p-arrestin ADP
Downregulation
plasma membrane
Cycle back cytosol to membrane
P-arrestin
Degraded phosphoprotein P-arrestin phosphatase
Figure 1.2 Desensitization and Downregulation of the P-Adrenergic Receptor
11 1.3 Specifics of the Cardiac Pi-Adrenergic Receptor
As previously mentioned, the Pi-AR is both structurally and functionally similar to the Pi-AR. Recently, the Pi-AR has been implicated as a possible mediator of ventricular arrhythmias (3,11). It is known that specific pi-AR stimulation with the agonist zinterol elicited significantly larger calcium transient amplitudes in ventricular myocytes fi-om failing dog hearts as compared to normal dog hearts (2). As mentioned previously, alteration in intracellular calcium can provoke oscillations in membrane potential thus leading to EADs and DADs. Therefore, these Pz-AR mediated changes in cytosolic calcium could contribute significantly to the induction of ventricular fibrillation and the incidence o f sudden death. Furthermore, dogs that are susceptible to VF can be protected by the administration of a selective P^-AR antagonist, ICI 118,551 (11). In a mouse model of Pz-AR overexpression. An et al. (3) found that the calcium current density was greatly increased as compared to controls while the slow delayed rectifier potassium current (Iks) was not significantly altered. The Iks current is largely responsible for the repolarization of the cardiac myocyte following a stimulus. With an increase in heart rate, as commonly seen with sympathetic stimulation, an increase in the
Iks current is necessary in order to ensure adequate diastolic filling time. These authors
(3) found an increase in calcium current with increased Pz-AR expression as expected.
However, Pz-AR expression failed to increase Iks current suggesting that Pz-AR stimulation may not control Iks- As previously mentioned, this increase in calcium without an increase in Iks may set the stage for EADs. Therefore, the Pz-AR may contribute to the advent of ventricular arrhythmias by increasing calcium without
12 decreasing action potential duration. As a consequence, during myocardial ischemia if
P2-AR activation further potentiates the increase in intracellular Ca"^ without stimulating
Iks this could increase the amount of time the cell remains depolarized causing inhomogeneities in the repolarization of the tissue. Inhomogeneities of the repolarization state can form a substrate for reentrant arrhythmias. This contrasts to the Pi-AR which simultaneously increases intracellular Ca^^ while increasing Iks (19). Although the increase in Ca^^ has been proven to be detrimental, the inhomogeneities in the refiractory periods may not be present. These data implicate a more predominant role for the Pz-AR. activation in the induction of ventricular arrhythmias.
Additional studies suggest that the Pz-AR may elicit different cellular responses than the Pi-AR. Although it is known that the Pz-AR tightly couples to Gs and thereby increases adenylate cyclase, the specific Pz-AR agonist, zinterol, failed to elicit an increase in cAMP in isolated ventricular myocytes from chick (58), rat (72), and dog (2).
However, stimulation of canine whole hearts with zinterol caused an increase in cAMP similar to that of the Pi-AR agonist, norepinephrine, which was later attributed to non muscle cells (42,43). Interestingly, Kuschel et al. (42) using isolated myocytes blocked the calcium current induced by both norepinephrine and zinterol with RP-cAMP, a cAMP inhibitor. The authors (42) state that there exists the possibility of a highly localized subsarcolemmal microdomain in the vicinity of the L-type calcium channels where cAMP and PKA are activated. This concept of a localized Pz-AR cAMP/PKA signaling pathway has previously been detailed in the frog cardiomyocyte (64). The frog cardiomyocyte is unique in that approximately 80% of its P-AR population is Pz-AR.
13 Pi-AR stimulation with zinterol in the frog increases calcium current which is completely blocked with the addition of a selective inhibitor of PKA. Studies using frog cardiomyocytes demonstrate that there is a local site directed increase in cAMP with
Pz-AR. stimulation which the authors state would be less visible to biochemical assays for cAMP (64). Skeberdis et al. (64) suggest that the Pz-AR activation is less efticiently coupled to the more distant proteins.
The lack o f an increase in cAMP at the myocyte level, however, does not always alter the other downstream cellular events. In isolated rat myocytes, stimulation by both a pi-AR agonist (norepinephrine) and the Pz-AR agonist, zinterol, provoked similar changes in contraction amplitude (43,72). However, pi-AR activation elicited a greater calcium transient amplitude (72). Furthermore, isolated rat cells when stimulated by zinterol did not show an increase in phospholamban phosphorylation which may account for the lack of a lusitropic effect (43). Interestingly, when rat ventricular myocytes were pretreated with pertussis toxin (PTX), a Gi sensitive toxin, Pz-AR stimulation provoked a lusitropic response and phospholamban phosphorylation similar to that achieved by pr
AR stimulation (43). In a similar manner, mouse cardiomyocytes were not responsive to
Pz-AR stimulation unless PTX was also present (75). Xiao et al. (75), using mouse cardiomyocytes and a photoaffinity labeling technique, found that Pz-ARs couple to the
Gi protein. A slightly different response to the pz-AR activation was noted in isolated myocytes from a healthy canine heart. Zinterol elicited an increase in the calcium transient amplitude in a dose dependent manner (2). However, Kuschel et al. (42), also using canine cardiomyocytes, foimd that the contraction amplitude was significantly
14 smaller when stimulated by zinterol as compared to norepinephrine. This zinterol
mediated increase in contraction once again occurred without an increase in cAMP.
These same authors (42) also reported that the substantial Pi-AR mediated chronotropic,
inotropic, and lusitropic réponse observed in the whole heart occurred in the absence of
PKA activation, phospholamban phosphorylation, or troponin I phosphorylation.
Interestingly, when examining the calcium transient amplitude, cells from failing dog hearts had a much greater Pz-AR responsiveness than cells from healthy dog hearts (2).
This coincided with the tipping of the Pi/Pz-ARs ratio in favor of Pz-AR that occurs during heart failure. Together, these data point to the possibility of a unique signaling pathway for the Pz-AR.
The mitogen activated protein (MAP) kinase pathway has been implicated as a possible alternate signaling pathway for the Pz-AR. Daaka et al. (22) found that stimulation of HEK293 cells with isoproterenol, a mixed Pi-AR/Pz-AR agonist, resulted in an increase in MAP kinase. Likewise using cultured neonatal rat cardiac myocytes,
Bogoyevitch et al. (14) found that isoprenaline and noradrenaline were both capable of increasing MAP kinase activity. Norepinephrine appears to activate MAP kinase in cultured rat cardiac myocytes through activation of both at- and Pi-ARs as the P-AR blocker, propranolol, decreased but did not completely diminish the MAP kinase activity
(76). Therefore, in cultured rat myocytes MAP kinase appears to be activated by both the at- and Pt-ARs. Unfortunately, MAP kinase activation has not been examined in the adult heart. In order to better examine the possible Pz-AR activation o f MAP kinase, one must first perform a review of this signaling mechanism.
15 1.4 Mitogen Activated Protein Kinase
Mitogen activated protein kinases are ubiquitous signaling molecules whose
activation transduces extracellular signals into intracellular responses. The signals may
be as diverse as growth factor receptor activation, cytokine presence, or a physical stress.
Recently, three distinct MAP kinases each having a unique signaling mechanism have
been identified (18). Briefly, the three pathways are named the extracellular signal-
regulated protein kinases (ERKs), the stress activated protein kinases or c-jun kinases
(SAPK/JNK class), and the p38 MAP kinase pathway. Regardless of the specific kinase
activated, they share some common features. From yeast to mammals, MAP kinases are
genetically conserved with activation achieved by dual phosphorylation of a conserved
threonine-XX- tyrosine residue with the separating amino acid (XX) varying with the
particular pathway (56). The MAP kinase pathways are a three kinase cascade where
each kinase phosphorylates and thus activates the next kinase in sequence. The dual
phosphorylation of both the threonine and the tyrosine residues is essential for the
activation of the particular MAP kinase. Phosphorylation of one residue does not activate
the enzyme although it may prime it for the second phosphorylation event (ie., an
allosteric interaction) (20). A MAP kinase cascade first involves phosphorylation of a
MAP kinase kinase kinase or MEKK (MAP or ERK kinase kinase). This enzyme in turn
phosphorylates a MAP kinase kinase (MEK) which finally phosphorylates the specific
final MAP kinase. The specific name of the MEK or MEKK varies depending on the activated pathway with some degree of cross talk occurring between the individual cascades. The activated MAP kinases go on to phosphorylate a number of downstream substrates at either a threonine or serine residue each with an adjacent proline (Figure
16 1.3)(56). These substrates can have substantial regulatory functions throughout the cell
including: other protein kinases, transcription factors, cytoskeletal proteins and other
enzymes. MAP kinases are turned off through the action of several different dual
specificity protein phosphatases that can dififerentially dephosphorylate the ERK kinase,
JNK/SAPK, and p38 enzymes.
The exracellular signal-regulated protein kinases (ERKs) were the first members of the MAP kinase superfamily to be discovered and are thus the most extensively studied. They are phosphorylated at a tyrosine and a threonine residue separated by a glutamine. The p42 activated MAP kinase has the approximate molecular mass of 42 kD and is known as ERK-2 while the p44 activated MAP kinase (44 kD) is known as ERK-1
(34,68). With an appropriate signal, both are activated simultaneously. The ERK-1 and
ERK-2 protein kinases are activated by various extracellular stimuli such as growth factors and tumor promoters, and therefore they play a key role in cell growth and regulation of various genes expression. Activation of the ERKs is commonly initiated by the binding of an agonist to a receptor. Potent activators of ERK 1 and ERK2 can be receptors with intrinsic tyrosine kinase activity or a G protein coupled receptor. In either case, the eventual activation of Ras, a small G protein binding protein, is necessary for the continuation of the cascade. If a receptor with intrinsic tyrosine kinase activity is stimulated, then there is the subsequent association of adaptor proteins (eg. Grb2).
17 Upstream Signal
ME
MEK
MAP I ^ a s e
Downstream Substrates
Figure 1.3. Typical Three Kinase Pathway Observed for the Family of MAP Kinases
18 Adaptor proteins fimctioa to bring the required signal transduction proteins into close proximity. The adaptor protein, Grb2, links Ras with SOS, a guanine nucleotide exchange factor. SOS catalyzes the exchange of GDP for GTP on Ras. However, if the initiating stimulus binds to a G protein coupled receptor, then activation of Ras is achieved through the association of the Src family of tyrosine kinases (51). The Src family phosphorylates adaptor proteins which then go on to link SOS and Ras.
Regardless of the initiating stimuli, Ras then goes on to mediate the translocation of c-
Raf to the plasma membrane (18). c-Raf translocation is accompanied by an MEKK.
Along with translocation, c-Raf and MEKK undergo phosphorylation by another kinase, most likely a tyrosine kinase (18). c-Raf acting as an MEKK activates MEKl and MEK2 which in turn phosphorylate ERK-1 and ERK-2. The activated p44/42 MAP kinases translocate to the nucleus and activate transcription by phosphorylation of transcription factors such as Elk-1 and Stat3 (Figure 1.4)(20,56). They are also capable o f phophorylating various extranuclear substrates such as the mitogen activated proteins:
MAP-1, MAP-2, and MAP-4. When phosphorylated by the ERKs, the MAPs play an important role in microtubular rearrangements and cellular morphological changes (31).
The p38 MAP kinase pathway is composed of four members: p38a, p38(3, p38y, and p385. In keeping with the genetic conservation of the MAP kinase family, there is
60% homology within the p38 family and 40-45% homology with other MAP kinase pathways. The p38a and p38(3 are present in all tissues studied to date while p38y is present only in skeletal muscle and p385 is present in the lungs, kindney, testes, pancreas. and small intestines (56). Activation of p38 is through pro inflammatory cytokines, TNF, interleukin-1 and H 2O2 (56). All of the p38 isoforms are activated by these stimuli, 19 however, differences are observed in the kinetics and the level of activation. Activation of the p38 pathway often occurs in conjunction, with the JNK pathway and sometimes the
ERK pathway. The initiating steps of the p38 family are not as well defined as the ERK pathway. The Rho family of low molecular weight G-proteins (eg. Rac) are believed to interact with small G protein activated kinases (PAKs) (56). PAKs then activate the first kinase of the three kinase pathway, MEKK5 (55). The MAP kinase kinase kinase,
MEKK5, then phosphorylates MEK3 and MEK 6 . MEK3 and MEK 6 are responsible for the phosphorylation of all of the p38 family members at a threonine- glycine- tyrosine residue motif (Figure 1.4)(22,56). In turn, the substrates for p38 are MAP kinase activated protein kinases 2 and 3 (MAPKAPK-2 and MAPKAPK-3) (50,53). Both the
MAPKAPK 2 and 3 go on to phosphorylate small heat shock proteins (53,60) while
MAPKAPK2 is known to activate glycogen synthase ( 6 6 ). There are also a number of transcription factors that are potential substrates for p38. These include: ATF-2, ATF-l,
Elk-1, SRF (serum response factor), CHOP 10, and myocyte enhance factor 2C (MEF2C)
(56).
2 0 ERK/MAP JNK/SAPK p38 Pathway Pathway Pathway I i Ras Ras I Rac/Rho Rac/Rho i I PAKs PAKs I i C-Raf and MEKK MEKKI and MEKK3 MEKK5 I I 0 - © - S MEKl and MEK2 MEK4 and MEK7 MEK3 and MEK6 0- 0 ^ © ^ T I © '~T_ i _ ERKI and ERK2 JNK/SAPK — p38, a, P, y, 5
© '
Downstream Substrates
Figure 1.4. Detailed Descriptiou of the Three MAP Kinase Signaling Pathways.
2 1 The c-jun kinase (JNK) is the stress activated protein kinase (SAPK) pathway.
This family contains many isoforms, including: p46JNKI, p54JNK2, p49JNK3, and
SAPK2 (45). It is similar to the p44/42 MAP kinase pathway in that it involves a three enzyme cascade with the terminal JNK enzyme being phosphorylated at a tyrosine and a threonine residue separated by a proline. As indicated by the name, the JNK/SAPK pathway is primarily activated by stress stimuli such as: cytokines, UV light, inhibitors of protein synthesis, osmotic stress, and inflammatory agents such as TNFa and IL 1
(18,56). Activation of the JNK/SAPK cascade appears to be similar to that of the p38 pathway with the initiating step being activation of the Rac/Rho family (rather than c-
Raf) by Ras (31). Interestingly, UV light and inhibitors of protein synthesis activate the
JNK/SAPK cascade but they are capable o f bypassing the Rac/Rho family (31).
Activation of Rac/Rho leads to phosphorylation of MEKKI and MEKK3 which, in turn, phosphorylates SEK (also known as MEK4) and MEK7. MEK 4 and MEK7 phosphoryltes the JNK/SAPK isoforms (56). JNK/SAPK binds tightly to and activate the transcription factors: c-Jun, ATF-2, and EIk-1 (Figure 1.4).
1.5 Role of MAP Kinases in Cardiac Muscle
The ventricular myocyte is a terminally differentiated cell that withdraws from the cell cycle shortly after birth. Pathophysiological conditions that require the heart to increase its workload can cause enhanced contractile capacity by increasing the myofibrillar content. While in the short term this may be advantageous allowing the heart to increase its cardiac output, in the long term it may lead to heart failure.
Typically, the myofibrillar content increases without an increase in the capillary density 22 or mitochondria. Therefore, there is an increased amount of myofibrils using energy
supplied by relatively fewer mitochondria thus potentiating the energy starvation that
occurs with heart failure (36). Furthermore, there are relatively fewer capillaries
supplying a larger tissue area thus causing underperfused regions of the heart potentially
contributing to pockets of ischemia (36). Myocardial ischemia and subsequent infarction also cause stress to the heart. Ischemia can lead to tissue damage resulting in the necrosis of myocytes and other cells. The surviving myocytes outside of the ischemic zone may undergo hypertrophy in order to compensate for the myocyte loss. The ERK pathway has been implicated as a possible mediator of this hypertrophy signal (79). With myocardial ischemia and subsequent reperfiision, the heart is also subjected to oxygen deprivation, the formation of reactive oxygen species, the release of proinflammatory cytokines
(TNFa and ILp) and ionic imbalances; all of which have been cited previously as stimuli o f the JNK/SAPK and p38 pathways. The role that the MAP kinase superfamily plays in these processes has yet to be completely elucidated.
There are a variety o f agents that stimulate the p44/42 MAP kinase cascade in the heart. In the heart, the ERKs are primarily activated by Gi-protein dependent pathway
(pertussis toxin sensitive) (14). However, recent evidence in neonatal rat cardiomyocyte culture has shown a cAMP/PKA dependent activation of ERK, and as such, suggest a role for the Gs subunit (76,79). As mentioned, activators of ERKI and ERK2 (such as phenylephrine and endothelin- 1) tend to provoke a powerful hypertrophic response. In contrast, bradykinin is also known to increase p44/42 without mediating hypertrophy
(6 8 ). Stress inducing agents such H 2O2, II-ip and TNFa that are not G protein linked also strongly activate ERK in neonatal rat ventricular myocytes ( 6 8 ). The age of the 23 heart may also play a role in the activation of the p44/42 and JNK pathways. Izumi et al
(34) found, when Looking at rat hearts, that the p44/42 MAP kinase levels decreased with
age. This decrease with age was true in both hypertensive and age matched ( 6 months)
controls. However, it should be noted that the hypertensive rats at each age level showed
significantly higher p44/42 levels than the non-hypertensive controls.
The activation of p38 MAP kinase in cardiac tissue is slowly being elucidated.
Studies in isolated hearts revealed that the p38 pathway is stimulated by ischemia and
activation is maintained if reperfiision is applied (14). In contrast, the ERK pathway is
not activated by either ischemia or reperfiision while the JNK pathway is only activated
by reperfiision following ischemia (14,56). It has been implied that p38 may play a protective role in myocardial preconditioning since inhibition of p38 abolishes the protective benefits of preconditioning (71). However, p38 is also known to increase inflammatory cytokines and adhesion molecules that might enhance post ischemic injur>".
As mentioned earlier, the cardiac tissue is known to contain both p38a and p38(3. In a mouse model of pressure overload, p38P was shown to be involved in the hypertrophy response causing an increase in cell size and sarcometric organization where as p38a was involved in apoptosis (70,77).
The three classes of MAP kinases have all been implicated in the pathophysiology of cardiac tissue. Interestingly, these pathways are not independent of each other.
Frequently, agents primarily responsible for activating one pathway are also capable of weakly activating a second pathway. For example, strong activators of JNK/SAPK pathway (H 2O2 , TNFa) also weakly activate the ERK pathway. These pathways are also known to share subsequent substrates. Unfortunately, most, if not all, of the studies have 24 been performed on neonatal cardiac myocyte cultures (14,76,79). The use of cultured
cells for these studies is advantageous in that many manipulations may be made to
investigate the down stream events. However, the role that these pathways play in a whole heart should also be investigated. The use of a whole heart, in vitro, model represents a novel approach to examine the stimuli activating the various pathways as well as potential interactions of these pathways. Furthermore, it is not known if activation of these pathways alters the electrical activity of the heart thus predisposing the heart to the formation of lethal arrhythmias. As mentioned previously, p38 has been implicated as a potential protective agent in the preconditioning phenomenon (71), but any alterations due to p44/42 MAP kinase activation are not known.
1.6 Proposed Studies
As mentioned previously, the p44/42 MAP kinase pathway may represent an alternate signaling pathway for the Pz-AR. It appears that the Pz-AR can activate a cAMP independent pathway thus leading to increases in intracellular Ca^^ without changing Iks which may be pro-arrhythmic, and/or it may activate the p44/42 MAP kinase pathway which has not been implicated in the development of arrhythmias to date. In addition, previous results from this lab ( 1 1 ) have shown that dogs that are susceptible to ventricular fibrillation can be protected by the administration of a selective pz-AR antagonist, ICI 118,551. ICI 118,551 did not alter the hemodynamic response to the coronary occlusion, yet it prevented VF (11). These results demonstrate an indirect link between Pz-AR activation and the incidence of lethal arrhythmias. Furthermore, isolated cardiac myocytes from the susceptible animals showed an increase in calcium transient 25 amplitude compared to the dogs that were resistant to VF when stimulated by both the selective pi-AR agonist, zinterol, and the combination of isoproterenol and CGP, a selective Pi-AR antagonist (11). ICI 118,551 abolished the isoproterenol increase in calcium transient amplitude of the susceptible but not that of the resistant dogs. Similar studies have not been performed in a whole animal preparation. Zinterol stimulation also failed to increase cAMP in the isolated myocytes from all dogs (11,2). However, whole canine heart preparations have been shown to increase cAMP with Pz-AR stimulation
(42). Therefore, these data suggest that the susceptible animals have an increased dependence on the Pz-AR for inotropic support. Furthermore, the calcium transient data have not been correlated with unloaded single cell twitch amplitude. This increase in apparent Pz-AR activation cannot be attributed to a difference in receptor number between the two groups, as both the susceptible and resistant animals had equal number of surface receptors (unpublished results). Therefore, it is possible that Pz-AR stimulation during ischemia provokes an increase in intracellular calcium via a cAMP dependent yet highly localized signaling mechanism leading to the formation of the lethal arrhythmias in the susceptible animals. Pz-AR stimulation in the resistant dogs may alternately lead to activation of the p44/42 MAP kinase signaling mechanism thus sparing these dogs from the development of arrhythmias.
26 Therefore, the purpose of this study was to test the following hypotheses:
1. Activation of p i- and Pz-AR. with isoproterenol would elicit a larger dose-dependent
increase in the inotropic state in the susceptible as compared to resistant animals.
Contractility was evaluated by echocardiography before and after anterior wall
myocardial infarction. (Ho=Isoproterenol elicits a similar increase in force in both
susceptible and resistant animals.)
If this hypothesis is correct, then one would predict the pz-AR antagonist, ICI
118.551, would elicit a greater reduction in the isoproterenol response in the
susceptible animals as compared to the resistant anim als. (Ho= The pz-AR
antagonist, ICI 118,551, elicits similar decreases in the isoproterenol response in both
the susceptible and the resistant animals.)
2. Activation of P i- and pz-AR with isoproterenol would also elicit a greater response in
the single cell shortening of cardiomyocytes obtained from susceptible versus
resistant animals. (Ho= Isoproterenol elicits similar increases in single cell
shortening in the cardiomyocytes obtained from susceptible and resistant animals.)
If this hypothesis is correct, then one would predict the pz-AR antagonist, ICI
118.551, would elicit greater reduction in the single cell unloaded shortening
isoproterenol response in the susceptible animals as compared to the resistant
animals. (Ho= The Pz-AR antagonist, ICI 118,551, elicits similar decreases in the
isoproterenol response in both the susceptible and the resistant animals.)
3. If the above hypotheses are correct and the resistant animals (those that do not
develop lethal arrhythmias) appear to have a decreased dependence on pz-AR
27 activation, then one would predict that in the resistant anim als the cardiac Pz-AR. is coupled to Gi subunit and thus preferentially activates the p44/42 MAP kinase pathway. (Ho= p44/42 activation by pz-AR stimulation is similar in the resistant and the susceptible animals).
28 CHAPTER 2
MATERIALS AND METHODS
2.1 Surgical Preparation of the Canine Model of Sudden Death
A total o f 45 heartworm free mongrel dogs, weighing 14.3-20.4 kg, were used in
this study. Twenty-four hours prior to surgery a transdermal fentanyl patch that delivers
75 pg/hour for 72 hours (Duragesic, Jansen Pharmaceutical Inc.) was placed on the left
side of the animal’s neck and secured with tape. On the day of surgery, the dogs received
500 mg amoxicillin (po), 15 mg (1 cc) Morphine (im) and sodium thiopental (Pentothal
2% , 20 mg/ml, iv) to induce anesthesia. Each dog was given between 17-20 mg/kg of
the sodium thiopental depending on the individual response. The dogs were then
intubated and a surgical plane of anesthesia was then maintained by inhalation of
isofluorothane (1-1.5%). Using strict aseptic techniques, a left thoracotomy was made in the fourth intercostal space. The heart was exposed and supported by a pericardial cradle.
The left circumflex coronary artery was dissected free of the surrounding tissue. A 20
MHz pulsed Doppler flow transducer and a hydraulic occluder were placed around the vessel. Insulated silver-copper wires were sutured to the epicardial surface of the left and right ventricles for the later recordings of a ventricular electrogram. A two stage occlusion of the left anterior descending coronary artery was performed approximately
29 one third the distance from the vessel’s origin in order to produce an anterior wail
myocardial infarction. The vessel was partially occluded for 20 minutes and then tied
off. All o f the leads to the cardiovascular instruments were tunneled under the skin to
exit on the back of the animal’s neck.
In addition to the fentanyl patch described above, morphine sulfate (1.0 mg/kg,
s.c.) was given as needed for postoperative pain. In addition, 0.5 ml of the long lasting
local anesthetic, 0.25% bupivacaine hydrochloride (Marcaine, Winthrop-Breon Lab), was
injected in each of three sites to block the intercostal nerves in the area of the incision to
minimize discomfort to the animal. Each animal was placed on antibiotic therapy
(amoxicillin 500 mg Bums Veterinary Supply) twice daily for seven days.
The animals were then placed in an “intensive care setting” for the first 24 hours.
To minimize the incidence of arrhythmias, the dogs received 100 mg lidocaine HCl (im)
(xylocaine, Astra Laboratories) before surgery, which was supplemented (60 mg iv)
before each stage of the two stage occlusion. The dogs also received 5 ml of
procainamide HCl (im) prior to the surgery.
2.2 Exercise plus Ischemia Test: Classification of the Dogs
An exercise plus ischemia test was performed in order to determine which dogs were susceptible or resistant to VF. Approximately 3-4 weeks after the production of the myocardial infarction, the animals were trained to walk on a motor-driven treadmill. For several days prior to the classification, the animals were brought to the laboratory in order to familiarize them with the setting. The cardiac response to submaximal exercise was then evaluated. The response to exercise was assessed by a protocol previously 30 described by Stone (67). Briefly, the treadmill exercise lasted a total of 18 minutes and was divided into 3-minute blocks. The protocol began with a 3-minute warm-up period during which the animal ran at 4.8 kph and 0% grade. The speed was then increased to
6.4 kph and the grade of the treadmill was increased every 3 minutes as follows: 0%, 4%.
8 %, 1 2 %, and 16%.
The susceptibilty to ventricular fibrillation was assessed in all of the animals by the combination of exercise and acute ischemia (6,63). Briefly, the animals ran on a motor-driven treadmill while the workload increased every 3 minutes, as described above. During the last minute of exercise, the left circumflex coronary artery was occluded. The treadmill was then stopped, and the occlusion was maintained for an additional minute. The total occlusion time was two minutes. Large metal plates (11 cm diameter) were placed across the animal’s chest so that electrical defibrillation could be achieved with minimal delay but only after the animal was unconscious. Of the 45 animals that underwent surgery, 11 died acutely within 48 hours of surgery, 2 were unable to be classified due to loss occluders, and 1 other was excluded due to adhesion of the heart to the chest wall. Of the 31 remaining dogs that were classified, 12 were susceptible and 19 were resistant. Electrocardiogram, heart rate, and left circumflex coronary blood flow were recorded throughout the exercise plus ischemia test. Left circumflex coronary blood flow was measured to confirm that the coronary occlusion was complete.
31 2.3 Echocardiography studies
The echocardiography studies described below were performed approximately one week prior to surgery. O f the 45 animals that eventually underwent surgery, 37 dogs were echoed prior to surgery and eventually classified (n=19, susceptible and n=16, resistant). One dog died acutely during surgery due to anesthesia overdose and one dog^s heart adhered to the chest wall and could not be classified. If the animals died during surgery due to VF or within 48 hours post surgery, it was classified as susceptible. The studies were again performed two weeks post surgery on the animals surviving surgery
(n=31 : n=l2 susceptible, n=19 resistant). All of the studies were performed and the data analyzed prior to the classification test. The dogs were lightly sedated with acepromazine
(0.5 mg/kg, im) prior to the studies. A conventional M-mode echocardiogram was obtained using a Sonos 1000 system (Hewlett Packard) with a 5.5 MHz transducer. The velocity of circumferential fiber shortening (Vcf) of the left ventricle was determined in order to provide a load independent measure of contractility incorporating both the extent and the velocity of fiber shortening (13). The Vcf was calculated from M-mode echocardiograms according to the formula:
(LVIDd-LVIDs)/(LVIDd x ET) where LVIDd is the short axis of the left ventricle in diastole (in cm), LVIDs is the short axis of the left ventricle in systole (in cm) and ET is the ejection time (in sec). Dividing by the square root of the R to R interval from a simultaneously collected electrocardiograph corrects for changes in heart rate (13).
The total P-adrenergic receptor response was quantified by infusing increasing doses of isoproterenol (ISO)(0.006 pg/min/kg, 0.017 pg/min/kg, 0.06 pg/min/kg, 0.18 32 Hg/min/kg, and 0.66 (xg/min/kg). The echocardiogram was obtained when a steady state heart rate response was achieved at each dose. Some of the dogs (pre surgery, n = 6 for susceptible and n=2 for resistant; post surgery, n = 6 for susceptible and n=5 for resistant) did not receive the highest dose of ISO if the maximum response appeared to be reached at a lower dose. After obtaining the mixed P 1/P2 in vivo response a bolus injection of either the Pi-AR antagonist (bisoprolol 0.6 mg/kg) (42) or the P 2-AR antagonist (ICI
118,551 0.2 mg/kg) was given (42). The ISO dose response infusion was then repeated.
The study was repeated one week later with the alternate P-AR antagonist.
Approximately half of the dogs received the bisoprolol first while the other half received the ICI drug first.
To test the hypothesis that the decrease in isoproterenol response was due to the presence of a p-AR antagonist and not desensitization and downregulation, the echocardiogram studies were repeated in 1 susceptible and 5 resistant animals approximately 2 weeks after surgery. In this set of experiments, the isoproterenol dose response was performed as described above and then repeated a few minutes later without the addition of the P-AR antagonist.
2.4 Isolation of Ventricular Myocytes
Dogs (n= 6 , susceptible and n=9, resistant) were anesthetized with sodium pentobarbital (10 mg/kg, i.v.). The chest cavity was opened and the beating heart was excised (approximate removal time 3-5 minutes). Upon excision, the beating heart was immersed in ice cold normal saline. The left circumflex coronary artery was perfused
33 with approximately 200 mis of ice cold cardioplegic solution (pH 8.5) containing: 55 mM glucose, 1.1 mM mannitol, 22.4 mM NaHCOs. and 30 mM KCl. Unless otherwise stated all the reagents were purchased from Sigma. A noninfarcted section o f the right ventricle was cut free of the whole heart. The right circumflex artery was cannulated and transferred to a Langendorf apparatus. This section of the heart was perfused in non- circulating mode at 37°C at a rate o f 20 ml/min with a perfusion buffer containing: 118 mM NaCl, 4.8 mM KCl, 1.2 MgS 0 4 , 1.2 mM KH2PO4, 6 . 8 mM glutamine, 4 mM glucose, 5 mM pyruvate 2 mM mannitol, 10 mM taurine, IpM insulin, amino acids
(GIBCO: Eagles minimum essential medium (MEM) and basal minimtun Eagle (BME)), vitamins (Basal Medium Eagle Vitamin Solution) and penicillin-streptomycin (GIBCO) gassed with 9 5 %0 2 —5% CO2 (pH 7.4). The perfusion continued until all of the blood had been washed out (approximately 500 ml). Collagenase (1 mg/ml, combination Type
I and IV, Worthington Biochemicals) and bovine serum albumin (BSA) (1 mg/ml, fraction V, Miles Lab) were then added to the perfusate and recirculation begun. The perfusion rate was gradually increased to 60 ml/min.
When the tissue showed signs of softening (approximately 20 minutes), calcium was added in small increments until reaching a final concentration of 0.5 mM. Upon completion of tissue digestion (approximately 60 minutes), the softened tissue was abraded with a scapel and the slurry suspended in the perfusion buffer plus collagenase and BSA and incubated for 15 minutes at 37°C. The myocytes were dispersed with an enlarged transfer pipet, filtered through cheesecloth, centriftjged at 50 g and resuspended in incubation buffer containing 5 mM NaHCOs, 118 mM NaCl, 4.8 mM KCl, 1.2
34 MgSO#, 1.2 ECH2PO4 , 0.68 mM glutamine, 1 mM CaClz, 4 mM glucose, 5 mM Na pyruvate, 20 mM HEPES, amino acids, vitamins, insulin, peniciliin-streptomycin, and
2% BSA.
Cell viability (exclusion of 0.3% trypan blue) was assessed by standard cell counting techniques using bright field light microscopy and a hemocytometer.
2.5 Edge Detection Protocol
Single cell unloaded shortening was recorded in the isolated myocytes from 9 resistant and 6 susceptible dogs. The visible motion of a single myocyte was collected through the use o f a low light video camera (Phillips CCD Video Camera) and a mirror system attached to an inverted microscope. The video image was analyzed by a video edge detector (Crescent Electronics). Motion of the cell was tracked through the use of voltage differences that occur in creating light or dark video images. By filtering out all but the area of interest, specific points along the edge of the myocyte (area of greatest contrast) were tracked and changes in length were recorded. Images were collected and stored using a standard VCR for later analysis. Digital conversion of output for quantitative analysis was accomplished through the use of an IBM-PC and edge detection software (Globelab).
Myocytes were placed in a flow through chamber and were allowed to adhere to the chamber surface for approximately 10 minutes. At the end of that time, the myocytes were field stimulated at a frequency of 0.5 Hz (Crescent Electronics Stimulator) in the presence of a gassed (95% Oz-5% CO 2) 25 mM bicarbonate buffer (pH 7.3 —7.5) containing: 121 mM NaCl, 4.85 mM KCl, 1.21 mM MgS 0 4 , 1.21 mM KH 2 PO4 , 1.8 mM 35 Ca^^, and 5mM pyruvate. This bicarbonate buffer, with and without the drug of interest, was gassed (95% O 2 and 5% CO 2) and heated to 37°C before being pumped across the flow through chamber containing the myocytes. The pump speed was set at 2 ml/min
(Masterflex). Edge detection was determined in the presence of the control bicarbonate buffer, 100 nM ISO and 100 nM ISO supplemented with either the Pi- or P 2- adrenergic receptor antagonists, ICI 118,551 (lOOnM) or bisoprolol (200 nM) (II). As done in the echocardiogram studies, to test that the decreases noted with the P-adrenergic receptor antagonists were not due to receptor desensitization and downregulation, cells from 4 resistant animals and 1 susceptible animal were treated with 100 nM isoproterenol a total of three times in a row.
2.6 Western Blot Detection of p44/42 MAP kinase or p38 MAP kinase
Two sections of the left ventricle (n=6 for resistant dogs and n=6 for susceptible dogs) from a noninfarcted region were cannulated and perfused with a buffer containing:
25 mM NaHCO], 118 mm NaCl, 4.8 KCl, 1.2 MgS 0 4 , 1.2 KH2PO4 , 0.68 mM glutamine.
4 mM glucose, 5 mM pyruvate, 2 mM mannitol, 10 mM taurine, IpM insulin, amino acids, penicillin-streptomycin, and 0.5 mM CaC^. After flushing out all of the blood
(approximately 300 ml), the perfusate was supplemented with either 10 pM zinterol (P 2-
AR agonist) with 300 nM CGP (Pi-AR antagonist) or 10 pM phenylephrine (ai-AR agonist) with 10 pM propranolol (P r and P 2-AR antagonist) and allowed to perfuse for 8 min at a pump speed of approximately 30 ml/min. After which, the perfusate was switched back to the control bufier and allowed to wash the section of myocardium for
36 30 minutes. At the end of the initial buffer wash, 8 minute drug perfusion and 30 minute
wash period, a section of the perfused heart was cut free, frozen in liquid nitrogen, and
stored at -80°C until further analysis.
On a subsequent day, the frozen samples were ground and solubilized in IX Cell
Lysis buffer (New England Biolabs) containing: 20 mM Tris (pH7.5), 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 nM sodium pyrophosphate, 1 nM P-
glycerolphosphate, 1 mMNa3V0 4 , Ipg/ml leupeptin, and 1 mM PMSF. The protein
content was assayed by the method of Lowry et al. (49) using Bio-Rad reagents and
bovine serum albumin as standard. Protein separation was accomplished using SDS
polyacrylamide mini gel electrophoresis. The mini gel consisted of a 10% separating gel
(lower) with a stacking gel (Bio-Rad). Samples containing 30 pg protein were diluted
2:1 with Laemmli sample buffer (BioRad) supplemented with 350 nM DTT (total volume
did not exceed 30 pi). The samples were heated to 100°C for 5 minutes allowed to cool
and microcentrifuged for 5 minute before being loaded onto the gel. SDS polyacrylamide
gel electrophoresis was carried out for approximately 30 minutes at 200 V until the
bromophenol blue tracking dye had migrated to the bottom of the gel. A mixed
molecular weight standard and a positive MAP kinase control (New England Biolabs)
were used for comparison.
After completion of electrophoresis, the gels were transferred to a piece
nitrocellulose by a Semi-dry system (BioRad) for 30 minutes at 15 V using a transfer
buffer containing: 25 mM Tris base, 0.2 M glycine, and 20% methanol (pH 8.5). The nitrocellulose was incubated at 4°C overnight in blocking buffer containing: IX Tris
37 buffered saline, 0.1% Tween-20, and 5% nonfat dry milk. On the second day, the nitrocellulose was incubated in 1° antibody (either p44/42 antibody or p38 antibody) at a
1:1000 dilution in blocking buffer at 4°C overnight with rocking. On the third day, the nitrocellulose was washed three times with 1% TEST buffer (IX Tris buffered saline and
0.1% Tween-20) at room temperature. Following washing, the nitrocellulose was transferred to the 2° antibody at a 1:2000 dilution in blocking buffer for one hour at room temperature. At the end of this incubation, the nitrocellulose was again washed three times with 1% TEST buffer at room temperature before being washed with LumiGLO chemiluminescent reagent (New England Eiolabs). The nitrocellulose was then exposed to Hyperfilm ECL (Kodak, Amersham) before being developed.
The developed films were digitized using Desk Scan software and quantified using Image Quant software.
2.7 Mitogen Activated Protein Kinase Activity Assay
The ground and solubilized samples firom 12 dogs (6 resistant and 6 susceptible) were used. To measure MAP kinase activity, a commercially available monoclonal phosphospecific antibody to the p44/42 MAP kinase was used to immunoprecipitate the active MAP kinase from the cell lysate (New England Biolabs). In order to immunoprecipitate the active p44/42 MAP kinase, 200 pi of a Ipg/pl protein sample was incubated overnight at 4°C with immobilized phospho-p44/42 antibody. On the second day, the immobilized antibody-MAP kinase complex was washed two times in IX lysis buffer at 4°C. This was followed by two washings with IX kinase buffer containing: 25 mM Tris (pH 7.5), 5 mM p-glycerolphosphate, 2 mM DTT, 0.1 mM Na 3V0 4 , and 10 mM 38 MgClz. The complex was then incubated in IX kinase buffer supplemented with cold
ATP and EIk-1 fusion protein for 30 minutes at 30°C. The reaction was terminated with
the addition of 3X SDS sample buffer (187.5 mM Tris-HCI, 6% w/v SDS, 30% glycerol.
150 mM DTT, and 0.3% w/v bromphenol blue). The sample was heated to 100°C for 5
minutes followed by vortexing and microcentrifuged for 2 minutes. The sample (20 pi)
was loaded on a 10% mini SDS gel for protein separation. The western blot detection
was carried out as described above. A phosphorylated Elk-1 control supplied in the kit
was used for comparison.
2.8 Statistical and Data Analysis
Results are expressed as means ± Standard Error (SE). Statistical significance of
the echocardiogram Vcf data was determined by performing a three way ANOVA [group
(2 levels) x drug (3 levels) x dose (6 levels)] with repeated measures on two factors (drug
and dose). If the F ratio was found to exceed a critical value (p<0.05) then the difference between the means was determined using Scheffe’s test. Similar comparisons were made before and after myocardial infarct.
For the single cell unloaded shortening data, cells from a given animal were averaged and the mean values were used in the ANOVA. Statistical significance of the single cell unloaded shortening data was determined by performing a two way ANOVA
[group (2 levels) x drug (3 levels)] with repeated measures on one factor (drug). Cells from a given animal were averaged and these mean values were used in the ANOVA.
Again, Scheffe’s test was used when the F ratio exceeded a critical value (p<0.05).
39 Statistical significance of the MAP kinase western blots as well as the MAP kinase activity assay was done using a two way ANOVA [group (2 levels) x drug (3 levels)] with repeated measures on one factor (drug). Scheffe’s test was used when the F ratio exceeded a critical value (p<0.05).
40 CHAPTER 3
RESULTS
3.1 Classification of Dogs
A total of 45 mongrel dogs, weighing 14.3-20.4 kg, underwent surgery. Of these
45 dogs, 11 died acutely within 48 hours of surgery, 2 were unable to be classified due to loss of the occluder, and 1 was excluded due to adhesion of the heart to the chest wall.
The 31 remaining animals underwent the exercise plus ischemia test, in order to be classified as either susceptible to ventricular fibrillation (VF) or resistant to VF. Of these
31 dogs, 12 were susceptible and 19 were resistant.
3.2 Analysis of Pre-Infarct Echocardiograms
Pre-infarct echoes were obtained from 37 of the 45 dogs used in this study. O f the 37 dogs that had pre-infarct echoes done, two dogs were unable to be subsequently classified and therefore were excluded from the study. O f the 35 remaining dogs, 19 were eventually found to be susceptible to VF while 16 were resistant.
Pre-infarct representative M-mode echocardiograms of the left ventricle for both a resistant and a susceptible dog are shown in Figures 3.1 and 3.2. Analysis of the velocity of circumferential fiber shortening (Vcf) obtained from these echocardiograms is displayed in Figures 3.3-3.9. The susceptible and resistant animals had similar Vcf 41 values when challenged with increasing doses of isoproterenol (Figure 3.3). If given the pi-adrenergic receptor antagonist, bisoprolol, the Vcf value was significantly decreased in both the resistant and the susceptible animals (Figures 3.5 and 3.8). Likewise, the P 2- adrenergic receptor antagonist, ICI 118,55, significantly decreased the Vcf value in both groups of animals (Figures 3.6 and 3.9). In both groups of animals, the attenuation caused by bisoprolol was significantly greater at the highest dose of isoproterenol than that achieved by ICI (Figures 3.4 and 3.7).
Increasing doses of isoproterenol were also capable of altering the heart rate response in these animals. Both the susceptible and the resistant animals experienced a significant increase in heart rate with increasing isoproterenol, however there were no differences noted between the groups (Figure 3.10). As seen with the Vcf calculation, bisoprolol (Figures 3.12 and 3.15) and ICI (Figures 3.13 and 3.16) significantly decreased the heart rate response in both groups of animals. Also, at the highest dose of isoproterenol, bisoprolol caused a greater decrease in the heart rate than did ICI in both groups of animals (Figures 3.11 and 3.14).
3.3 Analysis of the Post-Infarct Echocardiograms
Of the 45 dogs that underwent surgery, post-infarct echocardiograms were obtained from 31 dogs. Of these 31 dogs, 12 were susceptible and 19 were resistant.
Representative M-mode echocardiograms of the left ventricle are shown for a resistant
(Figure 3.17) and a susceptible dog (Figure 3.18). These echocardiograms were obtained approximately 2 weeks after the surgery.
42 Increasing doses of isoproterenol caused a significant increase in the Vcf value as compared to the baseline reading in both the susceptible and the resistant animals (Figure
3.19). However, the susceptible animals showed a much larger Vcf than the resistant animals at about every dose (Figure 3.19). In comparing the pre-infarct isoproterenol increases in Vcf to those seen post-infarct, the resistant animals showed the same level of increase, while the susceptible animals had a significantly larger increase at doses 1-4
(Figures 3.3 and 3.19).
As observed on the pre-infarct echoes, the Vcf value achieved with isoproterenol was attenuated by both bisoprolol and ICI 118,551 in both groups of animals (Figures
3.20-3.25). Interestingly, in the resistant animals bisoprolol caused a greater decrease in the Vcf achieved with the highest dose of isoproterenol than did ICl. Susceptible animals, however, showed an equal amount of decrease with both P-blockers (Figures
3.20 and 3.23). In fact, ICl decreased the isoproterenol Vcf response at dose 5 by 48% in the susceptible animals while only decreasing it by 20% in the resistant animals (Figure
3.26). To be sure that the decreases noted by both P-AR antagonists were due to the drug rather than receptor desensitization and down-regulation, back to back isoproterenol dose response curves were generated for 6 animals. When looking at the percent change of the
Vcf values calculated from the second isoproterenol infusion study as compared to the
Vcf values from the dose response generated immediately preceding it, it was found that there was no change in Vcf response for doses 1-3 and 5, but at dose 4, there was a significant increase in the isoproterenol response (Figure 3.27). These results indicate that the isoproterenol was in fact not causing desensitization and down-regulation and the differences noted with the P-AR antagonists were due to the action of those drugs. 43 As observed with the post-infarct Vcf values, there was a greater increase in the heart rate of the susceptible animals as compared to the resistant animats with increasing doses of isoproterenol. Both groups of animals experienced a significant increase in their baseline heart rate levels post surgery (Figures 3.10 and 3.28). In both the susceptible and the resistant animals, bisoprolol caused a significant decrease in the heart rate response at all of the doses of isoproterenol (Figures 3.30 and 3.33). The susceptible animals also experienced a significant decrease in heart rate at every dose of isoproterenol when ICI 118,551 was present; while the resistant animals saw decreases at doses 2 and 4 only (Figures 3.31 and 3.34). Again, the decreases noted by ICI were greater in the susceptible animals as compared to the resistant (Figures 3.29 and 3.32).
3.4 Analysis of Single Cell Unloaded Shortening
The unloaded single cell shortening was analyzed from cardiomyocytes from susceptible (n=6) and resistant (n=9) animals. Representative tracings from the edge detector for a single mycoyte from both a resistant and a susceptible dog may be found in
Figures 3.35 and 3.36. Percent cell shortening was calculated for each cell at a control baseline level, in the presence of 100 nM isoproterenol, and in the presence of 100 nM isoproterenol with either 100 nM ICI 118,551 or 200 nM bisoprolol (Figure 3.37). Both groups of animals had a significant increase in percent cell shortening when isoproterenol was added as compared to control. However, there were no difierences noted between the two groups. ICI 118,551 was capable of significantly decreasing the isoproterenol response of the susceptible animals but not in cells from resistant animals. Bisoprolol caused a significant reduction in the isoproterenol response in both groups of animals. 44 When converting the data to examine percent change from control, the
isoproterenol response was significantly greater in the susceptible animals as compared to
the resistant animals (Figure 3.38). Compared to control, the attenuation caused by ICI
was not different between the two groups; however, when compared to the response from
isoproterenol, ICI caused a significant reduction in the cells from susceptible animals.
Again, compared to control the isoproterenol plus bisoprolol response was significantly
greater in the susceptible animals. In both groups of animals, there was a significant
decrease in the isoproterenol response when bisoprolol was present.
The +dL/dt max for the single cell myocyte experiments examines the velocity of
contraction along with the extent of contraction. In examining the percent change from control, both groups of animals showed a significant increase with the addition of
isoproterenol (Figure 3.39). Again, the susceptible animals showed a significantly greater response to isoproterenol. This increase was attenuated by ICI 118,551 in the susceptible animals. Bisoprolol plus isoproterenol showed a greater response in cells from susceptible dogs (Figure 3.39).
The time to 50% relaxation was significantly decreased in both groups of animals when isoproterenol was present (Figure 3.40). However, there were no differences between the groups; i.e. neither P-blocker appeared, to alter the time for 50% relaxation.
As done in the echocardiogram studies, to ensure that the decreases noted with bisoprolol and ICI were due to their actions and not desensitization and down-regulation of the P-AR, cells from 5 dogs were treated with isoproterenol three times in a row. The percent cell shortening did not decrease, and in fact showed a non-significant trend towards an increase (Figure 3.41). 45 3.5 Analysis of the p44/42 and p38 MAP kinase Western Blots
Representative radiograms from the p44/42 MAP kinase western blots from a susceptible and a resistant dog are shown in Figure 3.42. The p44/42 MAP kinase levels were analyzed in 6 resistant and 6 susceptible dogs. For data analysis, the p44/42 MAP kinase levels were standardized to a commercially available positive control. The susceptible animals showed a significant increase in phosporylated p44/42 MAP kinase when stimulated by zinterol and this significant increase was maintained after a 30 minute wash out period (Figure 3.43). Perfusion of the cardiac tissue with phenylephrine also caused a significant increase in phosphorylated p44/42 MAP kinase compared to control levels in the susceptible animals (Figure 3.44). Neither phenylephrine nor zinterol were capable of increasing the level of phosphorylated p44/42 MAP kinase above control levels in the resistant animals (Figures 3.43 and 3.44). With both drugs, the p44/42 MAP kinase levels were significantly greater in the susceptible animals as compared to the resistant animals (Figures 3.43 and 3.44).
If the data is converted to percent of control, there are no significant differences between the susceptible and resistant animals with zinterol stimulation (Figure 3.45).
However, perfusion with phenylephrine caused a significant increase in p44/42 N'lAP kinase levels in the susceptible animals as compared to the resistant (Figure 3.46). Even though differences were not noted between the two groups with zinterol perfusion, the susceptible animals showed a significant increase above their control values when stimulated by zinterol that was not seen in the resistant animals (Figure 3.45). Perfusion of phenylephrine, however, caused a significant increase above control levels for both the susceptible and resistant animals (Figure 3.46). 46 Western, blot detection o f p38 was done to see if this stress activated MAP kinase increased during the perfusion procedure. Interestingly, the radiographs showed absolutely no activation o f p38 MAP kinase in either the susceptible or the resistant animals (data not shown).
3.6 Analysis of the p44/42 MAP kinase Activity Assay
The p44/42 MAP kinase activity assay was performed on both resistant (n=6) and susceptible (n=6) dogs. Representative radiographs firom this assay are shown in Figure
3.47 for both a susceptible and a resistant animal. Again, the values for p44/42 MAP kinase activity were normalized to a commercially available standard for data analysis.
There were no significant increases above control in p44/42 MAP kinase activity with either zinterol or phenylephrine in both groups of dogs (Figures 3.48 and 3.49).
Likewise, there were no differences between the two groups of animals.
If the data are converted to examine percent of control changes, then the resistant animals did show a significant increase with zinterol that was not seen in the susceptible animals (Figure 3.50). This increase was still seen after a 30 minute wash out period.
Phenylephrine, however, was not capable of causing this same level of increase (Figure
3.51).
47 3.7 Summary of Findings
• Pre-infarct, the isoproterenol Vcf values for both the susceptible and resistant
dogs were similar (Figure 3.3).
• Pre-infarct, bisoprolol reduced the isoproterenol Vcf response by
approximately 65% in both groups of animals (Figures 3.5 and 3.8).
• Pre-infarct, ICI 118,551 reduced the isoproterenol Vcf response by
approximately 25% in both groups of animals (Figures 3.6 and 3.9).
• Pre-infarct, there were no significant différences between the two groups of
animals with any of the drugs and doses studied.
• Post-infarct, the isoproterenol Vcf values were significantly higher in the
susceptible animals as compared to the resistant animals (Figure 3.19).
• Post-infarct, bisoprolol reduced the isoproterenol Vcf values in both the
susceptible and the resistant animals by approximately 52% (Figures 3.21 and
3.24).
• Post-infarct, ICI 118,551 reduced the isoproterenol Vcf values by 48% in the
susceptible animals and by 20% in the resistant animals (Figure 3.26).
• The percent change of single cell unloaded shortening as compared to control
was significantly greater with the addition of isoproterenol in the susceptible
animals as compared to the resistant animals (Figure 3.38).
• In the single cell unloaded shortening studies, ICI 118,551 elicited a
significantly greater decrease in the isoproterenol response in myocytes from
susceptible dogs as compared to those from resistant dogs (Figure 3.38).
48 • In the single cell unloaded shortening studies, the bisoprolol plus
isoproterenol response was significantly greater in myocytes from susceptible
animals as compared to those from resistant animals (Figure 3.38).
• The western blot p44/42 MAP kinase detection assay showed that susceptible
animals had a greater increase in MAP kinase with the addition of both
zinterol and phenylephrine (Figures 3.43 and 3.44).
• The p44/42 MAP kinase activity assay, however, showed that in the resistant
animals ERK 1 and ERK 2 were more active with the addition of zinterol as
compared to the susceptible animals (Figures 3.50).
49 Figure 3.L Representative M-mode echocardiograms of the left ventricle from a resistant dog prior to surgery. A. Baseline echocardiogram prior to the infusion of drug. B.
Echocardiogram taken during the infusion of 0.66 pg/min/kg isoproterenol (ISO) C.
Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence of 0.2 mg/kg of ICI 118,551 (ICI). D. Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence of 0.6 mg/kg of bisoprolol (BIS).
50 A. Baseline B. Isoproterenol Dose 5
C. Isoproterenol + ICI 118,551 D. Isoproterenol + Bisoprolol
Figure 3.1 Representative M-mode Echocardiograms from a Resistant Dog Prior to
Infarct
51 Figure 3.2. Representative M-mode echocardiograms of the left ventricle from a susceptible dog prior to surgery. A. Baseline echocardiogram prior to the infusion of drug. B. Echocardiogram taken during the infusion of 0.66 pg/min/kg isoproterenol
(ISO) C. Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence o f 0.2 mg/kg o f ICI 118,551 (ICI). D. Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence of 0.6 mg/kg of bisoprolol (BIS).
52 A. Baseline B. Isoproterenol Dose 5
C. Isoproterenol + ICI 118, 551 D. Isoproterenol + Bisoprolol
Figure 3.2 Representative M-mode Echocardiograms from a Susceptible Dog Prior to Infarct
53 Figure 3.3. The velocity of circumferential fiber shortening (Vcf) as calculated from M- mode echocardiograms in the presence of increasing doses of isoproterenol (ISO) for susceptible (n=19) and resistant (n=16) dogs prior to the surgical production o f the infarct. Dose 0 is the baseline Vcf value prior to the addition of drug, dose I is with the infusion o f0.006 pg/min/kg ISO, dose 2 is the infiision of 0.017 pg/min/kg ISO, dose 3 is 0.06 pg/min/kg ISO, dose 4 is 0.18 pg/min/kg ISO, and dose 5 is 0.66 pg/min/kg ISO.
54 Pre-Infarct Isoproterenol Response
15
—O— Resistant T5 > — Susceptible
0 1 2 3 4 5
Isoproterenol dose
Figure 3.3 Vcf Isoproterenol Dose Response for Susceptible and Resistant Dogs
Prior to Infarct
55 Figure 3.4. The Vcf values for increasing doses of isoproterenol alone and in the presence o f either 0.6 mg/kg BIS or 0.2 mg/kg ICI from resistant animals (n=16) prior to the surgical production o f an infarct.
56 Pre-Infarct Resistant
15
—o — ISO
ICl
—A — BIS
0 1 2 34 5
Isoproterenol dose
Figure 3.4 Pre-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and
Absence of p-Adrenergic Receptor Blockade for Resistant Dogs
57 Figure 3.5. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.6 mg/kg B IS for resistant animals (n=I6) prior to the induction of the infarct. The * indicate significant differences (p<0.05) between the Vcf values from the
ISO alone and ISO in the presence of BIS.
58 Pre-Infarct Resistant
15
12
9 —O— ISO o > — BIS 6
3
0 0 1 2 3 54
Isoproterenol dose
Figure 3.5 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without Bisoprolol for Resistant Dogs
59 Figure 3.6. The Vcf values for increasing doses o f isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for resistant animals (n=16) prior to the production of the infarct. The * indicate significant differences (p<0-05) between the Vcf values from the
ISO alone and ISO in the presence of ICI.
60 Pre-Infarct Resistant
15
12
9 —O — ISO o > — ICI S
3
0 0 1 2 4 53
Isoproterenol dose
Figure 3.6 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without the ICI 118,551 for Resistant Dogs
61 Figure 3.7. The Vcf values for increasing doses of isoproterenol alone and in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI from susceptible animals (n=I9) prior to the surgical production of an infarct.
62 Pre-Infarct Susceptible
15
— O— ISO
— - ICI
— A— BIS
0 1 2 3 4 5
Isoproterenol dose
Figure 3.7 Pre-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and
Absence of (3-Adrenergic Receptor Blockade for Susceptible Dogs
63 Figure 3.8. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence o f 0.6 mg/kg BIS for susceptible animals (n=I9) prior to the induction o f the infarct. The * indicate significant cüfîèrences (p<0.05) between the Vcf values from the
ISO alone and ISO in the presence o f BIS.
64 Pre-Infarct Susceptible
15
12
g —O — ISO ■s > — BIS 6
3
0 0 1 2 3 4 5
Isoproterenol dose
Figure 3.8 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without Bisoprolol for Susceptible Dogs
65 Figure 3.9. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for susceptible animals (n=l9) prior to the production of the infarct. The * indicate significant differences (p<0.05) between the Vcf values from the
ISO alone and ISO in the presence o f ICI.
66 Pre-Infarct Susceptible
15
12
g —O— ISO •s > - ICI 6
3
0 0 1 2 3 4 5
Isoproterenol dose
Figure 3.9 Pre-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without the ICI 118,551 for Susceptible Dogs
67 Figure 3.10. The heart rate response for susceptible (n=I9) and resistant (n=l6) dogs prior to the surgical production of the infarct. Dose 0 is the baseline heart rate prior to the addition of drug, dose I is with the infusion o f0.006 pg/min/kg ISO, dose 2 is the infusion of 0.017 pg/min/kg ISO, dose 3 is 0.06 pg/min/kg ISO, dose 4 is 0.18 pg/min/kg
ISO, and dose 5 is 0.66 pg/min/kg ISO.
68 Pre-Infarct Isoproterenol Response
175
150 c E S cs 125 •O— Resistant Ao (D Susceptible I 100 cs XID 75
50 0 1 2 3 4 5
Isoproterenol dose
Figure 3.10 Heart Rate Isoproterenol Dose Response for Susceptible and Resistant
Dogs Prior to Infarct
69 Figure 3.11. The heart rate response for increasing doses of isoproterenol (ISO) in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI from resistant animals (n=l6) prior to the surgical production of the infarct.
70 Pre-Infarct Resistant
175
150 c Ê —O — ISO S mcs 125 a . — ICI 0 1 100 —A— BIS 3 75 -
50 2 3
Isoproterenol dose
Figure 3.11 Pre-Infarct Heart Rate Isoproterenol Dose Response Curves in the
Presence and Absence of P-Adrenergic Receptor Blockade for Resistant Dogs
71 Figure 3.12. The heart rate response for increasing doses of isoproterenol (ISO) alone and in the presence of 0.6 mg/kg BIS for resistant animals (n=16) prior to the production
o f the infarct. The * indicates significant differences (p<0.05) between the heart rate values from the isoproterenol alone and ISO m the presence of BIS.
72 Pre-Infarct Resistant
175
150 c
125 — O — ISO 0 — BIS 1 100 o«S r 75
50 0 1 2 3 4 5
Isoproterenol dose
Figure 3.12 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Resistant Dogs
73 Figure 3.13. The heart rate values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for resistant animals (n=16) prior to the production of the infarct. The * indicate significant differences (p<0.05) between the heart rate values from the isoproterenol alone and ISO in the presence o f ICI.
74 Pre-Infarct Resistant
175
150 c S s O«B 125 —O— ISO
ID - ICI i 100 s 75
50 1 2 3
Isoproterenol dose
Figure 3.13 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Resistant Dogs
75 Figure 3.14. The heart rate response for increasing doses of isoproterenol (ISO) alone and in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI from susceptible animals
(n=19) prior to the surgical production o f an infarct.
76 Pre-Infarct Susceptible
175
150 c E —O — ISO 2 m0 9 125 a ICI 0 3 X
$ — $ 50 0 1 2 3 4 5
Isoproterenol dose
Figure 3.14 Pre-Infarct Heart Rate Isoproterenol Dose Response Curves in the
Presence and Absence of P-Adrenergic Receptor Blockade for Susceptible Dogs
77 Figure 3.15. The heart rate response for increasing doses o f isoproterenol (ISO) alone and in the presence of 0.6 mg/kg BIS for susceptible animals (n=19) prior to the production of the infarct. The * indicates significant differences (p<0.05) between the heart rate values firom the isoproterenol alone and ISO in the presence of BIS.
78 Pre-infarct Susceptible
175
150 c E £ 125 —O— ISO AS 0 — BIS 1 100 o«s r 75
50 0 1 2 3 4 5
Isoproterenol dose
Figure 3.15 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs
79 Figure 3.16. The heart rate values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for susceptible animals (n=19) prior to the production of the infarct. The * indicate significant differences (p<0.05) between the heart rate values from the isoproterenol alone and ISO in the presence of ICI.
8 0 Pre-Infarct Susceptible
175
150
—O — ISO
o — ICI s 100 o«a X 75
50 0 1 2 3 4 5
Isoproterenol dose
Figure 3.16 Pre-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs
81 Figure 3.17. Representative M-mode echocardiograms of the left ventricle from a resistant dog approximately 2 weeks after surgery. A. Baseline echocardiogram prior to the infusion of drug. B. Echocardiogram taken during the infiision of 0.66 pg/min/kg isoproterenol (ISO). C. Echocardiogram taken during the infusion of 0.66 pg/min/kg
ISO in the presence of 0.2 mg/kg o f ICI 118,551 (ICI). D. Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence of 0.6 mg/kg of bisoprolol (BIS).
82 A. Baseline B. Isoproterenol Dose 5
C. Isoproterenol + ICI 118,551 D. Isoproterenol + Bisoprolol
Figure 3.17 Representative M-mode Echocardiograms from a Resistant Dog Post
Infarct
83 Figure 3.18. Representative M-mode echocardiograms of the left ventricle from a susceptible dog approximately 2 weeks after surgery. A. Baseline echocardiogram prior to the infusion of drug. B. Echocardiogram taken during the infusion of 0.66 pg/min/kg isoproterenol (ISO). C. Echocardiogram taken during the infusion o f 0.66 pg/min/kg
ISO in the presence of 0.2 mg/kg of ICI 118,551 (ICI). D. Echocardiogram taken during the infusion of 0.66 pg/min/kg ISO in the presence of 0.6 mg/kg of bisoprolol (BIS).
84 A. Baseline B. Isoproterenol Dose 5
C. Isoproterenol + ICI 118,551 D. Isoproterenol + Bisoprolol
Figure 3.18 Representative M-mode Echocardiograms from a Susceptible Dog Post
Infarct
85 Figure 3.19. The velocity of circumferential fiber shortening (Vc£) as calculated from M-
mode echocardiograms in the presence of increasing doses of isoproterenol (ISO) for
susceptible (n=I2) and resistant (n=I9) dogs approximately 2 weeks after the surgical
production of an infarct. Dose 0 is the baseline Vcf value prior to the addition of drug,
dose I is with the infusion o f0.006 pg/min/kg ISO, dose 2 is the infusion of 0.017
pg/min/kg ISO, dose 3 is 0.06 pg/min/kg ISO, dose 4 is 0.18 pg/min/kg ISO, and dose 5
is 0.66 pg/min/kg ISO. An * denotes significant differences (p<0.05) between the susceptible and resistant dogs.
86 Post-Infarct Isoproterenol Response
15
12
9 —O— Resistant 73 > — Susceptible 6
3
0 0 1 2 3 4 5
Isoproterenol dose
Figure 3.19 Vcf Isoproterenol Dose Response for Susceptible and Resistant Dogs
Post Infarct
87 Figure 3.20. The Vcf values for increasing doses of isoproterenol alone in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI for resistant animals (n=I9) approximately 2 weeks after surgery.
88 Post-Infarct Resistant
15
— o — ISO
— Id
— A — BIS
0 1 2 3 4 5
Isoproterenol dose
Figure 3.20 Post-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and Absence of p-Adrenergic Receptor Blockade for Resistant Dogs
89 Figure 3.21. The Vcf values for increasing doses o f isoproterenol (ISO) alone and in the presence of 0.6 mg/kg BIS for resistant animals (n=19) approximately two weeks after surgery. The * indicate significant differences (p<0.G5) between the Vcf values from the
ISO alone and ISO in the presence o f BIS.
90 Post-Infarct Resistant
15
12
9 —O— ISO 5 > — BIS 6
3
0 0 1 2 4 53
Isoproterenol dose
Figure 3.21 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without Bisoprolol for Resistant Dogs
91 Figure 3.22. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for resistant animals (n=19) approximately two weeks after surgery. The * indicate significant differences (p<0.05) between the Vcf values from the
ISO alone and ISO in the presence of ICI.
92 Post-Infarct Resistant
15
12
9 —O — ISO o > ICI 6
3
0 0 1 2 3 4 5
Isoproterenol dose
Figure 3.22 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without ICI 118,551 for Resistant Dogs
93 Figure 3.23. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI from susceptible animals (n=12) approximately two weeks after surgery.
94 Post-Infarct Susceptible
15
12
—O — ISO
75 - ICI >
— A — BIS
0 1 2 34 5
Isoproterenol dose
Figure 3.23 Post-Infarct Vcf Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Susceptible Dogs
95 Figure 3.24. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence the presence of BIS .of 0.6 mg/kg BIS for susceptible animals (n=12) approximately two weeks after surgey. The * indicate significant differences (p<0.05) between the Vcf values from the ISO alone and ISO with BIS.
96 Post-Infarct Susceptible
15
12
9 —O — ISO •s > BIS 6 J— i
3
0 0 1 2 3 4 5
Isoproterenol dose
Figure 3.24 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and
Without Bisoprolol for Susceptible Dogs
97 Figure 3.25. The Vcf values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for susceptible animals (n=12) approximately two weeks after surgey. The * indicate significant differences (p ISO alone and ISO in the presence of ICI. 98 Post-Infarct Susceptible 15 12 9 —O — ISO ■5 > — ICI 6 3 0 0 1 2 3 4 5 Isoproterenol dose Figure 3.25 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs 99 Figure 3.26. A comparison of the Vcf response for dose 5 (0.66 pg/kg/min) of isoproterenol (ISO) alone and in the presence of ICI 118,551 from both resistant (n=19) and susceptible (n=12) dogs. The values are taken from the echos recorded after surgery. The * denotes significant differences (p<0.05) in the ICI reduction of the isoproterenol response in the resistant as compared to the susceptible animals. 100 Vcf Response to ISO and ICI 120 cCO o Q. 100 CO oc O (O 80 R esutant 0E 60 ^ ^ 2 ^ ] SuAceptible O)m c £C8 Ü 40 1o 20 a. 0 ISO ICI Figure 3.26 Percent Change of the Isoproterenol Response Due to ICI 118,551 in Susceptible and Resistant Dogs 101 Figure 3.27. Percent change of the Vcf response from a second isoproterenol dose response as compared to the isoproterenol dose response immediately preceding it from dogs (n=6) approximately two weeks post surgery. The * denotes significant differences (p<0.05) between the second ISO dose response and the first. 102 Percent Change of ISO Response 1 1 0 o 100 CO 90 c «9 40 o 30 c ID 20 O 10 O Q. 0 II 1 1 I 1 2 3 4 Isoproterenol dose Figure 3.27 Percent Change of the Vcf Isoproterenol Dose Response from two Consecutive Infusions 103 Figure 3.28. The heart rate dose response for susceptible (n=12) and resistant (n=I 9) dogs approximately two weeks after surgery. Dose 0 is the baseline heart rate value prior to the addition of drug, dose 1 is with the infusion of 0.006 pg/min/kg ISO, dose 2 is the infusion o f 0.017 pg/min/kg ISO, dose 3 is 0.06 pg/min/kg ISO, dose 4 is 0.18 pg/min/kg ISO, and dose 5 is 0.66 pg/min/kg ISO. An * denotes significant differences (p<0.05) between the susceptible and the resistant animals. 104 Post-Infarct Isoproterenol Response 175 150 / .. s 125 •O— Resistani Susceptible I 100 (9 rID 75 - 50 1 2 3 4 Isoproterenol dose Figure 3.28 Heart Rate Isoproterenol Dose Response for Susceptible and Resistant Dogs Post Infarct 105 Figure 3.29. The heart rate response for increasing doses of isoproterenol (ISO) alone and in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI from resistant animals (n=19) approximately two weeks post surgery. 106 Post-Infarct Resistant 175 150 — O — ISO m 125 £i — ICI 5 100 — A— BIS s IIII 50 1 1 2 3 4 Isoproterenol dose Figure 3.29 Post-Infarct Heart Rate Isoproterenol Dose Response Curves in the Presence and Absence of P-Adrenergic Receptor Blockade for Resistant Dogs 107 Figure 3.30. The heart rate response for increasing doses of isoproterenol (ISO) alone and in the presence of 0.6 mg/kg BIS for resistant animals (n=l9) approximately two weeks after surgery. The * indicates significant difierences (p<0.05) between the heart rate values from the ISO alone and ISO in the presence of BIS. 108 Post-Infarct Resistant 175 150 125 —O — ISO aS (D — BIS I 100 «5 rID 50 0 1 2 3 54 Isoproterenol dose Figure 3.30 Post-Infarct Vcf Dose Response Curve for Isoproterenol With and Without Bisoprolol for Resistant Dogs 109 Figure 3.31. The heart rate values for increasing doses of isoproterenol (ISO) alone and in the presence of 0.2 mg/kg ICI for resistant animals (n=19) approximately two weeks after surgery. The * indicate significant differences (p<0.05) between the heart rate values from the ISO alone and ISO in the presence o f ICI. HO Post-Infarct Resistant 175 150 - £ S «s 125 - —O — ISO £i(D — ICI 100 - eso 75 - 50 _L _L _L 1 2 3 Isoproterenol dose Figure 3.31 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Resistant Dogs 111 Figure 3.32. The heart rate response for increasing doses of isoproterenol (ISO) alone and in the presence of either 0.6 mg/kg BIS or 0.2 mg/kg ICI for susceptible animals (n=12) two weeks after surgery. 112 Post-Infarct Susceptible 175 150 c* E S. «S 125 u> A 3 ir 100 sa (5 (D r 75 50 Isoproterenol dose Figure 3.32 Post-Infarct Heart Rate Isoproterenol Dose Response Curv es in the Presence and Absence of p-Adrenergic Receptor Blockade for Susceptible Dogs 113 Figure 3.33. The heart rate response for increasing doses o f isoproterenol (ISO) alone and in the presence of 0.6 mg/kg BIS for susceptible animals (n=I2) two weeks after the surgery. The * indicates significant differences (p<0.05) between the heart rate values from the isoproterenol alone and ISO in the presence of BIS. 114 Post-Infarct Susceptible 175 150 c a a 125 —o — ISO am (B — BIS I 100 S X 50 0 1 2 3 4 5 Isoproterenol dose Figure 3.33 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without Bisoprolol for Susceptible Dogs 115 Figure 3.34. The heart rate values for increasing doses of isoproterenol (ISO) alone and in the presence o f 0.2 mg/kg ICI for susceptible animals (n=12) two weeks after surgery. The * indicate significant difierences (p<0.05) between the heart rate values firom the ISO alone and ISO in the presence of ICI. 116 Post-Infarct Susceptible 175 150 c E 2 cs 125 —O — ISO £>o 0 — ICI 1 100 50 0 1 2 3 4 5 Isoproterenol dose Figure 3.34 Post-Infarct Heart Rate Dose Response Curve for Isoproterenol With and Without ICI 118,551 for Susceptible Dogs 117 Figure 3.35. Representative unloaded single cell shortening tracings from a resistant dog taken at baseline prior to the exposure o f drug, as well as with the addition of 100 nM isoproterenol (ISO), and 100 nM ISO in the presence of either 200 nM bisoprolol (BIS) or 100 nM ICI 118,551 (ICI). A single tracing represents the average of 16 individual tracings taken from a single myocyte. 118 Resistant dog ISO ICI A l\ 20 - bis contro( A 10 I 1 sec Figure 3.35 Representative Edge Detection Tracings from a Resistant Dog 119 Figure 3.36. Representative unloaded single cell shortening tracings from a susceptible dog taken at baseline prior to the exposure of drug, as well as with the addition of 100 nM isoproterenol (ISO), and 100 nM ISO in the presence of either 200 nM bisoprolol (BIS) or 100 nM ICI 118,551 (ICI). A single tracing represents the average of 16 individual tracings taken from a single myocyte. 120 ISO 20 i ! ICI bis 3 (1 10 - Sub control 0 1 sec Figure 3.36 Representative Edge Detection Tracings from a Susceptible Dog 121 Figure 3.37. Percent cell shortening for single cardiomyocytes from both resistant (n=9) and susceptible (n=6) dogs prior to the exposure of drug, with the addition of 100 nM isoproterenol (ISO), and 100 nM ISO in the presence of either 200 nM bisoprolol (BIS) or 100 nM ICI 118,551 (ICI). 122 Unloaded Cell Shortening cO) oc o Resistant £QO Ü I I Susceptible c 8 o 0. control Figure 3.37 Percent Unloaded Cell Shortening for Resistant and Susceptible Dogs 123 Figure 3.38. Percent cell shortening normalized to control for single cardiomyocytes from both resistant (n=9) and susceptible (n=6) dogs with the addition of 100 nM isoproterenol (ISO), and 100 nM ISO in the presence o f either 200 nM bisoprolol (BIS) or 100 nM ICI 118,551 (ICI). The * denotes significant differences (p<0.05) between susceptible and resistant myocytes. 124 Cell Shortening 500 o 400 c o Ü E o 300 Resistant Susceptible 200 c 2 Q. 100 control ISO ICI BIS Figure 3.38 Percent Change from Control for Unloaded Single Cell Shortening 125 Figure 3.39. Single cell +dL/dt max for myocytes from both susceptible (n=6) and resistant (n=9) animals with the addition of 100 nM isoproterenol (ISO), and 100 nM ISO in the presence o f either 200 nM bisoprolol (BIS) or 100 nM ICI 118,551 (ICI). The * denotes significant differences (p<0.05) between susceptible and resistant myocytes. 126 single Cell +dL/dt max 1000 o 800 oc O £ o 600 o 05 CSC 6 400 c 8 Q. 200 control ISO ICI BIS Figure 3.39 Single Cell +dL/dt max from Susceptible and Resistant Dogs 127 Figure 3.40. The time to 50% relaxation from myocytes isolated from resistant (n=9) and susceptible (n=6) dogs with no drug present, with the addition of 100 nM isoproterenol (ISO), and with 100 nM ISO in the presence of either 100 nM ICI 118,551 (ICI) or 200 nM bisoprolol (BIS). The * denotes significant differences (p<0.05) between control and the ISO response. 128 Time to 50% Relaxation 1.00 0.75 y y / / / A Resistant 8 0.50 o Susceptible CO 0.25 0.00 controlISO ICI BIS Figure 3.40 Time to 50% Relaxation for Stimulated Single Cardiomyocytes from Susceptible and Resistant Dogs 129 Figure 3.41. Percent cell shortening for single cardiomyocytes from post-infarct dogs (n=5) prior to the exposure of drug and with the addition of 100 nM isoproterenol (ISO) repeated three times. 130 Unloaded Cell Shortening 35 30 o* c 25 oc £10 01 20 ■$ 15 O m 10 Q. 5 0 (SOI IS02control IS03 Figure 3.41 Percent Unloaded Cell Shortening from Multiple Isoproterenol Treatments 131 Figure 3.42. Representative chemiluminescent radiographs taken from p44/42 MAP kinase western blots from both a resistant and a susceptible dog. The lanes are labeled + for the positive control, CZ is the baseline level from the section of the heart perfused with zinterol, Z is the tissue treated with lOpM zinterol and 300 nM CGP, Z30 is the tissue that was perfused with zinterol followed by a 30 minute wash with control buffer, CP is the baseline level from the section of the heart perfused with phenylephrine, P is the tissue treated with lOpM phenylephrine and lOpM propranolol, and P30 is the tissue that was perfused with phenylephrine followed by a 30 minute was with control buffer. 132 R e s is ta n t Susceptible Figure 3.42 Representative p44/42 MAP Kinase Western Blots from Susceptible and Resistant Dogs 133 Figure 3.43. Comparison of the presence ofp44/42 MAP kinase normalized to the positive control for resistant (n=6) and susceptible (n=6) dogs at baseline, in the presence of lOpM zinterol and 300 nM CGP, and after a 30 minute washout period. The * denotes significant differences (p<0.05) between the susceptible and resistant animals. 134 Presence of p44/42 MAP Kinase 5 4 o T3 C 2(a 3 Resistant O ■o N 1 0 control zinterol post Figure 3.43 Presence of p44/42 MAP Kinase with Stimulation by Zinterol in Susceptible and Resistant Dogs 135 Figure 3.44. Comparison of the presence of p44/42 MAP kinase normalized to the positive control for resistant (n=6) and susceptible (n=6) dogs at baseline, in the presence of lOpM phenylephrine and lOpM propranolol, and after a 30 minute washout period. The * denotes significant differences (p<0.05) between the susceptible and the resistant animals. 136 Presence of p44/42 MAP Kinase 7 6 o 5 ■oc 2 CO V y y /y A Resistant o 4 ■o q) ^ ! Susceptible N 3 ■5 E 2 1 0 control phenylephrine post Figure 3.44 Presence of p44/42 MAP Kinase with Stimulation by Phenylephrine in Susceptible and Resistant Dogs 137 Figure 3.45. Comparison of the presence of p44/42 MAP kinase normalized to the baseline control level for resistant (n=6) and susceptible (n=6) dogs in the presence of lOpM zinterol and 300 nM CGP, and after a 30 minute washout period. 138 Presence of p44/42 MAP Kinase 600 500 o 400 c 8 Resistant o 300 c Susceptible 8 (D a. 200 100 control zinterol post Figure 3.45 Presence of p44/42 MAP Kinase as Compared to Control with Stimulation by Zinterol in Susceptible and Resistant Dogs 139 Figure 3.46. Comparison of the presence of p44/42 MAP kinase normalized to the baseline control level for resistant (n=6) and susceptible (n=6) dogs in the presence of lOjiM phenylephrine and lOpM propranolol, and after a 30 minute washout period. The * denotes significant differences (p<0.05) between susceptible and resistant animals. 140 Presence of p44/42 MAP Kinase 800 700 2 600 i 500 y /Z /y A Resistant 0 ^ 400 N I I Susceptible 1 300 o z 200 100 control phenylephrine post Figure 3.46 Presence of p44/42 MAP Kinase as Compared to Control with Stimulation by Phenylephrine in Susceptible and Resistant Dogs 141 Figure 3.47. Representative chemiluminescent radiographs taken from p44/42 MAP kinase activity assay western blots from both a resistant and a susceptible dog. The lanes are labeled + for the positive control, CZ is the baseline level from the section of the heart perfused with zinterol, Z is the tissue treated with lOpM zinterol and 300 nM CGP. Z30 is the tissue that was perfused with zinterol followed by a 30 minute wash with control buffer, CP is the baseline level from the section of the heart perfused with phenylephrine, P is the tissue treated with lOpM phenylephrine and lOpM propranolol, and P30 is the tissue that was perfused with phenylephrine followed by a 30 minute was with control buffer. 142 Susceptible Resistant Figure 3.47 Representative Radiographs from the p44/42 MAP Kinase Activity Assay for Susceptible and Resistant Dogs 143 Figure 3.48. Comparisoa of p44/42 MAP kinase activity assays normalized to the positive control for resistant (n=6) and susceptible (n=6) dogs at baseline, in the presence of lOpM zinterol and 300 nM CGP, and after a 30 minute washout period. 144 p44/42 MAP Kinase Activity 2.00 ■o 1.50 73«S oC 0.00 control zinterol post Figure 3.48 p44/42 MAP Kinase Activity for Susceptible and Resistant Dogs Following Stimulation by Zinterol 145 Figure 3.49. Comparison of p44/42 MAP kinase activity assays normalized to the positive control for resistant (n=6) and susceptible (n=6) dogs at baseline, in the presence of 10|xM phenylephrine and lOpM propranolol, and after a 30 minute washout period. The * denotes significant differences (p<0.05) between the susceptible and resistant dogs. 146 p44/42 MAP Kinase Activity 3.00 2.50 2 'OCD c 2.00 2 GO Resistant o 1.50 Susceptible CO E 1.00 o z 0.50 0.00 control phenylephrine post Figure 3.49 p44/42 MAP Kinase Activity for Susceptible and Resistant Dogs Following Stimulation by Phenylephrine 147 Figure 3.50. Comparisoa of p44/42 MAP kinase activity assays normalized to the baseline control level for resistant (n=6) and susceptible (n=6) dogs in the presence of lOpM zinterol and 300 nM CGP, and after a 30 minute washout period. The * denotes significant differences (p<0.05) between susceptible and resistant animals. 148 p44/42 MAP Kinase Activity 400 o 300 c 8 o Resistant % 200 N I I Susceptible Œ E o ^ 100 control zinterol post Figure 3.50 p44/42 MAP Kinase Activity Normalized to Control for Susceptible and Resistant Dogs Following Stimulation by Zinterol 149 Figure 3.51. Comparison of p44/42 MAP kinase activity assays normalized to the baseline control level for resistant (n=6) and susceptible (n=6) dogs in the presence of lOjiM phenylephrine and lOjjM propranolol, and after a 30 minute washout period. 150 p44/42 MAP Kinase Activity 300 o co 200 o v / / y y A Resistant o •o(D N I k Susceptible CO E o 100 z phenylephrinecontrol post Figure 3.51 p44/42 MAP Kinase Activity Normalized to Control for Susceptible and Resistant Dogs Following Stimulation by Phenylephrine 151 CHAPTER 4 DISCUSSION Sudden cardiac death is an unexpected natural death from a cardiac cause within a short period ( (VF) with the exercise plus ischemia test. The resistant animals are spared from these lethal arrhythmias. Since all of the animals used in this model undergo identical surgeries to produce the myocardial infarct, it is not known why the two classes of dogs respond differently. Outside of their arrhythmic tendencies, these two groups of animals also have a disparity in their hemodynamic responses to exercise. In particular, the susceptible animals experience left ventricular dysfunction during exercise (7). They have a significantly greater increases in heart rate and left ventricular 152 diastolic pressure with smaller increases in left ventricular systolic pressure (7). Exercise, an intervention that increases the sympathetic drive to the heart, is also accompanied with withdrawal of vagal tone (32). In an attempt to meet the physiological demands of exercise, the susceptible animals withdraw vagal tone to a greater extent as compared to the resistant dogs thus potentiating the increase in heart rate. Furthermore, the susceptible animals have a slight but insignificant decrease in vagal tone prior to the onset of exercise (32). Outside of the hemodynamic differences noted with exercise. there appears to be alterations in the calcium fluctuations across the sarcolemma. Calcium channel antagonists are capable of protecting susceptible animals from the development o f VF while the calcium channel agonist. Bay K 8644 induces VF in resistant animals (8,9). Furthermore, if the calcium content of the sarcoplasmic reticulum is depleted by ryanodine prior to ischemia, VF is not prevented in the susceptible dogs (46). Interestingly, agents that directly stimulate adenylate cyclase and PKA production fail to produce arrhythmias in the resistant animals, therefore these compounds and their downstream substrates may not play a detrimental role (4). Together these data point to a strong correlation between abnormal calcium handling and the development of lethal arrhythmias in the susceptible animals that is not present in the resistant animals. Furthermore, it is this increase in intracellular calcium that results from calcium entering through the sarcolemmal calcium channels rather than the increased release of calcium from the sarcoplasmic reticulum which appears to be detrimental. The agent or agents that cause this abnormally high calcium overload in the susceptible animals is not known. As mentioned previously, P-AR stimulation coupled with ischemia can cause an increased amount of intracellular calcium. Presumably though, an increase in P-AR 153 activation would be occurring in both groups of animals. Therefore, the mechanism that potentiates the increased intracellular calcium levels in the susceptible animals is not known. Recently, there has been particular interest the in role that the P%-AR plays in the development of these arrhythmias (II,47). In a meta-analysis of several clinical trials using P-AR antagonists in patients with congestive heart failure, Lechat et al. (47) found that P~AR blockade increased the ejection fraction and reduced the risk of death or hospitalization for heart failure. Although mortality was significantly decreased with all P-AR blockers used, the overall effect of the p-blockade varied according to the type of P-blocker used as well as the endpoint measurement. Metoprolol, a selective Pi-AR antagonist, had no effect on the incidence of sudden cardiac death. While carvedilol, a nonselective p- and a-AR antagonist, was successful at decreasing the incidence of sudden death. The authors (47) point out that blockade of the P%-AR with carvedilol may be an important factor as there is an alteration in the P 1/P2 AR ratio during heart failure. Furthermore, in the canine model of sudden death, the p%-AR antagonist, ICI 118,551 was able to protect the susceptible animals against the development of arrhythmias (11). Therefore, it was of interest to determine if differences in P 2 -AR activity account for some of the pathophysiological differences noted between the two groups of animals. 4.1 Analysis of Pz-AR Function in Susceptible and Resistant Dogs Billman etal. (11) previously reported that isolated cardiomyocytes from susceptible dogs showed a significantly greater calcium transient when exposed to 154 zinterol as compared to resistant animals. The echocardiography experiments, both pre and post infarct, were done to determine if these calcium transient amplitude differences noted in the isolated cardiomyocytes manifest as differences in contractility in the whole animal heart. It was also of interest to determine if physiological differences between the two groups are present prior to the development of the infarct. The use of M-mode echocardiography is a noninvasive technique for assessing cardiac function. The velocity of circumferential fiber shortening (Vcf) can be calculated by observing both the aortic valve and the left ventricular wall, and it is used as a reliable measure of load independent contractility (13). Isoproterenol was capable of significantly increasing the Vcf value in both the susceptible and resistant animals with a much greater increase noted in the susceptible animals (Figure 3.19). This disparity was not seen in the echocardiogram studies collected prior to the induction of the infarct (Figure 3.3). Since isoproterenol is both a Pi- and a P%-AR agonist, it was necessary to determine how selective Pi-AR or Pi-AR blockade would alter the response to isoproterenol. Therefore, further echocardiogram studies were done with isoproterenol in the presence of one of the two p-adrenergic antagonists, bisoprolol and ICl 118,551. As expected, both pre and post infarct, the isoproterenol Vcf response was reduced by both drugs in both groups of animals (Figures 3.4-3.26). However, post infarct ICl reduced the Vcf response with the highest dose of isoproterenol by 48% in the susceptible dogs while in the resistant dogs there was only a 20% reduction (Figure 3.26). This difference was not seen pre-infarct (Figures 3.6 and 3.9). In fact, the post infarct resistant animals showed a Vcf profile similar to that observed pre-infarct in both groups of animals (Figures 3.4. 3.7, and 3.20). 155 It has previously been determined that exercise elicits a greater increase in heart rate in the susceptible animals (32). This is true with the isoproterenol infusion as well. Prior to infarct, isoproterenol increased the heart rate similarly in both groups of animals (Figure 3.10). Interestingly, after infarct, both groups of animals had an increased resting heart rate indicating a slightly higher sympathetic drive to the heart after surgery (Figures 3.10 and 3.28). However post-infarct, the isoproterenol inftision elicited even greater increases in heart rate in the susceptible dogs suggesting an elevated p-adrenergic response in these animals which may also be coupled with a decreased parasympathetic input (32) in the susceptible animals post infarct (Figure 3.28). Post infarct, bisoprolol was able to reduce the heart rate response equally in both groups of animals (Figures 3.30 and 3.33). ICI, however, caused a greater decrease in the susceptible animals at most of the doses of isoproterenol studied (Figure 3.34). Again, this indicates a greater dependence on the P%-AR in the susceptible dog heart. The contractility changes noted in the whole animals were also present at the level of the single cell. As mentioned previously Billman et al. (11) reported that there was a difference in the calcium transient amplitude with the addition of 100 nM isoproterenol in myocytes from susceptible and resistant dogs. This discrepancy was also noted in the unloaded single cell shortening twitches. The absolute percent cell shortening data showed no significant differences between the two groups (Figure 3.37). However, when looking at the percent change from control, it was observed that the susceptible animals had a much larger increase in contraction amplitude with isoproterenol than did the resistant animals (Figure 3.38). Likewise, in the susceptible animals ICI 118,551 attenuated approximately 48% of this increase while in the resistant animals ICl only 156 decreased the response by 15%. This corresponds to the decreases noted in the echocardiogram studies with the addition of ICL Furthermore, the Pi-AR antagonist, bisoprolol, elicited different responses in the two groups of animals. It must be kept in mind that in the presence of isoproterenol and bisoprolol, the remaining activated P-AR is the P 2-AR. When bisoprolol was present, the percent cell shortening as compared to control was significantly greater in the susceptible animals than in the resistant animals (Figure 3.38). Single cell +dL/dt max takes into account the change in cell length as well as the rate at which this length change was achieved. These values should correspond to the post infarct Vcf values seen in the intact heart as Vcf also analyzes both the rate and amplitude of shortening. Figure 3.39 displays the +dL/dt max as a percent change from control for myocytes from both resistant and the susceptible dogs. As seen before, there was a significant increase in the isoproterenol response in both groups of dogs with the susceptible animals having a greater increase. With the addition o f isoproterenol and bisoprolol (the P 2-AR response) the suceptible animals have a significantly greater response. Interestingly, isoproterenol and ICI or the Pi-AR response was significantly less in the susceptible animals indicating an attenuated Pt-AR activation in the susceptible animals. All previous experiments in this present study indicate normal Pt- AR activation with an enhanced P 2-AR response in the susceptible animals. Since the Pt- AR amplitude of shortening was already shown to be equal in the susceptible and resistant dogs (Figure 3.38), this discrepancy would indicate an impairment in the velocity of cell contraction in the single cells that is not seen in the intact heart. Together, these data indicate a greater P 2-AR stimulation in the susceptible dogs. This 157 coincides with the increase in calcium transient amplitude noted in susceptible animals with the addition of zinterol (11). Likewise in the Billman et al. (11) study, the addition of a pi-AR antagonist, CGP, caused a greater reduction in the calcium transient amplitude of the resistant animals. In summary, ICI blocked a significantly greater amount of the isoproterenol response pointing to an increased dependence on the Pi-AR in the susceptible dogs, and it is this greater dependence on P 2-AR activity that may account for the increased isoproterenol response seen in the susceptible animals. The fact that the inhibition produced by bisoprolol was less in the susceptible animals further supports the concept of increased P 2-AR activation. As mentioned previously, these alterations in P 2-AR signaling are not due to an increase in the amount of P 2-ARS present. Previous results (Altschuld lab unpubUshed data) showed that the percentage of P 2-AR as well as the Kd value were similar between the two groups of animals. In a study using cardiomyocytes from failing and healthy dog hearts, Altschuld et al. (2) found that zinterol was capable of increasing the calcium transients in both groups of animals. However, the failing cells were significantly more responsive. During heart failure there is an “unmasking” of the P 2-AR due to a decrease in the Pi-AR number, but the P 2-AR number stays constant. Thus, even though the P 2-AR number may not be different between the two groups (failing dogs and healthy dogs), overall function or contribution of this receptor changes. Therefore, it is possible that the two groups of animals used in the present study (susceptible and resistant) have developed two different intracellular signaling mechanisms for the P 2-AR. Furthermore, it is of interest to determine if the P 2- AR may account for the arrhythmias noted in the susceptible animals. 158 4.2 Pz-AR Activation with Ischemia and the Development of Arrhythmias It is known, that the susceptible animals experience VF with ischemia and exercise while resistant animals do not, however it is not known exactly what is responsible for this difference between the two populations. The data detailed above, as well as from previous reports (11), points to possible alterations in Pz-AR stimulation and/or activation as one main difference between the two groups. Therefore, it must be determined if heightened Pz-AR stimulation is capable of causing arrhythmias during an ischemic challenge. As mentioned previously, with myocardial ischemia there is an increase in the activity of the cardiac sympathetc nerve fibers (62). This increase in sympathetic activity is due to cardiovascular reflexes as well as anxiety associated with ischemia (62). It appears that the catecholamine release during ischemia is grouped into time dependent stages (62). The first stage, which occurs within moments after ischemia begins, is regulated by central reflex mechanisms. With this first line of release (within the first 10 minutes), there are mechanisms in place to quickly take up the catecholamines thus sparing the tissue. Newton et al. (57) found that 60 seconds of ischemia was not enough to induce norepinephrine overflow from the human heart. McGrath et al.(52) using an in vivo dog model found that 5 minutes of ischemia was also not enough to cause a significant rise in plasma norepinephrine levels, however the epinephrine levels did increase. As the ischemic episode progresses though, local release mechanisms take over and the norepinephrine levels rapidly increase. This massive catecholamine release exceeds the uptake abilities. After 15 minutes of acute myocardial ischemia in a rat heart, there was a 100 fold increase in catecholamine concentrations in the extracellular 159 space of the ischemic zone which was coupled with a 30 % increase in P-AR number (62). Moreover, acute surgical cardiac denervation in an invfvo dog model failed to alter the norepinephrine levels during prolonged ischemia compared to control nondenervated but ischemic animals (25). These studies demonstrate the importance of local metabolic catecholamine release during an ischemic challenge, but again it must be emphasized that norepinephrine levels do not become significantly high until several minutes of ischemia has passed (62). More importantly, norepinephrine is primarily a Pi-AR agonist that does not usually activate the Pz-AR. Interestingly, Newton et al. (57) found that the 60 seconds of ischemia did significantly increase circulating epinephrine levels. Epinephrine is a dual Pi-AR/Pz-AR agonist. Again, the sudden death model used in the current set of experiments utilizes two minutes of ischemia. In summary, it has been reported that the 2 minutes of ischemia is not long enough to raise the norepinephrine level, but it can cause an increase in circulating epinephrine levels. It must also be kept in mind that although the levels of catecholamines may increase during ischemia there is no desensitization and down-regulation of the P-ARs. In fact in an in vivo dog model with a 30 minute left anterior descending artery occlusion, there was a significant increase in overall P-ARs in the ischemic zone, however when broken down into the receptor subtypes only the Pz-AR significantly increased (5). Since there is no change in Pz-AR mRNA in less than 60 minutes of ischemia (5) this increase must be due to a movement of receptors from a separate intracellular “light vessicle” compartment to the plasma membrane. This increase returned to baseline levels if reperfusion followed the ischemic incident. It is not known if such an increase in Pz-AR occurs within the 2 160 minutes of ischemia used in this model, but the possibility does exist. Therefore, preferential activation of the (3%-AR can occur during short bouts of ischemia. Also, it is not known if the P 2-AR number during ischemia differs between the two groups of animals used in the present study as the experiments to determine receptor number were done long after an ischemic episode. It must also be kept in mind that the 2 minutes of ischemia is coupled with submaximal exercise. Although changes in plasma catecholamine levels with exercise were not studied here, it is known that exercise will increase the levels of both norepinephrine and epinephrine (33). Furthermore, Stith (65), using the canine sudden death model, found that with exercise the epinephrine levels in both plasma and heart tissue significantly increased in both the susceptible and resistant animals, but a much greater increase was noted in the susceptible animals. Interestingly, no differences were reported for norepinephrine levels. The previous data by Stith (65) introduces the possibility of increasing epinephrine levels wdth exercise in the susceptible dogs thus contributing to the increased P 2-AR activation noted in the susceptible animals. Baseline levels, however, were similar between the two groups of dogs. Therefore, with the increased levels of catecholamines that accompany submaximal exercise there is a fiither increase in epinephrine levels with ischemia and a possible increase in P 2-Aft. number. Together these actions set the stage for P 2-AR stimulation precipitating the development of lethal arrhythmias. It has been determined that calcium fluctuations across the sarco lemma may partially account for the VF seen in the susceptible dogs (8,9). Furthermore, these arrhythmias do not appear to be dependent on cAMP or PKA production and activation 1 6 1 (4). Pa-AR activation phosphorylates the L-type calcium channel (3,42) thus increasing the intracellular calcium content of the cell. However, activation of the Pz-AR. does not increase whole cell cAMP content (2). Furthermore, it has already been determined that in this animal model it may be the calcium entry across the sarcolemma and not the increase in calcium released from the sarcoplasmic reticulum or the activation of adenylate cyclase that contributes largely to the ventricular fibrillation. These findings are consistent with the Pz-AR mode of signaling. Therefore, it is quite possible that Pz- AR through activation of the L-type calcium channel increases the intracellular calcium levels thus leading to the development of arrhythmias. p-AR activation also acts to increase the heart rate. In doing so, it must also increase the repolarization rate of the cardiomyocytes. In the normal healthy dog heart, repolarization is largely due to the fast component of the delayed rectifying current, Ikt, with a smaller contribution from the slow component of this current, the Iks current. As the heart rate increases either due to exercise or ischemia, the contribution of the Iks current becomes more important. Pt-AR stimulation does not alter Ikt but does phosphorylate and thus increases the Iks current (19). The phosphorylation of Iks appears to be PKA dependent and as such is primarily due to Pt-AR activation (19). Furthermore, as seen in a mouse model of Pz-AR overexpression an increase in calcium is not accompanied by an increase in the Iks current (3). Therefore, it is possible that Pz-AR activation alters potassium handling and as a result increases the action potential duration. This increase in the amount of time that a cell remains depolarized could produce inhomogeneities in the repolarization rates of the cardiac tissue. These inhomogeneities may, in feet, act as a substrate for the 162 development of arrhythmias. The possibility, therefore, exists that P 2-AR activation is increased in the susceptible animal, and it is this Pz-AR that contributes to the development of arrhythmias. The increased circulating epinephrine levels with exercise coupled to the heightened Pz-AR activation in the susceptible animals may contribute to the arrhythmias that develop with exercise and ischemia. However, the mechanism behind the heightened Pz-AR activation which is seen in both the in vivo echocardiogram studies and the in vitro single cell studies in the susceptible animals is not known. Therefore, it is possible that the Pz-AR is capable o f different signaling mechanisms in the two groups of animals with one mechanism leading to the generation of arrhythmias while the other spares the heart. 4.3 Intracellular Signaling of the pz-AR As previously noted, there have been numerous studies that have reported differences in the intracellular changes noted with pi-AR stimulation versus Pz-AR stimulation (29,42,43,72). To recapitulate, the Pi-AR manifests its intracellular actions through the traditional cAMP/PKA pathway. In doing so it causes phosphorylation of the L-type calcium channel, as well as phosphorylation of the Iks channel, troponin 1, and phospholamban. Together these actions cause an increase in the amount of calcium in the cell and subsequently an increase in the amplitude of the contraction. At the same time there is an increase in calcium sequestration by the sarcoplasmic reticulum, as well as, an increase in the repolarization rate. As a consequence, there will be an increase in the rate and strength of a contraction. The Pz-AR, however, appears to follow a different 163 yet somewhat similar activation. As mentioned, the P 2-AR is known to couple to Gs and thus activate adenylate cyclase, however the subsequent activation of cAMP appears to be more complex than with Pi-AR stimulation. p%-AR stimulation of isolated ventricular myocytes from chick, rat or dog produced no increase in cAMP with the addition of zinterol (2,58,72). However, Pz-AR stimulation with zinterol in an intact dog heart was capable of significantly increasing cAMP (42). These authors (42) explain that this increase may be due to P%-AR induced cAMP production in other cardiac tissues (ie. vascular smooth muscle cells or endothelial cells) which have higher levels o f pz-AR. Interestingly, in this same study Kuschel et al. (42) were able to block the calcium current induced by both norepinephrine and zinterol with the cAMP inhibitor, RP-cAMPs. However, neither canine cardiomyocytes nor canine whole heart samples showed an increase in global PKA activation, phospholamban phosphorylation or troponin I phosphorylation with the addition of zinterol (42). Similar results have been found using rat cardiomyocytes (43). Therefore this points to the possibility of a highly localized subsarcolemmal microdomain in the vicinity of the L-type calcium channels where cAMP/PKA are activated. Despite these obvious alterations in signaling, it has consistently been reported that Pz-AR stimulation causes an increase in calcium transient amplitude and single cell shortening at the level of the myocyte and a substantial increase in chronotropy, inotropy and lusitropy at the level o f the whole heart (11,42,43,72). In the present study, isoproterenol significantly and equally increased the relaxation rate in the susceptible and the resistant animals (Figure 3.40). This increase in relaxation rate was not altered by either ICI or bisoprolol. Kuschel et al. (42) report a significant 164 increase in the relaxation rate with both zinterol and norepinephrine in canine whole heart and isolated myocyte preparations with the relaxation rate being more sensitive to pi-AR stimulation. In the present study, however, there appears to be an equal dependence on both the Pi-AR and the P 2-AR. More importantly, there were no differences noted between the two groups of animals. Overall, it appears that the primary effect of Pz-AR stimulation is to cause phosphorylation of the L-type calcium channel thus increasing the overall intracellular calcium levels. The increased calcium due to L-type channel phosphorylation might account for the increases in calcium transient amplitude (II) seen previously as well as the increase in single cell shortening, and Vcf values seen with Pz- AR stimulation noted in the present study (Figures 3.19-3.39), but it still does not account for the differences noted between the two groups of animals. There is some evidence that points to a novel signaling mechanism by the Pz-AR. Multiple labs have implicated a role for the p44/42 MAP kinase cascade being activated via a Gi protein as a potential signaling pathway for the Pz-AR (14,22,76). In studies using cultured rat myocytes, norepinephrine, phenylephrine, and isoproterenol have been shown to increase the MAP kinase levels (14,76). Interestingly, the norepinephrine induced MAP kinase response is only slightly higher than the phenylephrine response (76). Furthermore, the addition of norepinephrine and propranolol decreased the MAP kinase, but not nearly as much as prazosin (76) indicating a larger dependence on a-A R activation for MAP kinase production with stimulation by norepinephrine. The isoproterenol response, however, was also greater than that of norepinephrine and prazosin, therefore indicating some Pz-AR contribution (76). Daaka et al. (22) using 165 HEK 293 cells report a significant increase in MAP kinase with the addition of isoproterenol which was almost completely blocked by propranolol. Although activation of the p44/42 MAP kinase pathway is a strong hypertrophic signaling pathway, there exists no evidence for it as a potential arrhythmic agent. Therefore, the hypothesis to be tested in this study was that the susceptible animals activate a pathway that causes an increase in calcium thus potentiating the arrhythmic potential of ischemia and exercise while the resistant animals preferentially activate a Gi protein mediated p44/42 MAP kinase pathway thus sparing them from the arrhythmias. However, perfusion of whole heart chunks with zinterol and phenylephrine from both susceptible and resistant animals showed a significant increase in p44/42 MAP kinase in the susceptible animals rather than the resistant animals (Figures 3.43 and 3.44). The baseline p44/42 MAP kinase levels appeared to be larger in the susceptible animals but this difference was not significant. Moreover, the susceptible animals had a slightly higher but insignificant increase in cell size. Thus, the trend towards an increased MAP kinase basal level could contribute to the greater cell size seen in the susceptible animals. How the P 2-AR couples to Gi has been extensively studied. Xiao et al. (73,75) reported that in rat ventricular myocytes, the P 2-AR dually couples to both Gs and Gi with inhibition of Gi enhancing the P 2-AR contractile and relaxation effects. More importantly, an inhibiton of Gi by PTX rescues P 2-AR mediated phosphorylation of phospholamban (44). Therefore, perhaps the function of the Gi protein pathway is to limit the magnitude as well as the quality of the P 2-AR/Gs/cAMP cardiac responses. The role that the dual coupling plays in the concept of susceptibility is of interest. Daaka et 166 al. (22) using HEK 293 ceils proposed that activation of PKA through a Pz-AR/Gs pathway causes phosphorylation of the Pz-AR itself The phosphorylation of the Pz-AR thus switches its subsequent activation to the Gi mediated pathway. In doing so, the Pz- AR may in fact “turn o ff’ some of its function while “turning on” other functions. Xiao et al (43) in a study using rat ventricular myocytes confirmed this concept of the two pathways, Gi and Gs, regulating each other. In this study (43), the hypothesis to be tested was that the activation of the Gi pathway causes rapid dephosphorylation of the phosphorylated proteins activated by the Gs pathway. Calyculin A, a nonselective protein phosphatase inhibitor, was capable of mimicking PTX and forcing the Pz-AR signaling down the Gs/cAMP/PKA pathway. It rescued the phosphorylation of the various intracellular proteins thus causing the cellular response to zinterol to be quite similar to that of norepinephrine. This was further substantiated by the fact that mouse ventricular myocytes were not fiinctional unless pretreated with PTX, therefore indicating a high level of Pz-AR Gi protein coupling (75). Zou et al. (79) state that the P-AR induced ERK 1 and ERK 2 were equally activated by both the Gs and Gi pathway in cultured rat cardiomyocytes. In this study (79), if PKA or cAMP were inhibited, then MAP kinase activity was also attenuated. However, pretreatment with PTX also decreased MAP kinase to the same extent. Therefore, these authors concluded that with stimulation of the P-AR receptor dual activation was needed. This conclusion supports the hypothesis that it is the Gs pathway that initiates the Gi pathway. Furthermore, in a series of experiments, Lefkowitz and coworkers (1,23,30,51) determined that Ras dependent activation of ERK 1 and ERK 2 requires the release of the Py subunit from the 167 Gi protein. It must be kept in mind that it is the Gpy subunit which is involved in terminating the Gs signal. With the binding of P-arrestin, the authors suggest that the Gs signal is terminated and a second wave of signal transduction may begin. Therefore, the activation of ERKs by P%-AR is dependent on desensitization and sequestration. In the present study we found no evidence of down-regulation or desensitization occurring in either the echocardiography studies or the single cell studies where tissue or cells were exposed to repeated doses of isoproterenol (Figures 3.27 and 3.41). Limas et al (48) showed that in isolated rat ventricular myocytes a 5-10 minute incubation with 1 pM isoproterenol is needed for significant down regulation. In our single cell experiments, 100 nM isoproterenol was exposed for 2 minutes therefore down regulation most likely did not occur, as well as, the switch from the Gs to Gi coupling. In the MAP kinase studies, the hearts were perfused for 8 minutes with 10 pM zinterol which may have been long enough for the switch from Gs/Gi to occur. Therefore, if the concept of switching from Gs to Gi is true, then it most likely would not have occurred in the echo or single cell studies. In summary, due to the greater calcium transient, cell shortening percent and Vcf values, it may be concluded ±at the susceptible animals couple more efficiently to the Gs proteins. Assuming that dual activation of Gs/Gi is achieved with pz-AR stimulation and that Gi stimulation is dependent on PKA production, then with the addition of zinterol the Gs/cAMP pathway is activated. This in turn will lead to an increase in the activation of the Gi subunit thus increasing MAP kinase. If the resistant animals do not couple as tightly to Gs as would be expected by the decrease in calcium, Vcf and cell shortening a 168 mutual decrease in Gi MAP kinase should be and was noted. Therefore, a stronger coupling to Gs in the susceptible animals would account for the increase in cell shortening and Vcf values in the present studies where desensitization and down regulation most likely did not occur, as well as the increase in MAP kinase production in the perfusion studies. Interestingly, the Pz-AR. signaling o f MAP kinase also appears to be dependent on intracellular calcium levels as well as cAMP levels. In a study using cultured neonatal rat cells, Bogoyevitch et al. (14) found that if calcium entry was blocked by either nifedipine or verapamil then the MAP kinase production induced by isoproterenol was completely abolished. Furthermore, if the cells were pretreated with agents that bypass the receptor and directly increase cAMP, then there also was no MAP kinase production. However, Daaka et al. (22) found that treatment of HEK 293 cells with forskolin caused an 8 fold increase in MAP kinase activity, thus pointing to possible differences in cell type response. Zinterol will increase the calcium levels in both susceptible and resistant, however the increase is greater in the susceptible animals (11). This greater level of calcium may contribute to the increased ERKs seen in the susceptible animals. The finding that increased cAMP production does not increase MAP kinase production coincides with the Pz-AR pattern of increasing calcium without increasing whole cell cAMP. Interestingly, the L-type calcium blockers were unable to block the MAP kinase phosphorylation due to norepinephrine suggesting that MAP kinase production due to a- or Pi-activity may bypass calcium sensitive steps (75). Therefore, the differences noted between the two groups of animals with phenylephrine are not as easily explained (Figure 3.44). Differences in the ai-AR in the two groups of animals are not known. Theai-AR 169 antagonist, prazosin, was found to protect susceptible animals from ventricular fibrillation (10). Therefijre, there may be a hightened responsiveness of the ai-A R in the susceptible dogs. The p44/42 MAP kinase western blot studies, however, do not coincide with the MAP kinase activity assays. In the latter studies the phosphorylation of Elk-1, a downstream substrate of p44/42 MAP kinase, was observed. In the resistant animals, zinterol significantly increased the amount of phosphorylated Elk-1 present while phenylephrine had no effect (Figures 3.50 and 3.51). Likewise, in the susceptible animals neither phenylephrine nor zinterol significantly increased Elk-1 levels. Taken together, it can be concluded that although the resistant animals had less p44/42 MAP kinase present, what was present appeared to be more active in phosphorylating Elk-1. Therefore, there exists the possibility that the immunoprécipitation procedure used in the activity assay may coprecipitate an inhibitor of ERK-1 or ERK-2, that may be more pronounced in the susceptible animals however this could not be confirmed. The present p44/42 MAP kinase studies were performed using whole heart tissue chunks thus allowing for the possibility of Pz-AR stimulation in other cell types. Attempts were made to study Pz-AR stimulation in isolated ventricular myocytes, but the results were unusable. The collagenase perfusion used to isolate the myocytes caused an upregulation of the p44/42 MAP kinase pathway to such a degree that further stimulation by drugs was not seen. MAP kinase is typically studied in cultured cardiac myocyte preparations where various manipulations are more easily achieved. One other study to date (personal communication with Wenjing Jiang) examined the presence of p44/42 MAP kinases in feline isolated myocytes. They also found that the isolation procedure 170 produced complete phosphorylation of the available p44/42 MAP kinase. Since all of the p44/42 MAP kinase was phosphorylated during the collagenase isolation procedure, these values serve as a measure of total p44/42 MAP kinase in the cells. In examining these data, it was observed that the MAP kinase pool was significantly larger in the susceptible animals, which may account for the greater activation seen in the susceptible dogs. From these data one may conclude that susceptible animals have an increased Pz- AR. response leading to an increase in contractility, increase in heart rate, increase in single cell twitch amplitude as well as as an increase in MAP kinase activity. Since the p 2-AR number and the Kd value appear to be similar in both groups the reasons for a larger response in the susceptible animals is not clear. A report by Turki et al. (69) points to the possibility of a Pz-AR heterogeneity in the human population. Four different polymorphisms at position 16,27,34 and 164 have been detected. In fact, the He-164 variant has a threonine amino acid substituted for a He at site 164. The results of this substitution is a substantial uncoupling from Gs. Transgenic mice overexpressing the He 164 Pz-AR show diminished adenylate cyclase activity, heart rate, and dP/dt with and without the presence of isoproterenol. The He 164 mutant showed similar results as their non-transgenic littermates. Although this dysfimction. associated with the He 164 mutant may be detrimental in pathophysiological states such as heart failure where the pumping efficiency of the heart becomes largely dependent on the pz-AR, it may appear to be beneficial in the onset of arrhythmias. Therefore, there exists the possibility that the susceptible and resistant animals are harboring different polymorphisms in their receptors. However, it would have been expected that differences in contractility would 171 have been noted in the animals prior to infarct. Although, it is possible that these differences may become “unmasked” during the remodeling process post-infarct. 4.4 Study Limitations Past experience with this animal model indicates that the dogs that are susceptible to ventricular fibrillation have a significantly larger surgically induced anterior wall infarct than those found to be resistant to VF. In the present study, the infarct size was not measured. It is possible that the larger more diffuse infarct may contribute to the susceptibility of the animals. However, since the enhanced P 2-AR was seen at the level of the intact heart (both the ventricles and the atria) as well as the single cell the role of the P 2-AR must also be considered as a potential variable in the induction of ventricular fibrillation. 4.5 Future Studies Future studies will need to focus on finding more differences between the two groups of animals in regards to the P 2-AR. Specifically, the possibility that there exists two different isoforms of the P 2-AR must be examined. The presence of the two different isoforms may help explain the existence of the different populations of animals seen post infarction. Studies are also currently underway to determine the coupling of the P 2-AR in the two groups of animals. It is of particular interest to determine if the susceptible animals couple more strongly to Gs while the resistant animals couple to Gi. 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Changes in contractility take into account both the rate and amplitude of shortening of the muscle fibers. Analyzing contractihty at the level of the intact heart is elusive at best. Common measures of ventricular function (eg. Ejection fraction, cardiac output, and fractional shortening) are often load dependent or only focus on one of the two variables: rate or amplitude. Clinically, the velocity of circumfirential fiber shortening (Vcf) is the preferred measure of contractility in the intact heart. Vcf also takes into account the extent of wall shortening as well as the velocity at which this was achieved. It is a non-invassive measurement that utilizes M-mode echocardiography. It is believed to be load independent although with examination of the below equation one could argue against this. However, in human studies it was foimd that acute alterations in preload and afterload did not alter the Vcf value (26). The Vcf value is calculated according to the following equation: LVIDd-LVIDs/LVrod*ET The above value is corrected for heart rate by dividing by the square root of the R-R interval taken from a simultaneously collected electrocardiogram. LVIDd is the left ventricle internal diameter during diastole (in cm), LVIDs is the left ventricle internal diameter during systole (in cm) and ET is the ejection time taken from the opening and closing of the aortic valve in seconds. Figures A. 1 and A.2 are representative M-mode 182 echocardiograms taken, from a cross-sectional view of the left ventricle and the aortic valve, respectively. These figures indicate where the above measurements were taken. The units for the Vcf value are arbitrary. The mathematical calculation prior to the heart rate correction leaves seconds**. However, few clinicians use this unit. The unit is generally expressed as circumferences/second or it is dropped altogether. In the clinic setting, physicians are more concerned with changes in contractility rather than the absolute V cf value (26). As mentioned previously, there is not one definitive measure of contractility at the level o f the intact heart. The velocity of circumferential fiber shortening was utilized in this set of experiments because it takes into account both the rate and amplitude of shortening but moreover due to its non-invassive technique. 183 Figure A. 1. Representative M-mode echocardiogram of the left ventricle demonstrating the LVIDd and LVIDs. 184 Figure A.2. Representative M-mode echocardiogram demonstrating the ejection time as measured from the aortic valve. 185