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Roles of Sarcoplasmic Reticular CA2+ -ATPase 2a and Action Potential Duration in Rat Normal and Hypertrophied Myocardium

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Title:
Roles of Sarcoplasmic Reticular CA2+ -ATPase 2a and Action Potential Duration in Rat Normal and Hypertrophied Myocardium
Creator:
TAYLOR, DAVID GLENN ( Author, Primary )
Copyright Date:
2008

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Action potentials ( jstor )
Animal models ( jstor )
Calcium ( jstor )
Dogs ( jstor )
Heart ( jstor )
Heart rate ( jstor )
Hypertension ( jstor )
Hypertrophy ( jstor )
Myocardium ( jstor )
Rats ( jstor )

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University of Florida
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University of Florida
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Copyright David Glenn Taylor. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2006
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72809768 ( OCLC )

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ROLES OF SARCOPLASMIC RETICULAR CA2+-ATPASE 2a AND ACTION POTENTIAL DURATION IN RAT NORMAL AND HYPERTROPHIED MYOCARDIUM By DAVID GLENN TAYLOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 By David Glenn Taylor

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I dedicate this work to my family, especially my parents Glenn and Vicki Taylor, and to my wife, Michelle, who have always given their love, support, and wisdom.

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ACKNOWLEDGMENTS First, I would like to acknowledge the Creator and Sustainer of all things, for this opportunity and the strength and support to accomplish the goal. To unlock one mystery and find ten more underneath is inspiring and humbling. I cannot thank Dr. Harm Knot enough for the honor and privilege of beginning my career in his laboratory and under his mentorship. Dr. Knot has been a kind, supportive, intelligent, and gifted example of what a scientist should yearn to be. Serving in his laboratory has been an education and a pleasure. I count him among my friends and look forward to being viewed as his colleague. Next I must thank Dr. Leonard Parilak, who served as my teacher and partner during this and many other projects. When the consequence of this work is widely accepted, our names will be placed together; and I would not have it any other way. A study of this breadth represents the teamwork of modern science. To those who contributed their sweat, I thank you—Dr. Yegia Song, Dr. Christopher Davis, Zhao Han—for your contributions. This work would not have been possible without the teaching and aid of many others: Dr. Yagna Jarajapu, Dr. Jeffrey Harrison, Dr. Sara Holmberg, Jason Gersting, Dr. Melissa Fleegal, Dr. Meg Davis, and the laboratories of Dr. M. Ian Phillips and Dr. Michael Katovich. I greatly appreciate the contributions of my committee—Drs. Stephen Baker, Maria Grant, Daniel Pauley, and Wilmer Nichols. iv

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I thank my family and friends, both in Mississippi and in Gainesville, who always believed in me. v

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABBREVIATIONS USED THROUGHOUT THIS TEXT............................................xiii ABSTRACT.....................................................................................................................xiv CHAPTER 1 INTRODUCTION........................................................................................................1 Epidemiology................................................................................................................1 The Aging Population............................................................................................1 Increased Prevalence of Peripheral Hypertension.................................................1 The Normal Cardiac Cycle...........................................................................................2 Systolic Contraction..............................................................................................2 Diastolic Relaxation..............................................................................................4 Force-Frequency Relationship...............................................................................5 Cardiac Hypertrophy....................................................................................................7 Cardiac Physiology of Aging and Hypertension........................................................10 Aging...................................................................................................................10 Hypertension........................................................................................................12 Rationale and Hypotheses...........................................................................................15 Rationale..............................................................................................................15 Hypotheses..........................................................................................................16 2 MATERIALS AND METHODS...............................................................................20 Background of Animal Models..................................................................................20 Experimental Animals................................................................................................21 Characterization of In Vivo Cardiovascular Parameters.............................................21 Measurement of Blood Pressure..........................................................................21 Measurement of Left Ventricular Function.........................................................22 Assessment of Cardiac Hypertrophy...................................................................22 In Vitro Isolated Muscle Experiments........................................................................23 Tissue Preparation...............................................................................................23 vi

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Forceand Ca2+-Frequency Relationship (FFR, CaFR)......................................24 Drug Studies........................................................................................................24 Post-Rest Potentiation (PRP)...............................................................................24 Quantification of Gene Expression.............................................................................25 Polymerase Chain Reaction (mRNA of SERCA2a and PLB)............................26 Immunoblotting (SERCA and PLB Protein).......................................................26 Measurements of Action Potential and L-Type Ca2+ Current....................................27 Statistical Analysis......................................................................................................28 3 QUANTIFICATION OF THE RAT LEFT VENTRICLE FORCEAND CA2+-FREQUENCY RELATIONSHIPS: SIMILARITIES TO DOG AND HUMAN......31 Introduction.................................................................................................................31 Animal Model.............................................................................................................33 Results.........................................................................................................................33 Systolic Force Frequency Relationships in Rat, Dog, and Human.....................33 Force and Calcium-Frequency Relationships in Rat LV Muscle........................34 Kinetics of Force and Ca2+ Transients as a Function of Frequency in Rat LV Muscle..............................................................................................................35 Effect of Isoproterenol and Cyclopiazonic Acid on the FFR, Ca2+, and Diastolic Force................................................................................................................36 Mechanistic Investigations..................................................................................36 Discussion...................................................................................................................37 Comparison of the FFR in Rat to that in Dog and Human..................................37 Kinetics of Force and Ca2+Transients as a Function of Frequency in Rat LV Muscle..............................................................................................................39 Pharmacological Effects on the Rat LV FFR......................................................39 Mechanistic Investigations/Limitations and Perspective....................................40 Conclusions.........................................................................................................42 4 ELECTRICAL CONTROL OF FORCE PRODUCTION IN NORMAL MYOCARDIUM: A SYNCHRONIZED ROLE FOR ACTION POTENTIAL DURATION AND CALCIUM PUMP ACTIVITY...................................................51 Introduction.................................................................................................................51 Animal Model.............................................................................................................52 Results.........................................................................................................................52 Forceand Ca2+Frequency Relationship...........................................................52 APDand ICa,LFrequency Relationship.............................................................52 Frequency Dependence of SERCA2a Activity...................................................53 Discussion...................................................................................................................53 5 ALTERATIONS IN CARDIAC FREQUENCY-AUGMENTED CONTRACTILITY AND CALCIUM-HANDLING PROTEINS DURING ADULT AGING.................62 Introduction.................................................................................................................62 Animal Model.............................................................................................................62 vii

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Results.........................................................................................................................62 Cardiovascular Parameters..................................................................................62 LV Contractility with Aging...............................................................................62 Ca2+-Handling With Aging..................................................................................63 Molecular.....................................................................................................63 Heart rate potentiation of SERCA activity...................................................64 Discussion...................................................................................................................64 Cardiovascular Parameters..................................................................................65 LV Contractility With Aging...............................................................................65 Ca2+-Handling with Aging..................................................................................66 Molecular.....................................................................................................66 Heart rate potentiation of SERCA activity...................................................67 Proposed Sequence/Role of APD........................................................................68 Early adult aging..........................................................................................68 Late adult aging............................................................................................68 APD and Cellular Hypertrophy...........................................................................69 Conclusions.........................................................................................................70 6 ELECTRICAL CONTROL OF FORCE PRODUCTION IN HYPERTROPHIED MYOCARDIUM: ROLES OF ACTION POTENTIAL DURATION AND CALCIUM PUMP ACTIVITY..................................................................................75 Introduction.................................................................................................................75 Animal Model.............................................................................................................76 Results.........................................................................................................................76 Cardiovascular Parameters..................................................................................76 Forceand Ca2+Frequency Relationships..........................................................76 APDand ICa-Frequency Relationships...............................................................77 Frequency Dependence of SERCA2a Activity...................................................78 SERCA and PLB Expression..............................................................................79 Discussion...................................................................................................................80 Cardiovascular Parameters..................................................................................80 Forceand Ca2+Frequency Relationships..........................................................80 APDand ICa-Frequency Relationships...............................................................80 Frequency Dependence of SERCA2a Activity...................................................81 SERCA and PLB Expression..............................................................................82 Contrast to Hypertrophy with Aging...................................................................82 Potential Mechanisms of Enhanced ICa...............................................................83 Consequences for SERCA Gene Therapy...........................................................83 Conclusions.........................................................................................................84 7 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................92 Conclusions.................................................................................................................92 Future Directions......................................................................................................102 viii

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REFERENCE LIST.........................................................................................................104 BIOGRAPHICAL SKETCH...........................................................................................118 ix

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LIST OF TABLES Table page 2-1. Nucleotide sequences of primers used in competitive PCR.......................................30 3-1. Amplitude and Shape of the FFR among species.......................................................44 3-2. Comparisons of amino acid sequences of major cardiac Ca2+-handling proteins in rat, human, and dog........................................................................................................50 5-1. Cardiovascular and hypertrophy parameters in aging WKY rat................................71 6-1. Cardiovascular and hypertrophy parameters of 2-month and 9-month WKY and SHR rats............................................................................................................................85 x

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LIST OF FIGURES Figure page 1-1. Typical ventricular cardiac action potential...............................................................18 1-2. Calcium ion movements during systole and diastole in cardiac muscle cells............19 2-1. Post-rest potentiation behavior in myocardium..........................................................29 3-1.Systolic force frequency relationships in rat, dog, and human....................................43 3-2. Forceand calcium-frequency relationships in rat LV muscle...................................45 3-3. Kinetics of force and Ca2+ transients as a function of frequency in rat LV muscle..47 3-4. Effect of isoproterenol and cyclopiazonic acid on the FFR, Ca2+, and diastolic force..48 3-5. Effect of time on SERCA activity..............................................................................49 4-1. Systolic forceand calcium-frequency relationships in isolated rat papillary muscle.57 4-2. Action potential duration and integral of Ca2+ current in rat ventricular myocytes as a function of stimulation frequency............................................................................58 4-3. Force and Ca2+ relaxation velocities in isolated rat papillary muscle........................59 4-4. Post-rest potentiation in isolated rat papillary muscle................................................60 5-1. Systolic force-frequency behavior in aging rat myocardium.....................................72 5-2. SERCA expression and activity in aging rat LV........................................................73 6-1. Systolic force-frequency relationships in isolated rat papillary muscle....................86 6-2. Representative forceand calcium-frequency relationships in 9-month-old WKY and SHR LV muscle from 0.2 to 6 Hz over 300 ms from stimulation pulse..................87 6-3. Action potential duration (to 80% repolarization) and integral of Ca2+ current in rat ventricular myocytes as a function of stimulation frequency...................................89 xi

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6-4. Measures of relative SERCA activity in hypertrophied and control in rat LV muscle.90 6-5. Expression of SERCA and pentameric PLB protein..................................................91 xii

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ABBREVIATIONS USED THROUGHOUT THIS TEXT [Ca2+]I Cytosolic Ca2+ concentration APD Action Potential Duration. Length of time myocyte is depolarized during contraction cycle. CamKII Ca2+/calmodulin-dependent kinase II. Regulator of several intracellular Ca2+-handling processes, including SERCA. CPA Cyclopiazonic Acid. Commercially available specific SERCA inhibitor. FFR Systolic Force-Frequency Relationship. ICa Inward Ca2+ Current through voltage-gated L-Type Ca2+ channels. KH Krebs-Henseleit solution. The physiological saline solution in which in vitro functional experiments were performed. LV Left Ventricle. S-D Sprague-Dawley Rat. Ubiquitous outbred laboratory rat model. SHR Spontaneously Hypertensive Rat. SERCA Sarcoplasmic (Endoplasmic) Reticular Ca2+-ATPase 2a. PLB Phospholamban. Inhibitor and regulator of SERCA activity. PRP Post-rest potentiation. (See Chapter 2). WKY Wistar Kyoto Rat. Normotensive control for the SHR. xiii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLES OF SARCOPLASMIC RETICULAR CA2+-ATPASE 2a AND ACTION POTENTIAL DURATION IN RAT NORMAL AND HYPERTROPHIED MYOCARDIUM By David Glenn Taylor August 2004 Chair: Harm Knot Major Department: Pharmacology and Therapeutics Myocardial force increases with stimulation rate from ~1-3 Hz (positive Force-Frequency Relationship, FFR) in larger mammals. The underlying mechanisms are Ca2+-related but not completely understood. I hypothesized that the rat is an appropriate mammalian cardiac model. Further, I hypothesized that sarcoplasmic reticulum (SR) intracellular Ca2+ stores work with extracellular Ca2+ influx (ICa) to provide contractile Ca2+ and that alterations in these mechanisms underlie the pathology of hypertrophy. Despite their scientific ubiquity, almost all cardiac research performed in small rodents and extrapolated to humans has been questioned because, despite similar contractile proteins, rodents traditionally do not display a positive FFR. The first set of experiments support the rat model and suggest that previous observations are due to a preponderant response to dissection trauma in rat than larger mammals. These results also further quantify the role of SR Ca2+-ATPase (SERCA) in the FFR. xiv

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The next set of experiments demonstrates that increased frequency lengthens the action potential duration (APD) and augments ICa. By increasing Ca2+-induced Ca2+ release over the range of the positive FFR, APD and ICa provide a link between increased SERCA-based SR Ca2+ load and systolic cytosolic Ca2+. A further set of experiments examines the roles and functions of SERCA, APD, and ICa in cardiac hypertrophy. During hypertrophy with aging, SERCA expression and activity decline, reducing FFR positivity. It appears the APD prolongation that accompanies senescence is a late electrical adaptation to maintain contractility despite this reduction. Peripheral hypertension induces cardiac adaptations believed to end in SERCA and systolic force depression. However, during the compensatory hypertrophy stage, SERCA expression is maintained. The myocardium adapts by early electrical remodeling, increasing basal APD and ICa to increase systolic force at lower frequencies. In summary, the two largest sources of systolic Ca2+—SERCA and ICa through the APD—work synergistically to produce greater developed force when metabolic demands increase. During aging, SERCA activity decreases due to both loss of protein and altered control mechanisms, and the FFR decreases. The APD prolongs to compensate. During compensation to peripheral hypertension, the APD prolongs at lower frequencies, yielding greater developed force with no change in SERCA expression. xv

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CHAPTER 1 INTRODUCTION Epidemiology The Aging Population In the United States, the proportion of persons aged 65 years of age or older is expected to increase from 12.4% in 2000 to 19.6% in 2030 or from 35 million people in 2000 to a projected 71 million people in 2030.1 Advanced age is a major risk factor for lethal infarction,2 arrhythmias,2 angina,2 and systolic2 and congestive heart failure.3 Whether this relationship is due to the natural changes during the aging process itself, those changes lowering tolerance for pathological stimuli, or if longer life merely represents longer exposure to other risk factors with little basal change remains debated.3 Although intensive and substantial research is being performed on the cardiac effects of aging, most studies concentrate on opposite extremes of the aging spectrum—the young and the very old. Little is known about the continuum of alterations and adaptations that accompany aging. Data at intermediate ages, as well as controlled longitudinal studies that remove as many risk-factor variables as possible, are noticeably lacking, making correlations to human conditions difficult. Increased Prevalence of Peripheral Hypertension Hypertension is broadly defined as the state of peripheral systolic blood pressure (SBP) of 140 mmHg or higher and/or diastolic pressure of 90 mmHg or higher4 and is a risk factor for infarctions5 and strokes,6 systolic7 and congestive heart failure,8 and 1

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2 retinal9 and renal disease.10 The risk of cardiovascular pathology is proportional to both the magnitude5 and duration11 of blood pressure elevation. SBP of 140-159 mmHg is classified as Stage 1, and 160 mmHg and above as Stage 2.12 In the United States alone, 23% of adults have hypertension and the percentage is rising. Of those affected, ~one-third (30%) are unaware of their condition, one-quarter are taking insufficient or ineffectual medication, and ~one-tenth (11%) are aware, but taking no medication.4 Despite large numbers of people living with elevated blood pressure, few studies have been done observing the cardiac effects of long-term, untreated hypertension. Most studies examining cardiac conditions instead rely on quicker alternate methods, such as genetically predisposed transgenic animal models, drugs, or surgeries, rather than “pure” systemic hypertension. Transitional phases are usually neglected and their particularities unknown. The Normal Cardiac Cycle Systolic Contraction The initiation of cardiac contraction is normally an electrical process (the action potential). Prior to the electrical stimulus, ventricular cardiomyocytes have a resting membrane voltage of ~-90 mV, largely created by the electrogenic Na+/K+-ATPase (which pumps three Na+ ions extracellularly for each two K+ ions brought into the cell13) and selective permeability of the plasma membrane to outward K+ current14 (action potential Phase 4). During the action potential originating in the sinoatrial node, the wave of depolarization flows through the myocardium, raising the membrane potential and opening some or many voltage-gated Na+ channels.14 At ~-70 mV the Na+ influx becomes self-sustaining, and the cell transiently depolarizes to ~20-40 mV13;14 (Phase 0). In most mammals, the transient outward K+ channel opens, releasing K+ ions (ITO)14 and

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3 creating a brief repolarization to ~20 mV13;15 (Phase 1). This is followed by the Phase 2 “plateau,” a period of balance between Ca2+ entry (ICa) through voltage-gated Ca2+ channels (which begin to open at ~-30 mV and open more slowly than the fast Na+15), and K+ exit through several types of channels14 (during which the cell maintains a declining voltage of 0-20 mV13;15). The voltage-gated Ca2+ channels are inactivated by elevated intracellular Ca2+ concentrations16 from the ryanodine receptor (see below) and subsequent cellular repolarization, leaving the outward K+ current, the Na+/K+ pump, and actions of other, more minor process (such as the plasma membrane Ca2+-ATPase) to repolarize the cell and complete the action potential cycle (Phase 3) (Figure 1-1). The process of raising cytosolic Ca2+ concentration that serves as the first step of the biochemical stage of cardiac contraction begins with Phase 2 (plateau) of the action potential. During normal diastole, [Ca2+]i lies at 100-300 nM, but rises to 1 M during systole.17;18 Extracellular Ca2+ (ICa) enters the cell through voltage-gated L-type Ca2+ channels (dihydropyridine receptors, DHPRs) located in the T-tubules.19 This Ca2+ is usually insufficient to begin contraction,19 but can contribute to developed force. Rather, this Ca2+ functions as a “trigger” for the process of calcium-induced calcium release. Local Ca2+ concentrations immediately near the DHPR rise rapidly, activating the closely located Ca2+-sensitive ryanodine receptor (RyR), the Ca2+ channel of the sarcoplasmic reticulum (SR). The RyR opens, and stored SR Ca2+ enters the cytoplasm. It is the common pool from these two Ca2+ sources (extracellular and SR) that instigate contraction (Figure 1-2). The extremely close geometric arrangement of the Ca2+ channel-RyR structure (dyad) is essential for proper contraction.20

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4 Mechanical contraction of cardiac myocytes occurs by the movement of the hinged myosin molecule (attached to the M-line in cardiomyocytes), along the thin filaments of actin (attached to the Z-line in cardiomyocytes). The myosin complex contains myosin ATPase in its globular head. In cardiac muscle, along the actin filaments lie the tropomyosin helixes and, at regular intervals, the troponin protein complexes. Troponin is made of three subunits—troponin I (TN-I), which inhibits ATP hydrolysis of myosin; troponin C (TN-C), which regulates troponin I; and troponin T (TN-T), a structural component.13;19 TN-C is regulated by the binding of Ca2+, with a Kd of roughly ~1 M, although affinity is related to myosin/actin crossbridge formation.18 Upon Ca2+ binding to TN-C, the myosin inhibition of TN-I is itself inhibited, and stacks of myosin molecules “walk” along actin, contracting the cell. [Ca2+]i required for myosin activation depends on many factors that influence myofilament Ca2+ sensitivity; but often begins at ~300 nM18;19 free calcium, although the true required Ca2+ input is much higher due to many Ca2+ buffering factors in the cell.17 Above this threshold, peak [Ca2+]i is directly and steeply related to peak systolic force production.18 The relative contributions of the two major sources of contractile Ca2+—ICa and SR—vary by species; there is a greater contribution of SR Ca2+ in the phylogenetically lower animals. In rabbits, the ratio is approximately 29% ICa/70% SR; in rats, 7% ICa/92% SR.17 Diastolic Relaxation Cardiac muscle relaxation is caused by removal of systolic Ca2+ from the cytosol, by removal from the cell or sequestration into the organelles. The largest component is

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5 returned to the SR, for “recycling” into the next contraction, by the action of the SR Ca2+-ATPase SERCA2a (SERCA). SERCA is regulated largely by its inhibitor phospholamban (PLB). Upon phosphorylation at Thr-17 by Ca2+/calmodulin-dependent kinase II (CamKII) and/or Ser-16 by protein kinase A,21 PLB dissociates from SERCA, lowering the Km of SERCA.19;21 SERCA itself can also be phosphorylated by CamKII, raising its Vmax, rather than altering its Ca2+ sensitivity.22 The vast majority (~7% in rat17) of ICa Ca2+ is removed by the Na+/Ca2+ exchanger, in a 3 Na+:1 Ca2+ ratio18. The remainder (1-2%) is removed by the plasma membrane Ca2+-ATPase or sequestered into the mitochondria17 (Figure 1-2). Force-Frequency Relationship In most mammals, cardiac systolic (“developed”) force increases with an increase in stimulation frequency, independently of neurohumoral control, a phenomenon known as the treppe effect or positive force-frequency relationship (FFR).23 In humans, this occurs between ~1 Hz (60 bpm) and ~3 Hz (180 bpm).24 The FFR allows increased cardiac output through stroke volume despite a reduction in endsystolic volume of the left ventricle (LV) and the resulting shift in the Frank-Starling relationship that occurs with higher heart rates.25 This relationship can be demonstrated in vivo by quantifications of the pressure-over-time differential (dP/dt) versus heart rate in the cardiac catheterization laboratory and fractional shortening by echocardiography. The FFR is also demonstrated in vitro as force generation in muscle strips and fractional shortening in isolated myocytes.26 Pieske et al.27 reported that the FFR of human non-failing myocardium results from frequency-dependent increases in the intracellular calcium transients. Additional supporting evidence among other mammals comes from cat papillary muscle28 and ferret

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6 ventricular myocardium.29 The molecular basis of the treppe effect has been attributed to a net gain of circulation of Ca2+ in the cell and increased calcium-induced calcium release, by either increased Ca2+ entry through the L-type Ca2+ channels and/or a reduction in Na+/Ca2+ exchanger activity during diastole due to more numerous depolarizations per unit time,30 both coupled to greater capturing and Ca2+ loading of the sarcoplasmic reticular Ca2+ stores by enhanced SERCA activity.25 The positive ascending limb of the force-frequency relationship is likely caused in part by higher SERCA activity due to increased phosphorylation of phospholamban and SERCA by Ca2+/calmodulin-dependent kinase II at rates above 1 Hz.31-34 Although SERCA levels/activity are a large determinant of absolute force development, phospholamban level/phosphorylation state has been theorized to be the “gain,” or determining factor of the FFR slope. Meyer et al.35 and Koss et al.36 proposed that the ratio of PLB/SERCA2a plays an important role in shaping the ascending limb of the FFR as part of an overall increase in Ca2+ cycling underlying enhanced force production. However, this may only be the case in near-normal stoichiometry, which may not exist in certain disease states. If PLB expression rises greatly, SERCA inhibition will probably be constant despite phosphorylation of a large fraction of PLB. Because it reflects so many basic contractile elements, an altered FFR is a commonality among disease states.25 Generally, it becomes flatter (less force augmentation with stimulation rate) as pathology progresses, with a great loss of basal developed force characteristic of systolic heart failure. A depressed FFR is both diagnostic, as +dP/dt and fractional shortening in vivo,26 and symptomatic, as exhaustion upon exertion.25

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7 The roles of molecular and electrical Ca2+-handling in the normal FFR will be explored in Chapter 3 (SERCA activity) and Chapter 4 (action potential and ICa). Cardiac Hypertrophy Cardiac hypertrophy, the enlarging of the heart or, most commonly, a discrete region such as a ventricle, is a common characteristic of the diseased heart due to many types of pathological insults.37 Although cardiac hypertrophy is a benign consequence of aerobic exercise, it is also an adaptation to a wide variety of pathological insults, including hypertension,38 ischemia,39 mitral regurgitation,40 and septal defects.41 For reasons not yet fully understood, exercise-induced hypertrophy appears to be almost entirely beneficial,41 but hypertrophy due to pathology is the leading risk factor for eventual heart failure.37 This difference may be partly due a greater shift from the to the form of myosin in pathological hypertrophy.42 Humans with left ventricular hypertrophy (by echocardiography) suffer a far greater risk of infarction, chronic congestive heart failure, and stroke and have much higher mortality with cardiac events.39 LV hypertrophy is a greater predictor of cardiovascular death than blood pressure, cigarette smoking, or total cholesterol level.39 Current theories state that hypertrophy is an adaptation to increased cardiac demand (e.g., pressure overload),43 wall stress (e.g., volume overload, pressure overload),44;45 or weakened myocardium (e.g., infarction).46 The “weak” heart responds by enlarging cardiomyocytes in both length and cross-sectional area to control rising wall stress and build more myofibrils to produce greater force. Also, greater LV thickness normalizes systolic wall stress, maintaining normal end-systolic volume and ejection fraction.47 This begins the state of compensatory hypertrophy, although larger cell mass requires more

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8 oxygen which the coronary arteries may eventually not be able to provide, weakening the heart further.48 Frequently, extracellular collagen deposits are increased, which sacrifices elasticity for strength.45 Also, the cytoskeletal protein titin may shift from the N2BA isoform to the less elastic N2B isoform.49 Thus it appears there are at least two stages of pathological hypertrophy: compensatory, in which the myocardium undergoes changes to adapt to the pathological stimulus; followed, if the stimulus is not corrected, by decompensatory, in which the previous changes adversely affect heart function.38 Cellular hypertrophy is an end result of many interconnected and still not fully-elucidated pathways that reactivate developmental genes50 and instigate hypertrophy, including protein kinase C (PKC),51 extracellular signal-regulated protein kinases (ERK),52 and mitogen-activated protein kinases (MAPK) cascades,50 and that can be activated by either ligands or mechanical stresses in the myocardium. A key thread is the renin-angiotensin-system (RAS). The heart local RAS is activated by hemodynamic overload, and antagonists to the angiotensin II (ANGII) type-1 receptor can reduce hypertensive hypertrophy.52-54 The PKC family contains several isoforms that are activated by various stimuli. ANGII appears to induce hypertrophy through this class of molecules.52 PKC can be activated by increases in Ca2+ by either an increased number of DHPRs55 or by elevated sustained [Ca2+]i through the Ca2+ sensitive phosphatase calcineurin.56;57 PKC is activated by stretch.51 However, activation of the PKC53 and calcineurin58 pathways have also been implicated in reduced SERCA expression. Catecholamines, especially norepinephrine, are powerful stimulants of hypertrophy, although the pathways are as yet unclear. PKC is activated by

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9 1-adrenoceptor activation, and consistent -adrenoceptor stimulation induces protein kinase A to activate ERK.52 Hypertrophy is generally divided into two major categories. In concentric hypertrophy, ventricular volume is reduced as the wall thickens and grows inward. This can include diastolic dysfunction as the thick walls may be less compliant, however ejection fraction (EF), the percentage of the blood entering the LV during diastole that is ejected during systole, may be preserved because total diastolic volume is less.59 Heart volume expands in eccentric hypertrophy. This is the form induced by exercise.37 However, in certain pathologies, the cardiomyocytes become as thick as metabolically possible but continue to grow longer,37;60 most likely due to diastolic wall stretch.37 Ventricular volume continues to grow, but the wall is remodeled and can no longer contract properly.37 This may be a point in the transition to “dilated” hypertrophy, in which ejection fraction is seriously compromised.37 For example, the Spontaneously Hypertensive/Heart Failure rat (SHHF), a substrain of the Spontaneously Hypertensive Rat (SHR), enters failure about 6 months earlier than the SHR, an attribute attributed to reaching maximum thickness at an earlier age but continuing to grow linearly.60 Heart failure (HF), a potentially lethal endpoint for hypertrophy, is a varied clinical syndrome with many symptoms37 representing the roles of the heart. Classic systolic heart failure is defined as reduced ejection fraction (typical definitions: normal 65-80%,61 failure 40%62) and diminished contractility.59 Low cardiac output weakens the body. There is an upper limit for cross-sectional area of cardiomyocytes, beyond which they grow only in length. At the endpoint of eccentric hypertrophy, the ventricular walls thin and lose their ability to contract properly, or even hold their structure against wall stress

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10 (dilated hypertrophy). Congestive heart failure is a chronic and sometimes fatal diagnosis based on a combination of criteria, including pulmonary edema, jugular venous distention, high venous pressure, ankle edema, dyspnea on ordinary exertion, and tachycardia.63 The ventricle fails to receive blood at sufficiently low pressures to allow proper diastolic filling, because of a reduced ventricle volume (i.e., concentric hypertrophy) and/or lack of compliance due to slowed relaxation, fibrosis, or thick walls. Fluid accumulates in the lungs and interstitial tissues.59 Low renal perfusion due to low cardiac output stimulates renin release and activates the renin-angiotensin-altosterone system, leading to fluid retention that contributes to further congestion.37;48 Cardiac Physiology of Aging and Hypertension Aging Senescence is accompanied by cellular hypertrophy,2;64 but no increase in LV wall thickness. This discrepancy is probably due to age-related loss of cells and hypertrophy of the remainder.64;65 Arteries, including the aorta, become stiffer and less compliant,66 leading some investigators to propose that in senescence a portion of the force of blood flow is reflected back into the ventricle as a potentially hypertrophy-inducing hemodynamic stress.64 Other known cardiovascular changes include an increase in sympathetic nervous system output66 and circulating catecholamines (notably norepinephrine67), but reduced sensitivity to -receptor stimulation (due to reduced downstream activation of adenylyl cyclase).65;66 Senescent myocytes are also more susceptible to Ca2+ overload, including arrhythmia and apoptosis.65;68 The relaxation phase of the contraction cycle is prolonged.69 A potential cause of slowed relaxation is slowed systolic Ca2+ extrusion, probably due to reduced SERCA expression/activity. SERCA message was greatly reduced in 30

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11 month-old rat ventricles compared to young adult,70 and expression was significantly reduced in 26-month-old.71 However, some studies have shown no change in SERCA or phospholamban expression in ~27-month-old rats,22 instead observing alterations in SERCA activity71 and SERCA and PLB phosphorylation states.22 These two results are not mutually exclusive, but may be based on choice of animal model and methodology. SERCA activity is closely tied to both its expression and phosphorylation state and the expression and phophorylation state of PLB, in a ratio.72 Insignificant changes in SERCA or PLB expression may become quite significant if measured as a ratio. The ratio of PLB:SERCA was shown to be very elevated with advanced aging by Lim et al.73 The roles of expression vs. ratio in aging require a longitudinal study to examine more fully.74;75 At low basal rates76 and higher 71 frequencies, ability of the myocardium to develop systolic force is not reduced with advancing age. Unfortunately, myocardial systolic force-frequencies relationships with aging have not been studied in detail. However, some studies have been done by other methods in animal models, and they have shown either a sustained or normal FFR in advanced aged animals compared to young adult. Rumberger et al.77 observed a downward shift in the frequency of maximum systolic force in 38-month-old guinea pigs, but the myocardium was able to produce the same basal force. In isolated LV myocytes from 34-month-old mice, Lim et al.76 observed no change in cell shortening or [Ca2+]i over the frequency range from 2-10 Hz. No change in LV systolic pressure or +dP/dt was observed by pressure transducer in the chest of 26-month-old rats.71 However, this study was in live animals at their normal pacing

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12 frequency (4-7.5Hz78), which in rats is far beyond the normal activation range of CamKII (1-4 Hz79), which appears to control the FFR within the range of larger mammals. Along with slowed relaxation, the contractile phase of the contraction cycle also lengthens. This relative prolongation is not eliminated even by near-depletion of catecholamines by 6-hydroxydopamine or blockage by propanolol,69 suggesting an innate change in the muscle cells rather than extracellular factors. Another fundamental change with senescence is the prolongation of the action potential duration (APD), probably the result of a decline in the transient outward potassium current (ITO), with an accompanying increase in ICa,L.80 Because varying animal models (e.g., rat, mouse, guinea pig, and human observations) have different lifespans, correlations between relative age and its effects on cardiac physiology among different models are difficult. Also, most studies compare young, healthy animals with senescent animals, at which time many changes have already occurred (e.g., cellular hypertrophy, APD prolongation, SERCA expression decline, depressed FFR). The sequence of events and their possible consequences on the others are largely unknown. To my knowledge, no previous study has investigated cellular alterations, major Ca2+ handling mechanisms, and contractility in a longitudinal fashion, examining the major stages in a consistent animal model. I perform such a study in Chapter 5. Hypertension When the heart pumps against a greater resistance, the body responds to the lowered cardiac output and flow to increase systolic force. Initially, neurohumoral factors work to quickly increase cardiac output, through such mechanisms as increased sympathetic tone, but can remodel the heart over the long-term. Chronic elevated

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13 sympathetic drive can also contribute to hypertension via vasoconstriction,81 increased stroke volume,82 and fluid retention83 and activates hypertrophic pathways. Also, increased afterload activates stretch receptors, which begins expression of hypertrophic and fetal development genes.84 Many of the known physiological changes that occur in the aging heart, including cellular hypertrophy, prolonged contraction and relaxation, and reduced -adrenergic response,47 also occur with peripheral hypertension, albeit more quickly, leading many investigators to view aging and disease as stimuli for a common adaptation.47;85 In experimental hypertension, maximum developed force and Ca2+:force ratio are unchanged.47 Unlike aging, the cellular hypertrophy of peripheral hypertension is not compensated by apoptosis, so the entire LV enlarges. Pressure-overload is probably the most-studied model of hypertrophy, and most information has come from this model. Although aging with hypertension is the most relevant for comparison to human pathology, many researchers substitute induced pressure-overload models that yield pathology more quickly, such as aortic banding which, in truth, better replicates aortic stenosis. Aging and pressure-overload also share similar molecular events. Notable is a reduction in SERCA expression and activity;86 although in the case of pressure-overload, most studies have been performed in decompensatory or HF models, where developed force is lessened. Thus the time course of SERCA reduction is unknown. During compensatory hypertrophy due to renal artery banding, there is no change in SERCA or PLB concentration.42 In moderate hypertrophy due to rat aortic banding, SERCA mRNA was not decreased, but its rate of Ca2+ uptake was reduced.47;86 Overexpression of

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14 SERCA in failing aortic-banded rats improved in vivo systolic function to near-normality.87 In human dilated cardiomyopathy, which has a flattened shape, the slope of the FFR is inversely related to SERCA concentration.88 In hypertensive hypertrophy, the APD prolongs, also probably due to a decline in ITO, and ICa,L integral increases accordingly.47 Whether DHPR density increases with hypertrophy appears to depend upon the model—unchanged in aortic constriction, but elevated in renal hypertension.47 Ahmmed et al.89 attributed APD prolongation in the aortic banded guinea pig (a species which does not exhibit ITO) to attenuated DHPR inactivation resulting from reduced SR Ca2+ release due to SERCA loss. Gomez et al.20 suggested that hypertrophy may impair the process of calcium-induced-calcium release by altering the geometric arrangement between the DHPR and RyR, and that this may also impair DHPR inactivation by SR Ca2+ released from the SR.89 These and other possible reasons for APD prolongation are not exclusive. One theory for the transition between compensatory and decompensatory hypertrophy is the aforementioned increased myocyte length. Others are that cellular hypertrophy occurs faster than SERCA production, lowering SERCA effective concentration,47 or SERCA expression actually diminishes.87 However, the frequent use of artificial models, which quickly enter the decompensatory stage confounds available data. The sequence in which cellular and molecular adaptation (i.e., hypertrophy, APD prolongation, SERCA reduction, PLB:SERCA elevation) occur in response to chronic systolic hypertension, and if each occurs during the compensatory or decompensatory stage, are unknown. Altered contractility is required to maintain cardiac output against resistance, and each of these factors can affect contractility. To my knowledge, no

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15 previous study has examined the changes in force production, action potential, and Ca2+ handling within the same hypertensive model of compensated hypertrophy to determine this. I perform such a study in Chapter 6. Rationale and Hypotheses Rationale The force-frequency relationship is a necessary component of enhanced cardiac output when required by the body. The magnitude of developed force is related to the magnitude of [Ca2+]i increase. By enhancing intracellular Ca2+ (re)cycling and reducing Ca2+ efflux, the frequency-regulated protein SERCA2a plays a central role in producing systolic force and augmenting contractility with heart rate. Despite the recognition of the importance of SERCA2, its precise contribution is poorly understood. Also, other factors are only now being recognized in the FFR, such as APD and ICa. Further study has been hindered by the widely disparate FFRs seen among animal models with very similar Ca2+ handling and mechanical proteins. Emphasizing the importance of the FFR is the observation that in many pathologies the FFR is depressed or even inverted, the lack of ability to increase force with heart rate thus contributes to symptoms such as exhaustion. A common correlate to disease and the altered FFR is enlarged cardiomyocytes, often accompanied by thickened, stiffer myocardial walls, which is the single greatest risk factor for serious complications such as heart failure and lethal infarctions. Characteristics of hypertrophied and diseased myocardium include reduced systolic force, diminished ventricular compliance, and slowed relaxation, that may be linked to the observation of reduced SERCA expression and/or activity. However, most of these studies investigated induced disease (e.g., aortic

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16 banding, salt hypertensive, surgical renal models) or late-stage pressure-overload (failing) models that do not examine earlier or more natural stages. Until recently, most studies ignored other possible factors in hypertrophy. The roles of APD and Ca2+ influx and efflux mechanisms, which have the potential for altering [Ca2+]i and thus developed force, are now questioned, but are studied in a “scattershot” fashion, in isolation of the others. For these reasons, the purpose of this study was to quantify the roles of the major Ca2+ influx and recycling mechanisms—SERCA2, APD, voltage-gated Ca2+ channel, and NCX—in the normal healthy FFR. I will then examine their contributions in two hypertrophied states, senescence and peripheral hypertension. This work will provide the first correlation of SERCA2 and APD with developed force; the first longitudinal study of FFRs and molecular mechanisms with aging; the first correlation of SERCA2, APD, and molecular mechanisms with developed force in hypertension; and provide a theoretical explanation for the unusual FFR frequently observed in small rodents. Hypotheses 1. The rat is an appropriate model for cardiac function and disease in the human. 2. The action potential duration contributes to the normal mammalian force-frequency relationship. 3. Reduced cardiac performance with adult aging is a result of a decline in SERCA expression. 4. Electrical remodeling is an early adaptation to cardiac hypertrophy secondary to peripheral hypertension. To test these hypotheses, hearts from young, aging, and systemically hypertensive rats will be examined both in function and at the molecular level. Hypertrophy will be assessed at both organ and cellular levels. To assess intrinsic myocardial contractility,

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17 developed force and SERCA activity will be measured in vitro in LV papillary muscles, isolated from the effects of neurohumoral influence. Action potential duration and inward Ca2+ current will be measured from isolated cardiomyocytes. SERCA2 and PLB will be quantified from LV tissue.

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18 Figure 1-1. Typical ventricular cardiac action potential.

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19 Figure 1-2. Calcium ion movements during systole and diastole in cardiac muscle cells.

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CHAPTER 2 MATERIALS AND METHODS Background of Animal Models Experimental protocols and baseline data were developed in the Sprague-Dawley rat strain (S-D). The S-D is a outbred albino strain used ubiquitously in research for which a generally healthy and consistent rodent model is required.90 The model for systemic hypertension for this work was the Spontaneously Hypertensive Rat (SHR) and its usual control, the Wistar Kyoto rat (WKY)—chosen because the SHR develops hypertension during maturation. Both the SHR and the WKY were developed from an outbred strain of Wistars from the Kyoto School of Medicine. In 1963 a hypertensive male was bred with a moderately hypertensive female to create hypertensive offspring, and brother/sister matings were continued until the new strain was stabilized. In 1971 the National Institutes of Health inbred representatives from the Kyoto line to develop a normotensive control for the SHR.91-93 Systolic blood pressure (SBP) in the WKY stabilizes at approximately 130 mmHg during adulthood.94. Systolic BP in the SHR often exceeds 150 mmHg by week 8 and frequently exceeds 200 mmHg in the adult.93 The SHR heart undergoes compensatory hypertrophy until a maximum of 18 months, at which time the animal begins to show evidence of heart failure.95 SHR HF is similar to the human, including cardiac hypertrophy, depressed myocardial function, extensive interstitial fibrosis, and apoptotic myocyte loss,96 killing the majority by age 24 months.95 Some studies suggest hypertension in the SHR may be because of enhanced sympathetic drive due to no 20

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21 reduction in the number of postganglionic neurons during development and/or diminished baroreflex control.97 Experimental Animals Male S-D, SHR, and WKY rats (Harlan, Charles River, and in-house breeding colony) were housed in conventional cages, provided standard rat chow and water ad libitum, and exposed to 12-h light/dark cycles. S-D were 2-3 months old, and WKY and SHR were of the following ages: 2 Months—WKY and SHR. Adolescent. SHR systolic blood pressure identical to WKY. 4 Months—WKY. Young adult. 9 Months—WKY and SHR. SHR hypertensive and with compensatory hypertrophy; prior to decompensatory hypertrophy. 16 Months—WKY. Immediately prior to action potential prolongation in WKY.80 24 Month—WKY. WKY considered senescent.98 31 Months—WKY. Advanced-age. Characterization of In Vivo Cardiovascular Parameters Measurement of Blood Pressure SBP was measured from nonanesthetized rats via the tail cuff method. Rats were placed into an opaque plastic cage and warmed by means of a heat lamp ~3 min to allow for vasodilation. SBP was measured by a Narco PE-300 pneumatic pulse transducer and sphygmomanometer coupled to a Narco MK-III-S physiograph (International Biomedical, Austin, TX) while the rat was held within a warming restrainer at 37C. The occlusion cuff was placed at the base of the tail to insure reproducibility. SBP represents the mean of four readings taken as the first pulsatile oscillation on the descending side of

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22 the pressure curve.99 SBP measurements were taken on subsequent days until multiple consistent readings were obtained. Measurement of Left Ventricular Function Left ventricular (LV) function was evaluated by LV ejection fraction (EF), as determined by echocardiography on a Hewlett Packard Sonos 5500 ultrasound system. Rats were lightly anesthetized with isoflurane and placed on their backs. If necessary, the chest anterior to the heart was shaved. A 12-MHz transducer probe covered with acoustic conductive gel was set at 2 cm depth. EF (%) was calculated by the cubed-method calculation100: [LV diameter]diastolic3-[LV diameter]systolic3 __________________________________ [LV diameter] diastolic 3. Assessment of Cardiac Hypertrophy Heart Weight/Body Weight Ratio. Rats were weighed while anesthetized prior to euthanasia. Upon removal and before subsequent dissection, hearts were weighed and compared to body weight. LV Wall Thickness. LV posterior wall thickness was measured during echocardiography (see Measurement of Left Ventricular Function). Myocyte Thickness. LV papillary muscles not used in in vitro experiments were fixed by immersion and storage in modified Karnovsky's fixative (2.5% glutaraldehyde, 1% formaldehyde in Millonig’s phosphate buffer containing (in mM): 13.04 NaH2PO4H20, 86.73 NaH2PO4H20, 85.56 NaCl, pH=7.2) at 4C. Histological sections were paraffin embedded, cut into both longitudinal and transverse 5 m sections, and stained with Masson’s trichrome. Cross-sectional area of cells was measured with Image-Pro Plus v4.5 (Media Cybernetics).

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23 In Vitro Isolated Muscle Experiments Isolated rat left ventricular papillary muscles have been shown to undergo hypertrophy similar to the LV wall101 and were used in these studies. Tissue Preparation Rats were anesthetized with isoflurane; the hearts were excised, placed and further dissected in Krebs-Henseleit (KH) solution at 4C containing (in mM): 127 NaCl, 2.3 KCl, 25 NaHCO3, 1.3 KH2PO4, 2.5 CaCl2, 0.6 MgSO4, 11 Glucose, 30 2,3-butanedione monoxime (BDM) (Sigma Chemical, St. Louis, MO; and Fisher Scientific, Fair Lawn, NJ), with 10 units/L insulin, saturated with 95% O2-5% CO2, pH 7.4. LV papillary muscles were removed and placed in the horizontal quartz flowchamber of a Muscle Research System (Scientific Instruments, Heidelberg, Germany). Both ends of the muscle were held by stainless-steel tweezers, one attached to a Grass SDJ9 stimulator (Grass Instruments West Arwick, RI), and the other to a force transducer (ground). The cross-sectional area between the tweezers was measured via a micrometer. Muscles wider than 1.6 mm were excluded from study.102 Muscle strips were perfused with oxygenated KH without BDM, pH=7.4, at 30C. Flow rate was ~1.5 mL/min. After 5 min in KH, stimulation voltage was set to ~30% above threshold. Muscle strips were stimulated isometrically with bipolar pulses of 5 ms duration. After 30 min equilibration at 0.15 Hz (S-D) or 0.05 Hz (WKY and SHR), the muscle was set at 0.2 Hz and gently stretched to the length of maximum systolic force. An initial FFR curve was performed at 0.2 Hz prior to Fura-2 loading. The muscle was stimulated at each frequency for 1 min prior to recording.

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24 Forceand Ca2+-Frequency Relationship (FFR, CaFR) The preparation was loaded with Fura-2 by recirculating oxygenated KH solution containing 5 M Fura-2 AM ester (Molecular Probes, Eugene, OR, or TefLabs, Austin, TX), 0.5% dimethyl sulfoxide, 0.2% cremophor (to increase Fura-2 solubility), and 10 mol/L N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a metal chelator that has been shown to enhance cardiac recovery after ischemia and reperfusion103;104) at 30C for 3.5-4 hr, while stimulated at 0.15 Hz (S-D) or 0.05 Hz (WKY and SHR). After Fura-2 loading, the muscle was re-equilibrated for 30 min in KH solution at 0.15 Hz (S-D) or 0.05 Hz (WKY and SHR), then 0.2 Hz for 5 min, and an FFR was performed as described above. The emission of Fura-2 excited alternately at 340/380 nm light was collected by a photometer unit and sampled by a signal sorter at 2-4 ms per ratio. Developed force (in mN) was recorded in OXC software and raw force and Ca2+ ratio transients in IonWizard v4.44 (IonOptix, Milton, MA) at 500 Hz. Forces were normalized to mN/mm2 tissue. Diastolic tension at 0.5 Hz was set as 0 mN baseline diastolic force. Force transients were analyzed with Ca2+ transients in IonWizard v5. Drug Studies After the 30-min Fura-2 washout/equilibrium, muscles were stimulated at 0.2 Hz for 5 min in KH, then in KH with the predetermined EC50 of drug until peak systolic force stabilized. The FFR was then performed as described above. Post-Rest Potentiation (PRP) Post-rest potentiations (PRP) are a qualitative measure of SR Ca2+ loading and, indirectly, SERCA activity.105 Once systolic force had stabilized at a given frequency (2

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25 5 min of equilibration), stimulation was stopped for given number of seconds, then restarted. During the rest, SERCA continues to fill the SR, leading to a greater SR Ca2+ release, and thus systolic force, upon restimulation than under steady-state conditions. The value of the first systole was compared to the mean systolic value of five contractions preceding the rest period. The PRP curve is multifactorial, reflecting total intracellular Ca2+ handling, and, including sufficient data points, bell-shaped (Figure 2-1). At short rest periods, SERCA Ca2+ sequestration predominates; the PRP contraction qualitatively reflects SERCA activity. However, after longer periods of rest, Na+/Ca2+ exchanger activity and SR Ca2+ leak become more prevalent. An inflection point appears, after which rest-potentiation declines.106 PRPs performed over a range of stimulation rates approximate the effects of frequency on SERCA activity and other intracellular calcium handling activities, which is augmentative within the normal physiological range in healthy mammals (Figure 2-1). However, as it is proportional to steady-state systolic force, a negligible PRP (~100% steady-state systolic force) will reflect lack of frequency potentiation for seemingly contradictory reasons--whether because SERCA remains at a low activity despite an increase in stimulation rate, or because SERCA activity is already elevated at lower frequencies. For the WKY and SHR studies, PRPs were performed at 3, 5, 7, 10, 15, 20, 30, 45, and 60 s near the base and peak of the normal mammalian FFR curve (1.5 Hz and 3 Hz). Quantification of Gene Expression After dissection, hearts were quick-frozen on liquid N2 and stored at C.

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26 Polymerase Chain Reaction (mRNA of SERCA2a and PLB) Heart tissue from left ventricle was collected and extracted by RNeasy Mini Kit and on-column DNaseI (QIAGEN, Valencia, CA). RNA quality and quantity was estimated by ethidium bromide staining on a 1% agarose gel and spectrophotometry at A260/280. Reverse transcription was done using Taqman Reverse transcription reagents (Roche, Branchburg, NJ) and random hexamers. Competitive polymerase chain reaction (competitive PCR) was performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) using 1 M of primers (Table 2-1), 50 ng cDNA, 10M dNTPs labeled with 32P-dATP, and 18S rRNA primer and competimer ratio of 2:8 (SERCA) or 3:7 (PLB) (Universal 18S Internal Standard kit, Ambion, Austin, TX). Initial step was 94oC 2 min followed by 36 cycles (SERCA) or 27 cycles (PLB) of 94oC 30 s, 60oC 30 s and 72oC 50 s. No-reverse-transcription control PCR was performed on all samples. PCR products were separated on a 6% polyacrylamide gel, imaged on a phosphorimager, and analyzed with the NIH Image program. Comparisons of PLB to SERCA expressions were calculated by dividing PLB value by SERCA value. Immunoblotting (SERCA and PLB Protein) Because common proteins used for quantitative western blot (e.g., actin, calsequestrin) might be altered during cellular hypertrophy, SERCA and PLB immunoassays were compared to a consistent external standard (STD) of the same quantity of protein pooled from LV of 4 adult outbred Sprague-Dawley rats. This method yields a larger uncertainty, reflected in SEM, than an internal standard, but allows trends to be visualized despite hypertrophy.

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27 LV wall tissue (~50 mg) was homogenized in 0.5 ml Homogenation Buffer (HB) (containing (in mM): 20 Tris, pH=7.4; 20 NaCl; 0.1 EDTA; 1% Triton X100; 0.5% sodium deoxycholate) by three 20-s burst with 20 s interval on ice. Homogenates were centrifuged 6 min at 10 000 rpm. Supernatant was stored at -80C. Protein concentration was determined in a 1:7 dilution with HB via the BCA Protein Assay Kit (Pierce, Rockford, IL). Sample and STD protein (15 g) were separated by 4-20% polyacrylamide SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked in 5% nonfat milk in TBS-T (20 Tris, 150 NaCl, 0.1% Tween, pH=7.4) for 30 min, cut by molecular weight, and incubated with primary antibody (PLB: mouse monoclonal IgG, Affinity Bioreagents MA3-922, 1:10 000 dilution; SERCA: goat polyclonal IgG, Santa Cruz sc-8094, 1: 2000 dilution) for 1 hr at room temperature. Membranes were washed three times (10 min/wash) with TBS-T and incubated in peroxidase-conjugated secondary antibody (PLB: sheep anti-mouse, Sigma A-6782, 1: 10 000 dilution; SERCA: donkey anti-goat, Santa Cruz sc-2033, 1:5000 dilution) for 1 hr at room temperature. Membranes were washed, then developed for 5 min with Supersignal West Pico chemiluminescent kit (Pierce). Developed films were scanned into a computer and analyzed with the NIH Image program. Mean band density of individual animals was compared to STD. Comparisons of PLB to SERCA expressions were calculated by dividing PLB value by SERCA value. Measurements of Action Potential and L-Type Ca2+ Current Isolated myocytes were placed into a recording chamber and superfused with Tyrode solution at 35oC. Transmembrane voltages and currents were determined using an Axopatch-200 amplifier, a DigiData-1200A interface and pCLAMP6 software (Axon

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28 Instruments, Foster City, CA). When measuring action potentials, microelectrodes (1-3 M) were filled with a solution containing (in mM): 120 K-aspartate, 20 KCl, 1 MgSO4, 4 Na2ATP, 0.1 Na3GTP, 1 EGTA and 10 HEPES, pH=7.2. To induce action potentials, a 5-ms depolarizing pulse was applied at a frequency of 0.2-6 Hz. The duration of the action potential was measured at 80% (APD80) repolarization. For recording ICa,L, both K+ and Na+ in the superfusate (Tyrode solution) were replaced with tetraethylammonium. The microelectrodes were filled with a solution containing (in mM): 120 Cs-aspartate, 20 CsCl, 4 ATP-Mg, 0.1 GTPNa3, 1 EGTA and 10 HEPES, pH=7.2. Myocytes were voltage-clamped at a holding potential of -85 mV. The electrode capacitance, whole-cell capacitance, and series resistance were maximally compensated. A train of 30-ms depolarizing pulses to -10 mV was applied at a frequency of 0.2-6 Hz. This voltage protocol could activate both Land T-type Ca2+ channels. However, T-type Ca2+ current was not detected in ventricular myocytes of normal, adult rat in previous studies.107 The magnitude of ICa,L in response to the 10th depolarizing pulse at each pulse frequency was determined, and was expressed as an area current (nA*ms) using the pCLAMP program. Statistical Analysis Student t tests or ANOVA were performed to assess statistical significance (at 95% confidence limit) in Prism v3. For APDand ICa-frequency relationships, if ANOVA revealed significance, a Newman-Keuls or Bonferroni posttest compared the 0.2 or 0.5 Hz value to subsequent frequency values. Graphs were made in Prism and Origin v6. Data are shown as mean S.E.M.

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29 01357101520304560100150200250300350400 % Pre-Rest Systolic ForceSeconds Rest 2 Hz 3 Hz 4 Hz 5 Hz Figure 2-1. Post-rest potentiation behavior in myocardium. Sprague-Dawley rat papillary muscle after 3.5-4 hr Fura-2 loading (See In Vitro Isolated Muscle Experiments). n=5-6. Mean S.E.M. (See Statistical Analysis).

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30 Table 2-1. Nucleotide sequences of primers used in competitive PCR. SERCA-Forward 5’-GGAAAGACCTTGCTGGAACTTGTG-3’ SERCA-Reverse 5’TTCAGCGTTTCTCTCCTGCCATAC-3’ PLB-Forward 5’-GAGCTCCCAGACTTCACACAACTA-3’ PLB-Reverse 5’-GGGCATTTCAAT-AGTCGAGGCTCT-3’

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CHAPTER 3 QUANTIFICATION OF THE RAT LEFT VENTRICLE FORCEAND Ca2+-FREQUENCY RELATIONSHIPS: SIMILARITIES TO DOG AND HUMAN Introduction In species whose contractile force more heavily depends on SR Ca2+ cycling for providing contractile Ca2+, such as the mouse and rat,17 the FFR regularly observed in experimental preparations has been negative in the range of 1-4 Hz.108;109 This is in contrast to the positive FFR observed in larger mammals such as dog and human in that frequency range, despite the remarkable similarity in structure and primary sequence of all key proteins involved in Ca2+ cycling and force production among mammals. Although the mouse and rat are the most common laboratory models, this fact has slowed acceptance of these animals as a model for research into cardiac diseases in humans, for which a depressed FFR is often both symptomatic and diagnostic.25;88 To explain the observations in rat, observers have postulated several parameters that can affect the in vitro FFR, including specimen size110 and solution composition. Nonetheless, a positive FFR could be induced in the rat by altering experimental conditions that impair SR Ca2+ release, such as reducing extracellular Ca2+ 111 or ryanodine treatment.112 Together, these results suggested that rat cardiac tissue is intrinsically capable of presenting the characteristic large mammalian FFR if an appropriate experimental protocol were applied. Recently, using newer protocols and approaches, positive FFRs are being observed in the rat113;114 and mouse115;116 in cardiac muscle preparations. The shape of the FFR curve differs somewhat but not greatly from 31

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32 that seen in larger mammals in slope and optimal frequency (frequency of greatest systolic force).25;117 Although the mouse has become a popular subject for genetic manipulation, its use is limited for obtaining quantitative physiological measurements in vivo and in vitro. In contrast, several rat strains, such as the SHR and Spontaneously Hypertensive/Heart Failure-Prone Rat (SHHF), that are currently available are increasingly revisited95 in cardiovascular research. In fact, these rat models have been the basis in the past for the development of many cardiovascular drugs that are on the market today. For these and other reasons, several new transgenic rats with disease profiles aimed at mirroring human pathology have been generated.118 Although most pathology lies in the left ventricle, most studies in rats focused on right ventricle tissue for perceived technical reasons and limitations associated with LV such as muscle strip dimension.110;113;115;116 Prior to beginning my studies in a diseased rat model, both the normal/healthy behavior of the rat LV had to be quantified and the relevance of this data to humans had to be ascertained. Also, a rationale for the negative FFR frequently observed in the rat should be developed to explain the earlier discrepancies. The goal of this study was to use a widely used protocol developed for human LV biopsy strips25 and apply this to the study of isolated rat LV muscle to investigate whether this would reveal a positive FFR that is similar in shape to that seen in humans and dogs. I hypothesized that based on the sequence and functional similarity of proteins involved in Ca2+ cycling, the FFR in mammals should be comparable if they were obtained under the same experimental conditions. To this protocol, I added loading of the cytoplasm with the Ca2+ dye Fura-2, in the time set aside for equilibration, which allows simultaneous quantitative

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33 measurements of systolic and diastolic Ca2+ transients with force. I further used this protocol to study the effects of cardioactive drugs with known defined action on Ca2+ cycling to further elucidate the nature of the mechanisms underlying the ascending limb of the FFR and particularly the likely cause of the persistent negative FFR found in rat when the experiments are performed within an hour of dissection of the preparation. I show that the rat FFR is remarkably similar to that in humans and dogs when the same experimental protocol is used. The data further suggest that the negative FFR in rat, when taken early after dissection, is due to high post-isolation/dissection Ca2+ cycling caused by high activity of the SR SERCA Ca2+ pump. Animal Model Male Sprague-Dawley rats, aged 2-3 months, were used in this study. Results Systolic Force Frequency Relationships in Rat, Dog, and Human The initial FFR in rat obtained within 60 min after dissection, following equilibration, is negative and characterized by very high forces as shown in Figure 3-1A. Peak force declined from 11.85 0.81 mN/mm2 at 0.2 Hz to 5.21 0.32 at 6 Hz. In contrast, after the 3-4 hour Fura-2 loading time, developed force had dropped at all frequencies, but particularly below the optimal frequency of 3-4 Hz. As a result, the FFR revealed a markedly positive ascending limb. The FFR had a steep increase in developed force from 0.5 Hz to 3 Hz (0.5 Hz: 2.47 0.24 mN/mm2, 1 Hz: 2.66 0.24, 2 Hz: 3.87 0.34, 3 Hz: 4.96 0.42, n=9). Developed force peaked at 3-4 Hz (4 Hz: 4.96 0.47), implying that the optimal frequency lay between 3 and 4 Hz, and declined slightly beyond 4 Hz (5 Hz: 4.53 0.45, 6 Hz: 4.01 0.39) (Figure 3-1A,B). Diastolic force is shown in Figure 3-1A with 2 mN force added for clarity. The FFR for diastolic tension

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34 increased slightly by 0.13 0.04 mN/mm2 from 0.5 to 2 Hz, remained unchanged from 2 to 4 Hz (4 Hz: 0.13 0.03), and slightly increased from 4 to 6 Hz (5 Hz: 0.23 0.05, 6 Hz: 0.41 0.09) compared to systolic force. To determine if this force decline was a consequence of the Fura-2 dye loading, which as a calcium dye has the potential to buffer cytosolic calcium, I performed experiments in which the KH physiological solution was recirculated without Fura-2 AM, but with TPEN and cremophor for 3.5-4 hr. The resulting FFR was positive with an optimal frequency at 4 Hz and not different from the FFR in Fura-2 loaded tissue (1-6 Hz, n=5, p<0.05, Figure 3-1B). The same protocol was used to measure the FFR in epicardial strips from dog and human left ventricle. In order to compare the FFR in rat to those obtained in dog and human, and because these strips originate from scalpel biopsies that are pared down, the FFRs were converted by normalization to the force at 1 Hz. The result is shown in Figure 3-1C. In humans, the index of the heart rate-induced increase in force between 60 and 180 beats per minute (bpm) is the physiologically most significant and has been termed chronotropic contractile reserve (CCR) by Alpert et al.25 The CCR for rat, dog and human were similar, yet differed in amplitude and heart rate of maximum force (Table 3-1). The relation between optimum heart rate and CCR was linear (Figure 3-1D). Force and Calcium-Frequency Relationships in Rat LV Muscle It is well accepted that force development closely mirrors the increase in cytosolic Ca2+ in cardiac muscle in health and disease. Figure 3-2A shows a representative original experiment of simultaneous measurement of developed force and cytosolic calcium at stimulation frequencies from 0.5 to 6 Hz in isolated adult rat papillary muscle.

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35 Each force transient was preceded by a cytosolic calcium transient with a relative peak height corresponding to the relative peak height of the force transient. As frequency increased, force and calcium transient amplitude increased as shown in Figure 3-2B while retaining their close relationship. The change in developed force was mirrored by a corresponding change in cytosolic calcium concentration. From 0.5 to 3 Hz, peak force doubled (196.6 22.9%), and calcium increased to 126.7 4.9% (n=8). From 4 to 6 Hz, developed force fell to 157.1 21.0% and calcium fell to 120.9 6.7% the 0.5 Hz value. As first shown by Brixius et al.119 and Gwathmey et al.,120 the relationship between peak force and peak Ca2+ is near linear and shown for all individual experiments in Figure 3-2C. Kinetics of Force and Ca2+ Transients as a Function of Frequency in Rat LV Muscle I performed further analysis of several components of the systolic force and calcium transients that are most likely to alter under pathological conditions. In all cases, calcium values are reflected in the subsequent force transient. Time from stimulus pulse to peak force declined significantly from 1 to 4 Hz and remained constant up to 6 Hz (0.5 Hz: 74 2 ms, 2 Hz: 67 2, 3 Hz: 62 2, 4 Hz: 60 1, 5 Hz: 60 1, 6 Hz: 59 1, p<0.0001), but was unchanged in the calcium transient (Figure 3-3A). The relaxation velocity of force increased with frequency and closely mirrored the systolic FFR whereas the [Ca2+]i removal velocity, a direct indicator of SERCA activity, remained constant up to 2 Hz but then markedly increased between 2 and 6 Hz (Figure 3-3B). The time of force and calcium transient to return to 50% of peak value (RT50) significantly accelerated with an increase in frequency (Figure 3-3C). Speed of decline in calcium was significantly faster than the preceding frequency at all experimental frequencies 2 Hz and greater. Speed of decline in force was only significantly faster than

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36 the preceding frequency at 2 Hz and above (RT50 1 Hz: 124 4 ms, 2 Hz: 114 3, p<0.05), which was also the frequency of greatest incremental increase in peak force. Effect of Isoproterenol and Cyclopiazonic Acid on the FFR, Ca2+, and Diastolic Force To further ensure that our results reflect normal mechanisms within the myocardium, I treated the muscle with drugs with defined effects on contractility and intracellular Ca2+ handling and proceeded to measure the FFR. These results are summarized in Figure 3-4 (each drug n=5). Representative developed force and calcium transient tracings of muscle treated with isoproterenol and cyclopiazonic acid stimulated at 2 Hz are shown in Figure 3-4A. The -adrenergic agonist isoproterenol, at a predetermined EC50 of 100 nM, increased peak developed force at all frequencies, but especially at low stimulation rates, thereby flattening and eliminating the ascending limb of the FFR. Peak force was significantly higher, particularly at frequencies below the optimal frequency of ~3 Hz. Isoproterenol also shortened the calcium and force transients (Figure 3-4). The specific SERCA inhibitor cyclopiazonic acid (CPA), at a predetermined EC50 of 10 M, reduced both absolute and proportional increase in peak force compared to controls (Figure 3-4B). Isoproterenol lowered (p<0.05, 1-4 Hz) and CPA raised (p<0.05, >3 Hz) diastolic force across most of the FFR (Figure 3-4C). Mechanistic Investigations To further investigate the possibility that high SERCA activity early post-dissection may represent one cause of the preload negative FFR, I performed two established measurements closely related to SERCA activity—force relaxation velocity (FRV) and post-rest potentiation (PRP). Post-loading FRV increased 2.9-fold with frequency,

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37 yielding a relationship qualitatively similar to the post-loading FFR (0.5 Hz: 22.9 3 mN/sec; 4 Hz: 66.2 8), whereas the pre-Fura-2-loaded FRV remained unchanged (0.5 Hz: 78.4 6.6; 4 Hz: 79.8 8.4) but significantly higher than post-loading at the lower frequencies. Isoproterenol in post-loading papillary muscles accelerated relaxation velocity at all frequencies (3.6-fold at 0.5 Hz: 82.5 9.3; 1.6-fold at 4 Hz: 110.1 11.6) (Figure 3-5A). Developed force was augmented by frequency in the post-Fura-2-loaded PRPs from 190% at 2 Hz to 320% at 5 Hz of the pre-rest steady-state force, whereas increasing frequency had no effect (from 150% at 2 Hz to 150% at 5 Hz) on potentiation prior to Fura-2 loading. These data strongly suggest that SERCA activity was already near maximal in pre-Fura-2-loaded muscle (Figure 3-5B). Discussion In this chapter, I present the first comprehensive qualitative and quantitative analysis of rat isolated LV muscle function, measured simultaneously as the FFR and CaFR for systolic and diastolic function, in small isolated papillary muscle strips in vitro using a protocol designed for larger mammals. My results reveal a positive FFR and suggest that the reason for the often-observed negative FFR is at least partly related to enhanced Ca2+ cycling, driven by the SERCA pump, in tissue early post-dissection. Comparison of the FFR in Rat to that in Dog and Human It is well accepted that the molecular bases for the shape and amplitude of the FFR are intimately related to the fate of intracellular Ca2. Therefore it has been surprising to see differences between smaller and larger mammals in the FFR despite the extraordinary molecular similarity at the DNA and protein level of proteins involved in Ca2+ cycling.

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38 To illustrate this point I performed a comparative analysis on the protein sequences available in GenBank as shown in Table 3-2. I hypothesized that based on the sequence and functional similarities, the FFR in mammals should be comparable if they were obtained under the same experimental conditions. I found that the FFR obtained in rat is strikingly similar to that seen in larger mammals with established positive force-frequency relationships, such as human and dog when done under the same experimental conditions (Figure 3-1). The steep ascending limb of the FFR occurs over the same stimulation range (~0.5 Hz to ~3.5 Hz) and shares a similar peak value (~200% of 1 Hz force). The rat FFR also has similar optimal frequency (~3 Hz) to the human FFR in this study and to other published human data25;117;121-123 taken from nonfailing control myocardium in Tyrode’s solution and are also similar to these canine data obtained in KH solution and similar to that from Bouchard and Bose.108 I examined stimulation frequencies within and beyond the normal physiological range of humans (~1-3 Hz) and rats (4-7.5 Hz78), where depression of the FFR in the 1-3 Hz (60-180 bpm) range is both diagnostic and symptomatic of disease in humans. Taken together, these data suggest that similar mechanisms may underlie this part of the FFR in all three species. Alpert et al.25 have previously shown a remarkable linear correlation between optimum heart rate of the FFR (frequency of peak developed force) and the amplitude of the FFR in humans with different degrees of cardiac disease using this protocol. I report here a similar finding for these parameters in human versus dog and rat using this protocol, further suggestive of a similarity in underlying mechanisms (Table 3-1).

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39 Kinetics of Force and Ca2+Transients as a Function of Frequency in Rat LV Muscle I used advanced transient analysis to further look into possible mechanisms underlying the positive FFR in rat heart. Kinetic analysis of the excitation phase of the force and Ca2+ transients revealed a marked frequency-dependent up-regulation of the amount of Ca2+ into the cytoplasmic space. As elegantly shown by Bers and others, in rat, the bulk of Ca2+ released for contraction is thought to originate from the SR.17 Previous studies in rat have demonstrated that the efficiency and gain of EC-coupling is near optimal and that release from the SR is quantal and directly related to the activity of the L-type Ca2+ channel (DHPR).124 I suggest therefore that the increased amount of Ca2+ may result from an increase in the amount of “trigger” Ca2+ supplied by the DHPR (see Chapter 4). Analysis of the relaxation phase of the force and Ca2+ transients reveal a marked increase in speed of lowering cytosolic Ca2+ above 2 Hz up to 6 Hz as well as speed of force relaxation as a function of stimulation frequency between 1 and 4 Hz. These observations most likely reflect the canonical belief that SERCA activity increases with frequency at least in part due to frequency-dependent activation of CamKII.25;33 Pharmacological Effects on the Rat LV FFR Consistent with the hypothesis that the high forces at low frequencies are a result of enhanced Ca2+ cycling are my observations on the effect of isoproterenol. Isoproterenol mostly affected the low-frequency side of the FFR. The increase in force resulted in a flattened FFR with a “gull-shape” and enhanced force at all stimulation frequencies. The most likely mechanisms are the effect on the DHPR,18 leading to increased Ca2+ channel activity, in tandem with the stimulatory effects enhancing Ca2+ efflux from the SR, via

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40 the ryanodine sensitive Ca2+ release channels, and most notably increased SERCA activity, by phosphorylation of phospholamban, resulting in higher SR Ca2+ load. Consistent with published data on the effect of CPA on the positive human FFR,125 CPA both lowered the peak and lengthened the decline of the calcium transient, indicating reduced SERCA pumping of cytosolic calcium into the SR resulting in depressed systolic force. CPA also depressed the systolic amplitude of the FFR and increased the diastolic FFR, consistent with previous studies in human heart muscle.125 Mechanistic Investigations/Limitations and Perspective Notwithstanding the present study, one remaining question is why earlier studies consistently showed negative FFRs. Several extrinsic causes for the negative FFR have been suggested, including hypoxia or metabolic waste accumulation due to tissue size in in vitro observations, decreased SR Ca2+ load (due to Ca2+ efflux or irregular spontaneous RyR Ca2+ release) or release with frequency113 and high [Na+]i slowing Ca2+ extrusion and causing the filling of the SR even at low frequencies.17 Tissue size was found to have an impact on the slope of trabecula FFR by Gulch and Ebricht.110 I observed inconsistencies in peak and diastolic force production in very wide muscles, but not below our limit of 1.6 mm. Adequate oxygenation of our preparation, even at higher rates, is inferred from the increase in SERCA activity (a major consumer of ATP) seen with frequency. Post-peak [Ca2+]i decline velocity (92% due to SERCA in the rat17) increased and RT50[Ca] quickened throughout the measured frequencies up to 6 Hz (Figure 3-3B,C). Absolute force at 6 Hz also stayed well above the force obtained at the lowest frequency of 0.5 Hz. While decreased SR Ca2+ release due to RyR or L-type Ca2+ channel inactivation between action potentials is a possible explanation for the descending FFR at 4 Hz and

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41 greater and was the subject of further investigation (see Chapter 4), it does not adequately explain the different observations regarding the rat FFR in previous studies. Both positive and negative rat FFRs have been observed in physiological solutions containing 135-152 mmol/L Na+ and 4.7-5.8 mmol/L K+,108;111-113;126 suggesting that [Na+] cannot be the exclusive cause, although it may still contribute substantially to force amplitude due to its effect on Ca2+ load. A likely candidate suggested by these present results is recovery from cold ischemia during excision of the heart and preparation of the muscle strips. Retrospectively, I examined the time from dissection to actual experiment in papers in which a positive FFR was seen in the rat. These times were found to be lengthy, exceeding 1-3 hr;113;116 and including Fura-2 loading, my experiments occurred almost 5 hr post-dissection. With recovery time, the rat in vitro FFR experiences a reduction in developed force, mainly at lower frequencies, which “unmasks” the positive slope. Spurred by the isoproterenol data, I realized that near-maximal SERCA activity with little room for frequency-dependent increase would produce such a shape and investigated SERCA activity preand post-Fura-2 loading. As direct Ca2+ measurements are impossible prior to dye loading, I performed two established measurements to assess SERCA activity—relaxation velocities and post-rest potentiations (Figure 3-5). Speed of force decline (which is largely due to removal of cytosolic Ca2+ by SERCA), was significantly faster pre-Fura-2 loading at frequencies up to 2 Hz. Post-rest systolic contractions have been shown to quantitatively measure SR Ca2+ loading and to augment with frequency in humans.106 This feature is readily observed in rat LV post-Fura-2 loading but absent in the pre-loading FFR (Figure 3-5).

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42 The rat heart is metabolically very active in situ, with heart rates often exceeding 400 bpm,78 and more dependent upon SERCA17 for force production. Whereas human and dog tissue were carefully excised from arrested and merely cooled (to ~15C) hearts on cardiopulmonary bypass and then further dissected at room temperature, rat hearts are typically quickly excised at regular heart rate and temperature and then immediately immersed in ice-cold KH buffer. The biochemical trauma of this brief warm/cold ischemia and the associated disruption of the Donnan equilibrium is unknown, but current investigations suggest that ischemia leads to increased SERCA activity. It is conceivable that more time is needed for the tissue to recover from this trauma and that caused by prolonged hypothermia. Conclusions Rat LV papillary muscle can augment force as a function of stimulation rate similar to larger mammals, such as dog and human when subjected to the same experimental protocol. The positive FFR is likely due to a corresponding increase in cytoplasmic Ca2+ and increased SR Ca2+ loading by increasing SERCA activity. In freshly isolated tissue, SERCA activity is very high and hence frequency-dependent increase is greatly diminished, but recovers with time. With the current protocol, the rat LV shares basic cardiac functional characteristics with dog and human that render it a better laboratory model for cardiovascular diseases in which an altered FFR is a distinguishing feature than previously assumed.

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43 012345624681012 Pre-Fura Loading Peak Developed Force Post-Fura Loading Peak Developed Force Post-Fura Loading Diastolic TensionA.Force (mN/mm2)Frequency (Hz) 01234562.02.53.03.54.04.55.05.56.06.5 Fura-2 Loaded Unloaded ControlB.Peak Developed Force (mN/mm2)Frequency (Hz) 01234566080100120140160180200220240 C.Developed Force (% of 1Hz/60BPM)Heart Rate, Hz Dog Human Rat 1.01.52.02.53.03.54.04.55.0150175200225250 D.RatDogHuman% Potentiation at peak Force(Fmax)Heart Rate at Fmax, Hz Figure 3-1. Systolic force frequency relationships in rat, dog, and human. (A) FFR of isolated papillary muscle after 45 min equilibration (prior to Fura-2 loading) and after 3.5-4 hr in recirculating KH with Fura-2 AM (post Fura-2 loading) (B) FFR in experimental vs. control conditions after 3.5-4 hr in Fura-free recirculating KH compared to Fura-containing KH. (C) FFR of isolated rat papillary and human (n=10) and dog (n=6) LV myocardial strips using same experimental protocol. (D) Plot of heart rate at maximal force versus percent potentiation of force over the force at 1 Hz.

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44 Table 3-1. Amplitude and Shape of the FFR among species. Comparisons of the heart rate at which force is maximal (Fmax), range (%) of optimum heart rate, and percent increase of force at optimum heart rate over force at 1 Hz. * p<0.05 human versus dog and rat. Heart Rate at Fmax Range, Fmax20% %Fmax/F1.0Hz Human 2.7 Hz; 162 bpm 2.2-3.2 215 * Dog 3.4 Hz; 204 bpm 3.0-4.5 185 Rat 3.7 Hz; 222 bpm 3.0-5.0 175

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45 0.5 HzA. 100 ms 200 Fura-2 AU 1 mN/mm2 1Hz 2 Hz 3 Hz 4 Hz 5 Hz 6 Hz Figure 3-2. Forceand calcium-frequency relationships in rat LV muscle. (A). Representative developed force (thick line) and cytosolic calcium (Fura-2 AM, thin line) tracings from 0.5 to 6 Hz over 335 ms from stimulation pulse. Systolic intracellular Ca2+ level increases with an increase in frequency from 0.5 to 3 Hz and is accompanied by an increase in developed force. Rate of decline in both force and calcium transients accelerates with an increase in frequency. Note that the Ca2+ ratio scale is exponential, where small changes at the top represent substantial changes in Ca2+. (B) Forceand calcium-frequency behavior shown as percentage of peak value at 0.5 Hz. (C) Peak developed force as a function of peak calcium at examined frequencies in individual experiments (experiments n=8).

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46 .5123456100120140160180200220 .5123456 100110120130140 Peak Developed ForcePeak Developed Force (% 0.5 Hz)Frequency (Hz) Peak CalciumB.Peak Calcium (% 0.5 Hz) 80100120140160100150200250300350400 C. r=0.981Peak Developed Force (% 0.5 Hz)Peak Calcium (% 0.5 Hz) Figure 3-2. Continued.

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47 0123456406080 Force CalciumA.Time to Peak Value (ms)Frequency (Hz) .5123456203040506070 .5123456 3500400045005000550060006500700075008000850090009500 ForceRelaxation Velocity (N/sec)Frequency (Hz) CalciumB.Ca2+ Return Velocity (AU/sec) 012345680100120140160180200 Force Calcium******C.Time to 50% From Peak Value (ms)Frequency (Hz) Figure 3-3. Kinetics of force and Ca2+ transients as a function of frequency in rat LV muscle. (A). Time from Stimulus to reach Peak Force and Ca2+. (B) Maximum Velocities of Force and Ca2+ in relaxation phase of the transients. (C) Time from peak to 50% relaxation from peak value. Force n=9. Calcium n=8. p<0.05 from 0.5 Hz value. * p<0.05 from value of preceding frequency.

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48 Isoproterenol (100nM) Cyclopiazonic Acid (10M) Control100 msec A.1 mN/mm2 Systolic Force 100 Fura-2 AU Systolic Calcium 01234561234567 Control Isoproterenol (100nM) Cyclopiazonic Acid (10M)********B.Systolic Force (mN/mm2)Frequency (Hz) 0123456-0.10.00.10.20.30.40.50.60.70.8 Control Isoproterenol (100nM) Cyclopiazonic Acid (10M)C.*Diastolic Tension (mN/mm2)Frequency (Hz) Figure 3-4. Effect of isoproterenol and cyclopiazonic acid on the FFR, Ca2+, and diastolic force. Force-frequency behavior of isolated papillary muscle in 100 nM isoproterenol (n=5) or 10 M cyclopiazonic acid (n=5), compared with controls (n=9) * p<0.05 from controls. (A) Representative systolic force and calcium tracings (Fura-2 AM) in papillary muscles in isoproterenol, cyclopiazonic acid, and controls at 2 Hz. (B) Systolic FFR (C) Diastolic FFR.

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49 .5123456255075100125 **********A. Pre-Fura Loading Post-Fura Loading Isoproterenol Post-Fura LoadingForce Relaxation Velocity (mN/sec)Frequency (Hz) 2345100150200250300350400 Pre-Fura Loading PRP Post-Fura Loading PRPB.% Steady-State Developed Force (PRP at 5s Rest)Pre-Rest Frequency (Hz) Figure 3-5. Effect of time on SERCA activity. Indirect SERCA activity measurements in isolated papillary muscle after 45 min equilibration (prior to Fura-2 loading) and after 3.5-4 hr in recirculating KH with Fura-2 AM (post Fura-2 loading) and in 100 nM Isoproterenol. (A) Relaxation velocity. Preload n=7. Postload n=9. Isoproterenol n=5 * p<0.05 from post-load controls. p<0.05 from pre-load. (B) Post-Rest Potentiation Behavior at 5 sec rest. Preload n=3. Postload n=5. p<0.05 from 2 Hz value.

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50 Table 3-2. Comparisons of amino acid sequences of major cardiac Ca2+-handling proteins in rat, human, and dog. As the ryanodine receptor type 2 has not yet been sequenced in rat, the mouse, a closely related rodent with a frequently negative FFR, has been substituted. Alignment achieved using the PILEUP algorithm of the GCG package.127 Genbank accession numbers: SERCA2a rat-NP_058986, human-NP_733765; RyR2 mouse-NP_076357, human-Q92736; Phospholamban rat-S37638, human-P26678; NCX rat-NP_062141, humanNP_066920; Ca2+ Channel subunit rat-P22002, human-Q13936; Calmodulin rat-NP_036650, human-AAB60644. Protein Species % Identical % Similar SERCA2a Rat Human 98 >99 Ryanodine Receptor 2 Mouse Human 97 >99 Phospholamban Rat Human 98 100 Na+/Ca2+ Exchanger Rat Human 95 98 L-Type Ca2+ Channel (1 subunit) Rat Human 91 93 Calmodulin Rat Human 100 100

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CHAPTER 4 ELECTRICAL CONTROL OF FORCE PRODUCTION IN NORMAL MYOCARDIUM: A SYNCHRONIZED ROLE FOR ACTION POTENTIAL DURATION AND CALCIUM PUMP ACTIVITY Introduction Although increased SERCA2a activity due to increased CamKII activity with frequency is widely viewed as the major component of the positive FFR,25;33 another possible mechanism for the enhanced force production with increasing heart rates is prolongation of the action potential duration (APD) via the frequency-dependent facilitation of the inward calcium current (ICa). Noble et al.128 were the first to report this frequency-dependent increase of ICa in frog atrial fibers. This observation has also been made in myocytes obtained from rats,129 guinea pig,130 and in human atrium.131 The effect of increased ICa would be to enhance the trigger for calcium-induced calcium release and to calcium-load the myocyte. Despite these observations and the acceptance of electrical remodeling as an integral part of the cardiac disease process, the possible role of the APD or ICa on cardiac force output is routinely ignored in in vitro studies measuring force or the FFR. In Chapter 3, I showed that the Ca2+ cycling mechanisms underlying the FFR in rat are similar to that in dog and human. In the current study, these measurements were extended to include APD and ICa to quantify the interaction of these electrical components with force, Ca2+, and SERCA2a. I hypothesized that the FFR ascending limb results from frequency-dependent increases both of ICa (to increase Ca2+ loading of the myocyte) and in SERCA2a activity. 51

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52 Animal Model Male WKY rats, aged 9 months, were used in this study. Results Forceand Ca2+Frequency Relationship The FFR for rat papillary muscle stimulated at 1-6 Hz was bell-shaped (Figure 4-1). Peak developed force was maximal when muscles were stimulated at 3-4 Hz (129.7 8.2% of 1 Hz at 4 Hz; n=12). The relationship between frequency and peak Ca2+ was similar to the FFR (CaFR, 117.2 8% of 1 Hz at 4 Hz, n=6). Together, these results are consistent with previous studies that have shown a close relationship between Ca2+ transients and force.132 APDand ICa,LFrequency Relationship The APD-frequency relationship was determined in isolated myocytes using the patch-clamp technique (Figure 4-2). The duration of the action potential (APD80) increased from 62 8 ms to 90 9 ms (n=10) as pacing frequency was increased from 1-4 Hz, then decreased slightly when pacing frequency was further increased from 4 to 5, then 6 Hz. To investigate the possibility that the prolongation of the APD with frequency was due to a frequency-dependent increase in ICa, the current-time integral of ICa,L was determined as a function of frequency (n=6). The shape of the relationship between APD and stimulation frequency, and ICa,L and stimulation frequency were similar (Figure 4-2), suggesting that changes in ICa,L underlie the observed changes in the frequency-APD relationship.133

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53 Frequency Dependence of SERCA2a Activity Figure 4-3 displays the maximum Ca2+ transient decline (CaRV) and force transient relaxation (FRV) velocities as a function of pacing rate. The characteristics of the relaxation phase of the calcium transient are governed by the ability of the cell to pump cytoplasmic calcium into the SR and into the extracellular space,134 as well as the release of calcium from troponin C. The relationship between [Ca2+]i decline and frequency was flat from 0.5-1 Hz. From 1-6 Hz, there was a linear relationship between CaRV and pacing frequency (n=6). Myocardial force relaxation velocity is governed by the rate of dissociation of myosin crossbridges (cross-bridge cycling rate) in addition to calcium extrusion by pumps/exchangers.135 In contrast to the linear relationship between CaRV and frequency, the relationship between FRV and frequency was biphasic (maximum of 196.3 16.7% of 1 Hz baseline at 4 Hz, n=10). Note the correlation between the velocity response to frequency measured as FRV and force as the FFR. It appears that SERCA2a activity increases with increasing heart rates from 1 Hz to 6 Hz, based on the analysis of the calcium transient relaxation velocities. Additional evidence was obtained by measurement of post-rest potentiation near the base and peak of the FFR ascending limb (1.5 vs. 4 Hz, Figure 4-4). Pieske et al.136 used the slope (time to plateau) rather than the plateau of the PRP curve as an indication of SERCA2a activity. The time to plateau at 4 Hz was much shorter than at 1.5 Hz (7 1, n=4, versus 36 5 sec, n=8, p<0.005) suggesting SERCA2a activity at 4 Hz was greater than at 1.5 Hz. Discussion The frequency-dependent potentiation of cardiac contractility is observed in nearly all mammalian ventricular muscle between approximately 1 and 4 Hz, including normal rat regardless of strain (Figures 3-1, 4-1). The exact molecular mechanisms underlying

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54 this phenomenon have remained elusive but recent attention has focused on the role of increasing SERCA2a activity with increasing heart rate.31;32;137 My data from WKY rat ventricle are consistent with enhanced SERCA2a activity with increasing heart rates above 1 Hz. From analysis of the calcium transient relaxation velocities, I infer that removal of calcium from the cytosol becomes faster with increasing heart rates starting at 1 Hz (Figure 4-3). This is supported by the PRP data at the 1.5 and 4 Hz stimulation rates (Figure 4-4). This enhanced SERCA2a activity leads to increased SR calcium loading and increased force production with increasing heart rates. An increase in SR calcium load was observed by Bers and Macleod138 and Bers105 when stimulation frequency was increased in rabbit ventricle, as assessed by rapid cooling contractures. A key question then is from where does the Ca2+ come to load the SR? Interestingly, these investigators observed extracellular calcium depletions with increasing heart rate.138 This observation, taken together with the observations by Noble and others referred to earlier, led me to explore the possibility that prolongation of the APD via a frequency-dependent increase in the Ca2+ current may supply this extra Ca2+. Shattock and Bers139 estimated the changes in the reversal potential of Na+/Ca2+ exchange that accompany the action potential and calcium transient in rabbit and rat ventricle. During the action potential, the membrane potential exceeds the reversal potential for the Na+/Ca2+ exchanger, and there is a modest driving force favoring calcium entry during the action potential. This is consistent with the transient extracellular calcium depletions seen by Bers and Macleod138 during contractions in rabbit ventricle. In rat ventricle, where the action potential is shorter compared to the rabbit, there is a driving force for calcium extrusion via the Na+/Ca2+ exchanger when

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55 intracellular calcium is high and the reversal potential for the sodium/calcium exchanger is positive to the membrane potential. This is consistent with the negative FFR at low pacing rates in the rat as I observed at frequencies up to 1 Hz here and in Chapter 3. However, if depolarization is prolonged in the rat myocyte near 0 mV by a voltage clamp pulse, then the negative frequency relationship can be converted to a positive frequency relationship.140;141 This is exactly what is observed in this study in the WKY rat at frequencies greater than 1 Hz where the APD is indeed increased. This is therefore the first direct correlation shown between force and APD response to changes in heart rate in the same experimental protocol. Although several investigators have confirmed the intrinsic positive FFR in the rat at physiologic pacing rates,113;114 they did not consider or measure the underlying electrical responses. Increasing the calcium loading of the myocyte to enhance and maintain elevated steady-state force can be achieved by two mechanisms: favoring net Ca2+ influx via Na+/Ca2+ exchange or by prolonging the duration of the action potential allowing prolonged Ca2+ entry through the L-type calcium channel, both coupled to an enhanced capturing ability of the SR via elevated SERCA activity. In terms of mechanisms underlying the positive limb of the FFR, these data are consistent with enhanced Ca2+ current with increasing heart rate.128;131;142-145 In terms of a control mechanism, the higher SERCA2a activity with increasing heart rate is most likely due to increased phosphorylation of its control protein phospholamban, by CamKII at rates above 1 Hz.31;32 An additional level of SERCA2a control may be at the level of Ca2+ available to the enzyme. During systole, when cytosolic calcium is elevated and SERCA2a is operating rapidly, the rate-limiting step is

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56 probably ATP binding to SERCA2a and the creation of the high-energy state of the enzyme. However, during diastole, low cytosolic calcium is rate-limiting.146;147 Concerning control of the inward calcium current, it has been demonstrated that several kinases, including CamKII, can phosphorylate cardiac calcium channels.148 Whether or not the frequency-controlled increase of the calcium current in the normal heart is through a phosphorylation mechanism or is an intrinsic property of the calcium channel is unclear at this time and the subject of further investigation. In summary, I provide the first combined direct measurements of electrical, calcium, and force data as a function of rate in rat ventricular muscle in order to explore the mechanisms underlying the positive FFR in mammalian heart. These data are consistent with the following model for enhanced contractility as a function of heart rate: The ascending limb of the FFR is controlled by two frequency-dependent synchronized mechanisms. With increasing heart rate, the APD lengthens via the increased inward calcium current. This leads to increased calcium-induced calcium release from the SR and greater force of contraction. The extra Ca2+ coming in through these pathways is taken up into the SR via concomitant enhanced SERCA2a activity that captures and maintains the increased calcium load of the cell allowing for sustained augmented contractility. Mechanistically, I therefore propose a tight coupling between the frequency-dependent increase in inward calcium current and increase in SERCA2a activity to underlie the rate-dependent net increase in force in the normal myocardium at frequencies greater than 1 Hz.

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57 0123456 95100105110115120125130 01234561.82.02.22.42.62.83.0 Peak [Ca2+]iPeak Calcium (% 1 Hz)Peak Developed Force (mN/mm2)Frequency (Hz) Peak Force Figure 4-1. Systolic forceand calcium-frequency relationships in isolated rat papillary muscle.

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58 0123456 95105115125135145 01234565060708090100 ICa (% 0.5 Hz) ICa **** Action Potential DurationAPD80 (ms)Frequency (Hz) Figure 4-2. Action potential duration and integral of Ca2+ current in rat ventricular myocytes as a function of stimulation frequency. APD (to 80% repolarization, n=10) and Ca2+ Current (n=6) were measured in isolated rat LV myocytes at steady state (10th stimulus after frequency change). ICa is shown as percentage of 0.5 Hz value. * APD; ICa p<0.05 from 0.5 Hz value.

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59 0123456 100120140160180200220240260 0123456100120140160180200220240260 Calcium***[Ca2+]i Removal Velocity (% 1 Hz)Force Relaxation Velocity (% 1 Hz)Frequency (Hz) Force Figure 4-3. Force and Ca2+ relaxation velocities in isolated rat papillary muscle. Maximum velocities of force (n=10) and Ca2+ (n=6) in relaxation phase of systolic transients as a function of stimulation frequency, shown as percentage of 1 Hz value. Original units were mN/sec (force) and AU/sec (Ca2+). * Force p<0.05 from 0.5 Hz value.

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60 0102030405060160200240280320360400 1.5 Hz 4 Hz% Pre-Rest ForceSeconds Rest Figure 4-4. Post-rest potentiation in isolated rat papillary muscle. Post-rest potentiation was measured at 1.5 Hz (n=8) and 4 Hz (n=4) stimulation rate after 3-60 sec rest phase. The plateau is obtained after 45-60 seconds rest at 1.5 Hz, but after just 3-7 seconds rest at 4 Hz.

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CHAPTER 5 ALTERATIONS IN CARDIAC FREQUENCY-AUGMENTED CONTRACTILITY AND CALCIUM-HANDLING PROTEINS DURING ADULT AGING Introduction During adult aging, there is an associated decrease in cardiac contractility and relaxation in response to increasing heart rate.71;73;76 These abnormalities have been correlated to a decline in the message74 and expression71 of SERCA that occurs with age and have been partially corrected by exogenous viral SERCA overexpression in rat ventricular myocytes.71 However, duration of the action potential prolongs with old age in all mammalian models studied, postulated to be a result of a decline in the transient outward potassium current (ITO).80 Lengthened APD is accompanied by an increase in inward Ca2+ current through the L-type Ca2+ channels (ICa).80 It is established that APD lengthens in the late-adult Wistar-Kyoto rat near 18 months of age, becoming significantly prolonged by 24 months.80;149 Most studies have observed these changes in either young adult or advanced aged animal models, paying little attention to stages of progression. In the previous chapter, I examined the correlation between SR Ca2+ loading (via SERCA activity) and Ca2+ release (via APD and ICa). It is likely the two mechanisms work in tandem to raise contractile Ca2+ with stimulation rate. In view of this theory, a decline in SERCA appears contradictory to APD prolongation and probably relates to the flattened FFR. I therefore undertook a longitudinal study of aging, performing the first correlation of the FFR and quantification of major Ca2+-handling proteins from the 61

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62 adolescent to the senescent rat. These data define the sequence of events that occur at both the functional and molecular levels in adult aging and support the hypothesis that the depressed FFR relates to a decrease in SERCA activity. Maintained intrinsic contractility may be due to increased ICa that accompanies prolonged APD in aging. Animal Model Male WKY rats, aged 2-31 months, were used in this study. Results Cardiovascular Parameters Basic cardiovascular parameters for the adolescent (2-month) and adult (4-31-month) rat ages are shown in Table 5-1. Systolic blood pressure did not change with age. Both heart (HW) and body weights (BW) increased with age, but the HW did not grow significantly in relation to BW until senescence. LV wall thickness assessed by 2-D echocardiography (LVWT) did not change throughout adulthood. However, myocyte thickness by histology increased with aging, becoming significantly different from the young adult (4-Month) age at 24 months. Time to 80% repolarization of the action potential (APD80) at 0.5 Hz did not significantly change from adolescent (2-Month) to 9 months of age, in keeping with prior data on the WKY which showed an insignificant lengthening in APD from 3 months to 18 months of age.80 LV Contractility with Aging In adolescence (2 months of age), the WKY rat displays a bell-shaped systolic FFR, positive from 1 Hz to 3 Hz, similar to the Sprague-Dawley rat and in the same physiological range of larger mammals (Chapter 3) and of CamKII activation.33;79 Peak force increased from 2.58 0.05 at 1 Hz to 5.38 0.60 at 3 Hz. From 3 Hz to 6 Hz, peak force declined to 4.20 0.60 mN/mm2. In early adulthood (4 months), the FFR remains

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63 bell-shaped (1 Hz: 3.13 0.42; 3 Hz: 5.14 0.54; 6 Hz: 3.58 0.57). At 9 months, the FFR remains positive, but with only a 48% rise over the same stimulation range (1 Hz: 2.99 0.28; 3 Hz: 4.43 0.39). By 16 months, the ascending limb of the FFR had flattened (1 Hz: 3.61 0.47; 3 Hz: 3.53 0.58). It remained flat at 24 (24 months 1 Hz: 3.67 0.44; 3 Hz: 3.49 0.47) and 31 months (31 months 1 Hz: 3.78 0.70; 3 Hz: 3.42 0.67), but developed force at ~1 Hz did not decline (Figure 5-1A). Relaxation of the papillary muscle was quantified by assessing the time from peak force to 50% reduction (RT50) at 3 Hz stimulation rate, the peak of the FFR curve and probable near-maximum SERCA activation.79 As expected, I noted a slowing in relaxation with aging (Figure 5-1B). Ca2+-Handling With Aging Molecular SERCA2a mRNA rose with the transition from adolescence to maturity (2 months: 0.12 0.02 AU; 4 months: 0.25 0.04). SERCA2a mRNA then declined with age throughout adulthood (31 months: 0.09 0.02). PLB mRNA rose with SERCA from adolescence to maturity and remained constant throughout adulthood (2 months: 0.18 0.01; 4 months: 0.30 0.04 AU; 31 months: 0.32 0.04 AU) (Figure 5-2A). The ratio of PLB:SERCA mRNA rose significantly from 4 to 9 months (4 months: 1.19 0.04; 9 months: 1.32 0.02; p<0.05), and rose thereafter throughout aging (31 months: 3.46 0.04) (Figure 5-2B).150 PCR data was corroborated with immunoblotting. Because proteins commonly used as internal standards in quantitative western blot (e.g., actin, calsequestrin) might be altered during cellular hypertrophy, I compared our SERCA2 and pentameric PLB immunoassay to an external standard of LV protein pooled from Sprague-Dawley rats

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64 (STD) (see Chapter 2). As expected, I found that protein expression generally followed transcript. SERCA expression rose slightly from adolescence to maturity (2 months: 83 7%; 4 months: 99 14%), but did not change (~100% STD) from 4 to 9 months, and declined at later ages (16 months: 71 9%; 24 months: 64 11%; 31 months: 71 8%, p<0.05 from 9-month age). PLB in the pentameric form varied but did not change dramatically throughout life (2 months: 98 3%; 31 months: 126 17%) (Figure 5-2C). Like mRNA, the PLB:SERCA protein ratio was greatly elevated in late adulthood (4 months: 0.73; 31 months: 1.77, as mean percentages of STD) (Figure 5-2D).150 Heart rate potentiation of SERCA activity As a measure of frequency-potentiated SERCA SR filling ability,106;151 I performed post-rest potentiations (PRP) and compared the rest period required to reach the maximum relative increase in systolic force. By comparing two frequencies, near the minimum (1.5 Hz) and maximum (3 Hz) of the FFR, I extrapolated the ability of the SR to increase its Ca2+-sequesteration with frequency. Rest duration required to reach maximum PRP (SR Ca2+ load) decreased with frequency to 9 months of age, although the difference in required rest duration between frequencies declined (2 months: 42 s; 4 months 35; 9 months: 31.7 8.8 from 3 Hz to 1.5 Hz maximum). At 16 months, the SR was filled during the same duration of rest at 1.5 and 3 Hz (1.5 Hz: 16.2 3.3 s; 3 Hz: 8.2 1.6 to maximum, p>0.05), displaying a lack of SERCA frequency potentiation. Frequency potentiation of SERCA returned slightly at 24 months (24 months: 14.5 2.8; 31 months: 15.0 3.9 from 3 Hz to 1.5 Hz maximum, p<0.05) (Figure 5-2E). Discussion To the extent similar studies have been performed in humans, the aging rat displays comparable physiology with normotensive aging in humans. In both species,

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65 cardiomyocytes enlarge, but ventricular hypertrophy is modest. Contractility of the myocardium is maintained at the lower heart rates;64 but the force-frequency-relationship, augmenting with frequency in youth (Chapter 3), becomes less positive, reducing maximum cardiac output with exertion.64 Cardiac relaxation slows,65 and action potential prolongs, manifested as a longer QT interval in humans.152 The study represents the most comprehensive exploration of the contractile response to frequency and how it is altered in aging to date. The focus of this study was to correlate age-related physiologic changes to their molecular basis, with special attention to intracellular Ca2+ handling. Cardiovascular Parameters In the aging WKY, systolic blood pressure did not change with age to 31 months, allowing me to study the age-related changes independently of pressure-overload effects. Senescent rats were hypertrophied at the cellular but not the organ level. LV myocyte thickness increased although LVWT did not, and HW/BW ratio did only at the oldest age. This observation is probably due to age-related loss of cells coupled to hypertrophy of the remainder.64 APD data are consistent with prior studies80;153 and are unchanged from 2 to 9 months (Table 5-1). LV Contractility With Aging The FFR represents the intrinsic ability of the myocyte to increase its contractility in response to frequency. In humans, the FFR accounts for up to 40% of the increase in cardiac output during exercise.154 During maximal work output, cardiac output is increased ~3x above resting level. An increase in heart rate is responsible for ~70% of this augmentation of cardiac output. The remaining 30% is the result of an increase in stroke volume despite a fall in preload that would normally reduce contractility through

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66 the Frank-Starling relationship. An impairment of the FFR response probably contributes to the progressive loss in maximum aerobic capacity that occurs with advanced aging.2 To my knowledge, I show the first sequence of FFRs performed over normal aging. The systolic FFR seen in the young adult WKY is strongly positive over the same physiological range as another rat strain (Sprague-Dawley), healthy humans, and dogs (Chapter 3). This suggests that the study of the FFR in rat and its molecular determinants may contribute to a better understanding of human physiology. The intrinsic contractility of the rat LV muscle is maintained through aging, but the ability to have an inotropic response to frequency is progressively diminished, as seen in Figure 5-1A. This finding is in keeping with others in the literature.71;73;76 Additionally, there is a continual decline in the ability of the rat papillary to relax after contraction as seen in Figure 5-1B. This, too, is supported by the literature.73 In fact, the evaluation of relaxation and its implication for diastolic dysfunction has been the major focus of studies on contractility in aging. Ca2+-Handling with Aging Molecular In terms of mechanisms for the depressed FFR with aging, it seems likely that the loss of SERCA activity by a decline in the absolute level of the protein and/or rise in the PLB/SERCA ratio may be responsible (Figure 5-2A,B). Meyer et al.35 and Koss et al.36 have reported that the uplimb of the FFR is controlled by SERCA activity as determined by PLB:SERCA ratio. The use of an external standard for SERCA and PLB western blots, while allowing us to visualize protein concentrations despite cellular enlargement, is not as precise as an internal standard, such as that used for mRNA expression. SERCA was significantly reduced with the flattening of the FFR, but a continued decline in

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67 SERCA after 16 months of age, as seen by PCR, may not be measurable by westerns. Many investigators have reported a decrease in the level of SERCA70;74;75 and/or an elevation in PLB73 with aging, but have not shown its physiologic significance to force in a longitudinal fashion. Reductions in phosphorylation of PLB and SERCA have also been observed in aged rats, which would have the effect of further reducing SERCA activity at ages at which I saw little further decline in SERCA expression or PLB:SERCA protein ratio. CamKII expression and activity, the kinase of PLB,21 SERCA,155 and the ryanodine receptor21 have been shown to be reduced in WKY at 24 months by western blot and phosphorylation assay.22 Heart rate potentiation of SERCA activity The augmentation of systolic force following a rest period is an index of the amount of contractile Ca2+ released from the sarcoplasmic reticulum,105 and the rest period required to reach a predetermined relative increase in systolic force has been used by Pieske et al.106 and Bluhm et al.151 as an indication of SERCA2a SR-filling activity. When performed at different frequencies, post-rest potentiation experiments are a qualitative measure of relative SR Ca2+ content, and thus indirectly the frequency potentiation of SERCA.105 To 9 months of age, less time of rest was required to load the SR with increased stimulation rate, indicating that SERCA Ca2+ sequestering activity was higher at greater frequencies. However, the difference between 1.5 and 3 Hz diminished with age. At 16 months of age, no further SR loading occurred with greater frequency, supporting an elimination of SERCA frequency-potentiation seen in the FFR. This may be due to reduced SERCA activity at higher frequencies or maximized activity at lower heart rates, although FFR data support reduced activity at higher rates. A small

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68 augmentation of SERCA activity with frequency returned at 24 months, after the lengthening of the APD80;149 (Figure 5-2E). Proposed Sequence/Role of APD Early adult aging I observed depressed SERCA mRNA at 9 months and protein at ages beyond 9 months. The ratio of SERCA’s inhibitor, PLB, to SERCA rises in both mRNA and protein at 9 months, the same age as the beginning of adult FFR depression (Figure 5-2A,B). This also correlates to the reduction of SR Ca2+ uptake at 9 months, as assessed by PRP (Figure 5-2C). I also show in this data that the APD at 0.5 Hz is unchanged through 9 months of age in the rat myocyte (Table 1). Other studies have shown that APD is unchanged through 18 months of age.80 I therefore conclude that the decline in the positive FFR that begins in adulthood (9 months) is likely due to reduced SERCA total expression and/or its ratio to its inhibitor with aging, leading to reduced frequency potentiation and dampened FFR. My key finding is the temporal relationship of a progressive loss in SERCA prior to electrical remodeling in the aged myocyte. Late adult aging As SERCA levels and activity continue to fall at ages beyond 9 months, there is total negation of force augmentation with increasing heart rate. Further, one would expect intrinsic contractility to fall, but it is maintained. It is well-established that the action potential in the WKY rat prolongs between 18 and 24 months.80;149 This is after SERCA’s ability to augment contractility with frequency begins to decline (9 months). The longer APD yields a greater voltage-gated inward Ca2+ current149 and augments contractility in the senescent animals.

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69 As shown by Janczewski et al.,149 the age-prolonged APD allows greater SR Ca2+ content, calcium-induced Ca2+-release, and Ca2+ transients than would otherwise be possible in senescent myocytes. They concluded that APD prolongation preserved intracellular Ca2+ handling despite reduced SERCA activity. This hypothesis may explain why absolute force production (mN/mm2) at lower heart rates is not diminished with advanced age (>16 months, Figure 5-1A) despite reduced SERCA expression and activity. Between 16 and 24 months, the APD lengthens.149 ICa prolongation is likely an adaptation to maintain contractility to compensate for reduced SERCA expression and activity. APD prolongation may provide increased Ca2+ influx over a longer duration which is able to be sequestered by the remaining SERCA activity. APD and Cellular Hypertrophy A decline in SERCA expression and activity would lead to a rise in diastolic Ca2+. A rise in cytosolic Ca2+, especially diastolic Ca2+, is a powerful regulator of gene expression,84 which we expect could affect APD by three possible, not mutually exclusive, mechanisms. First, [Ca2+]i could signal a downregulation of repolarizing IK+ channels, particularly ITO. Second, a reduction in SERCA SR loading could reduce SR Ca2+ release via the ryanodine receptor and thus affect Ca2+ channel inactivation,89 making intracellular Ca2+ handling self-regulating.149 Finally, cellular hypertrophy itself has been theorized to cause a geometric rearrangement between the Ca2+ channel and ryanodine receptor in severe hypertrophy due to hypertension, thus reducing Ca2+ channel inactivation.20;156 Cellular hypertrophy in the aged animal is often attributed to haemodynamic stresses related to a decrease in aortic compliance.64 However, cellular hypertrophy may

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70 partly be a direct consequence of elevated diastolic Ca2+ in the aged animal. The sustained-Ca2+ sensitive phosphatase calcineurin is activated by an elevation in Ca2+ plateau;56 the hypertrophy-linked PKC and JNK pathways by calcineurin;57 and the PKCpathway by increased DHPR density55 in transgenic mice. Calcineurin activity has been shown to be an early mediator of the hypertrophy effects of angiotensin II and phenylephrine157 and can activate the PKC pathway.57 Conclusions The FFR depression with adult aging into senescence is probably due to a correlated decline in SERCA and the corresponding rise in the PLB:SERCA ratio and fall in SERCA SR-filling activity. Eventually, this decline leads to total abolition of frequency-augmented contractility, yet systolic force is preserved. At this point, one would expect intrinsic contractility to fall, but I postulate it is maintained by the prolonged APD and ICa seen in advanced aging.

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71 Table 5-1. Cardiovascular and hypertrophy parameters in aging WKY rat. n=3-13. p<0.05 from 2-Month (adolescent); 4-Month (young adult) age. 2-Month 4-Month 9-Month 16-Month 24-Month 31-Month Ejection Fraction (%) 81 1 77 3 71 2 74 3 78 2 73 2 Systolic Blood Pressure (mmHg) 125 3 127 2 132 3 128 2 130 3 125 3 Heart Weight (mg) 972 106 1406 60 1658 80 1890 80 1785 60 1654 40 Body Weight (g) 184 18 341 16 397 10 458 7 421 17 448 12 Heart/Body Ratio (mg/g) 5.4 0.4 4.1 0.1 4.1 0.1 4.1 0.2 4.3 0.2 3.7 0.1 Wall Thickness (cm) 0.139 0.004 0.161 0.009 0.182 0.008 0.166 0.002 0.166 0.004 0.156 0.005 Mean Cardiomyocyte Cross-Sectional Area (m2) 197 16 289 15 299 15 357 32 396 46 436 43 APD80 (ms) (0.5Hz) 55 10 55 7 100 (estimated from Wei et al.153)

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72 0123456234567 ADeveloped Force (mN/mm2)Frequency (Hz) 2 4 9 16 24 31 249162431 40 45 50BAge (Months)RT50 Figure 5-1. Systolic force-frequency behavior in aging rat myocardium. (A) Systolic force-frequency relationships of isolated papillary muscle from aging rats. (B) Time from peak developed force to 50% relaxation from peak value at 3 Hz. n=5-12.

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73 249162431249162431 0.0 0.1 0.2 0.3 0.4 0.5A***SERCAPLBmRNA Level (vs. 18S) 051015202530351.01.52.02.53.03.5 BPLB:SERCA Ratio (AU)Age (Months) 249162431249162431 0 50 100 150 B***SERCAPLBProtein Level (% STD) 051015202530350.60.81.01.21.41.61.82.02.2 DPLB:SERCA Ratio (% STD)Age (Months) Figure 5-2. SERCA expression and activity in aging rat LV. (A) mRNA quantification of SERCA2a and PLB by PCR. n=4-6. * p<0.05 from 4-month age. (B) Ratio of PLB and SERCA2a and mRNA. (C) Protein quantification of SERCA2 and PLB by Western blot, compared to rat LV STD. n=3-6. * p<0.05 from 9-month age. (D) Ratio of SERCA2 and PLB protein. (E) Difference in rest required to maximum increase in systolic force (maximum PRP) from 1.5 and 3 Hz . n=4-7. Note: Duration required for 2and 4-month to reach maximum PRP at 1.5 Hz was greater than 60 s, the longest examined period, i.e. the difference was >45 s for 2-month-old and >35 s for the 4-month-old rats.

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74 249162431 10 20 30 40(See Note)(See Note)E*****Age (Months)Difference in time toMax. PRP,1.5 to 3 Hz (sec) Figure 5-2. Continued.

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CHAPTER 6 ELECTRICAL CONTROL OF FORCE PRODUCTION IN HYPERTROPHIED MYOCARDIUM: ROLES OF ACTION POTENTIAL DURATION AND CALCIUM PUMP ACTIVITY Introduction The preceding study in aged rats showed the molecular changes of SERCA expression/activity and APD and the concurrent elimination of the positive FFR occur simultaneously as enlargement of the cardiomyocytes. Another condition known to produce hypertrophy of cardiomyocytes is hypertension. Like aging, the positivity of the FFR is altered158 and contraction and relaxation are slowed.47 Unlike aging, however, hypertrophy secondary to hypertension is generally recognized as occurring as part of a continuum of adaptations leading to eventual systolic failure if the hypertension is left untreated. The time course of these changes and the pathway from compensatory to decompensatory hypertrophy are unknown because the majority of the few studies in unaltered hypertension examined young and failing animals. Reduced SERCA expression and activity could explain these characteristics and have been shown to be reduced in failure, but for the same methodological reason it is unknown what role Ca2+ sequestration decline plays in altered function. I next sought to correlate the FFR with APD, ICa, and activity of SERCA in hypertrophy secondary to hypertension. To facilitate comparisons to the adaptations of aging, I chose the 9-month-old hypertensive SHR rat, which displays cardiomyocyte size similar to the senescent WKY and no sign of the decompensatory trend to failure. 75

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76 Animal Model Male SHR (experimental) and WKY (control) rats, aged 2 and 9 months, were used in this study. Results Cardiovascular Parameters Basic cardiovascular parameters for the pre-hypertensive (2-Month) and hypertrophied (9-Month) SHR ages are shown in Table 6-1 with normotensive age-matched WKY. Cardiac systolic function, by LV ejection fraction, was preserved in all animals. Systolic blood pressure was normal in the 2-Month WKY, 2-Month SHR, and 9-Month WKY, but greatly elevated in the 9-Month SHR. Pre-hypertensive SHR were identical to age-matched WKY by all markers of cardiac hypertrophy, but 9-Month SHR had cardiac hypertrophy at the organ (HW/BW ratio), LV (wall thickness), and cellular (cell thickness) levels. Cell thickness in the hypertrophied SHR was not different from 24and 31-Month WKY (24 month WKY: 396 46 m2; 31 month: 436 43, p>0.05), but the SHR, like the senescent WKY, did not display any indication of decompensatory hypertrophy (i.e., lowered EF, physical weakness). Forceand Ca2+Frequency Relationships At 2 months of age, the SHR and WKY display similar bell-shaped systolic FFR curves, positive from 1 Hz to 3 Hz, which is the same physiological range as larger mammals. Peak force ~doubled from 1 to 3 Hz (WKY 1 Hz: 2.58 0.05, 3 Hz: 5.38 0.60; SHR 1 Hz: 3.05 0.19, 3 Hz: 5.62 0.68) (Figure 6-1A). At 9 months, the WKY FFR remains positive over the same stimulation range as the pre-hypertensive animals, but to a lesser degree (1 Hz: 2.99 0.28; 3 Hz: 4.43 0.39). The hypertrophied SHR displays greater systolic force than WKY in the lower

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77 range of 0.2 to 2 Hz (0.2 Hz; 7.04 0.61; 0.5 Hz: 5.51 0.57; 1 Hz: 4.57 0.48; 2 Hz: 4.66 0.40, p<0.05 at 1 Hz) but not at 3 Hz and above (3 Hz: 4.32 0.25; 4 Hz: 3.54 0.27; 5 Hz: 2.99 0.26, 6 Hz: 2.40 0.24). This elevation in force at lower frequencies produces a flattened FFR curve in which systolic force is not further enhanced by increasing stimulation rate (Figure 6-1B). Due to the linear relationship between systolic [Ca2+]i and developed force (Figure 3-2), the elevated developed force at low frequencies probably reflects greater systolic Ca2+ transients. Unfortunately, the experimental protocol employed allows measurement of changes in relative systolic [Ca2+]i (Chapter 3) but not absolute values. Thus, I was unable to statistically compare [Ca2+]i in hypertrophied SHR and age-matched WKY. Representative force and Ca2+ transient traces, however, are displayed in Figure 6-2. These two individuals were representative of their strains in the shape of their FFR curves and similar in developed force and Ca2+ transient heights at 3 Hz (3 Hz differences: force<10% mN/mm2; [Ca2+]i,<4% AU), at which frequency WKY and SHR mean systolic forces are ~equal. Like developed force, hypertrophied SHR displayed greater systolic [Ca2+]i at low stimulation rates and less increase in peak Ca2+ with frequency than WKY. APDand ICa-Frequency Relationships To understand the possible mechanisms involved in this Ca2+ and force elevation at lower frequencies in the hypertrophied SHR, I evaluated the APD and its largest contributor, the inward calcium current (ICa) with respect to frequency (APD-FR and ICa-FR) in isolated myocytes from the LV. APD increased with frequency (by posttest) in all non-hypertrophied animals in a manner similar to the FFR within the range of 0.2-4 Hz (2-Month WKY 0.2 Hz: 48 7 ms, 3 Hz: 75 14; 2-Month SHR 0.2 Hz: 56 6, 3 Hz:

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78 91 12; 9-Month WKY 0.2 Hz: 50 5, 3 Hz: 87 10). The APD in hypertrophied SHR cardiomyocytes, however, was prolonged at lower frequencies and did not lengthen with rate (9-Month SHR 0.2 Hz: 85 12, 3 Hz: 75 5), creating a flattened shape similar to the FFR (Figure 6-3A,B). Comparison of the ICa in the hypertrophied SHR to the age-matched WKY reveals a much greater ICa integral at all frequencies in the hypertrophied SHR (WKY 0.2 Hz: 13.6 3.5 –nA x ms, 0.5 Hz: 15.3 3.4, 1 Hz: 17.0 3.5, 2 Hz: 18.9 3.8, 3 Hz: 19.9 4.3, 4 Hz: 20.1 4.3, 5 Hz: 19.8 4.5, 6 Hz: 18.9 4.4; SHR 0.2 Hz: 27.4 1.0, 0.5 Hz: 29.4 1.5, 1 Hz: 32.2 1.4, 2 Hz: 34.0 1.4, 3 Hz: 34.2 1.1, 4 Hz: 33.2 1.2, 5 Hz: 33.0 1.2, 6 Hz: 31.8 1.2) (Figure 6-3C). Additionally, ICa is not as greatly enhanced by frequency in the hypertrophied SHR as in the non-hypertrophied WKY (WKY 3 Hz: 155 8%; SHR 3 Hz: 125 3% of 0.2 Hz value) (Figure 6-3D). Therefore, changes in the ICa response to frequency also paralleled the APD and FFR in the hypertrophied animals.159 Frequency Dependence of SERCA2a Activity Because of the prominent role of frequency-augmentation of SERCA in the positive mammalian FFR within the same range as developed force was elevated in the hypertrophied SHR (1-3 Hz) (Chapter 3), I performed three measurements closely related to relative SERCA activity—the maximum velocity of the systolic force and Ca2+ transient decline (FRV, CaRV) and PRPs. There is a close correlation between the FRV response to frequency and the FFR in the 9-month groups (Figure 6-4A). Relaxation velocities are higher at lower frequencies and augment less with frequency in the hypertrophied SHR (WKY 0.2 Hz: 49.68 8.06 mN/s, 1 Hz: 33.35 4.98, 3 Hz: 55.57 6.85, 6 Hz: 50.02 5.43; SHR 0.2 Hz: 73.13 11.90, 1 Hz: 42.14 6.80, 3 Hz: 50.44 5.80, 6 Hz: 37.87 1.64)

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79 I next measured the maximum rate of removal of systolic Ca2+ from the cytoplasm, which has been shown to be 92% due to SERCA pumping activity in the rat.17 Systolic Ca2+ transient decline velocities accelerate with frequencies above 1 Hz in all groups studied. Pre-hypertensive SHR and age-matched WKY exhibit a ~70% increase in CaRV from 1 to 6 Hz (6 Hz: SHR 174 2%, WKY 172 14% 1 Hz value). There is an age-enhancement of CaRV (9-Month WKY 6 Hz: 230 20%) that hypertrophy does not alter (9-Month SHR 6 Hz: 241 12%). (Figure 6-4B). The CaRV indicates an increase in relative SERCA SR-filling activity with frequency in all groups studied; however, it does not indicate absolute activity at the lower heart rates. PRP data at frequencies near the base (1.5 Hz) and peak (3 Hz) of the FFR are presented in Figure 6-4C. Rest duration required to obtain maximum PRP decreased with frequency in all non-hypertrophied animals studied (2-Month WKY 1.5 Hz: >60 s, 3 Hz: 11 2; 2-Month SHR 1.5 Hz: >60 s, 3 Hz: 18 7; 9-Month WKY 1.5 Hz: 41 6, 3 Hz: 16 5). Hypertrophied SHR reaches maximum PRP force (maximum SR Ca2+ load) at 1.5 Hz during a similar rest period as non-hypertrophied animals at 3 Hz, and time to maximum PRP does not further shorten with an increase in frequency (9-Month SHR 1.5: 18 3, 3 Hz: 15 6). SERCA and PLB Expression Protein expressions of SERCA and its inhibitor PLB (pentameric form) were not different between SHR and WKY at either age group (2-Month WKY SERCA: 92 725 7 544 densitometry units, PLB: 142 157 3 983; 2-Month SHR SERCA: 101 419 4 144, PLB: 157 563 8 181) (9-Month WKY SERCA: 62 705 5 006, PLB: 105 579 7 185; 9-Month SHR SERCA: 63 598 4 698, PLB: 127 561 12 296) (Figure 6-5A,B). The ratios of PLB to SERCA were not different between SHR and WKY at either age

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80 group (2-Month WKY: 1.57 0.14; 2-Month SHR: 1.56 0.13) (9-Month WKY: 1.61 0.17; 9-Month SHR: 2.00 0.09) (Figure 6-5C).159 Discussion Cardiovascular Parameters The young animals in both the WKY and SHR groups were not hypertensive, were identical in all measures of hypertrophy, and had preserved systolic function by ejection fraction. The 9-Month SHR were significantly hypertrophied by HW/BW ratio, LV wall thickness, and mean cross-sectional area of the myocytes compared to age-matched WKY but continued to exhibit normal systolic function. Forceand Ca2+Frequency Relationships The pre-hypertensive SHR and age-matched WKY exhibit identical FFR curves from 1 to 6 Hz. The FFR of the hypertrophied SHR displays augmented systolic force at the lower frequency range studied. This enhanced systolic force at the lower frequency ranges flattens the FFR, and the ascending limb is eliminated (Figure 6-1). Representative calcium transients parallel the changes in contractility. Hypertrophied SHR showed greater peak systolic calcium at low frequencies and less augmentation with frequency than age-matched WKY (Figure 6-2). Also, the lack of force reduction is evidence that this age is not in decompensatory hypertrophy. APDand ICa-Frequency Relationships In Chapter 4, I showed that systolic force is probably augmented by greater APD and ICa, possibly via enhancement of the trigger for SR Ca2+ release and increased systolic calcium loading of the myocyte. Analysis of the APD and ICa yields a possible mechanism for the enhanced calcium transients at low frequencies in the hypertrophied SHR. There is a strong correlation between the shape of the APD-FR and the FFR curves

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81 in all animals (Figure 6-3A,B). Stilli et al.160 noted an increased APD with hypertrophy in a rat model of left ventricular hypertrophy with aortic banding. Inward Ca2+ current through the L-type Ca2+ channel, the largest contributor to the APD, in hypertrophied SHR is much greater at all frequencies and shows less augmentation with frequency than age-matched WKY (Figure 6-3C,D). Wang et al.161 observed increased inward calcium current density in a mouse model with aortic banding. Frequency Dependence of SERCA2a Activity SERCA activity appears to be elevated and able to sequester most or all of this increased influx Ca2+. FRV data support an elevation in contractile Ca2+ removal at low frequencies (Figure 6-4A). However, the FRV has many contributing factors, such as Ca2+ efflux mechanisms and stiffness due to collagen deposits, in addition to SERCA and is not an ideal measure of SR filling. CaRV is likely a more direct measure of SERCA activity because 92% of Ca2+ removed from the myoplasm occurs by uptake into the SR in the rat.17 In hypertrophy, the CaRV keeps its ability to augment with frequency as the non-hypertrophied groups and is not different from the age-matched WKY (Figure 6-4B). A limitation of CaRV is it can only be calculated as a relative increase from a baseline, unlike FRV in which absolute values are available. It cannot be used to compare the basal activity of SERCA. The augmentation of isometric force following a rest period (PRP) is an index for the amount of activator calcium released from the sarcoplasmic reticulum.105 The rest period required to reach a predetermined relative increase in systolic force has been used by Pieske et al.106 and Bluhm et al.151 as an indication of SERCA SR-filling activity. PRP data suggest the SR reaches maximum Ca2+ loading faster at low frequencies in the hypertrophied than the non-hypertrophied groups. This may be due to either higher

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82 SERCA activity at lower rates or an inability to increase basal activity of SERCA in hypertrophy. FFR data indicates the higher SERCA activity at lower rates. Enhanced Ca2+ transport into the SR has been observed in mild cardiac hypertrophy in banded rats.162 One possible mechanism for enhanced activity is increased Ca2+ substrate available for the SERCA enzyme. During low Ca2+ states, such as diastole, the binding to SERCA of the two Ca2+ ions required for ATPase activation is the rate-limiting step of Ca2+ transport. During higher Ca2+ states, such as systole, transport rate is probably limited only by ATP availability and binding.146;147 SERCA and PLB Expression Although basal SERCA activity appeared elevated at the level of control, I also examined the amount of protein present. There was no change in hypertrophy compared to age-matched control in SERCA expression or in their ratio. This is further evidence that the 9-Month SHR is not in systolic failure, in which SERCA reduction has been reported. 86 88 During compensatory hypertrophy due to renal artery banding, there was no change in SERCA or PLB concentration observed by Chang et al.42 No difference in SERCA mRNA was seen in non-failing hypertrophied SHR compared to WKY by Boluyt et al.163 Contrast to Hypertrophy with Aging Although both hypertrophied SHR and hypertrophied WKY display a flattened FFR, these FFR and molecular data contrast to the preceding study of cardiac aging. Systolic force at the peak of the FFR (3 Hz) was progressively depressed with adult aging, probably partly due to a decline in SERCA and the corresponding rise in the PLB:SERCA ratio. Eventually, this decline leads to total abolition of frequency-augmented contractility. Both groups exhibit a lack of rate-enhanced SR loading, but for

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83 apparently opposite reasons. SR filling rate in the senescent WKY probably remains low, while in the hypertensive WKY it cannot rise much more. Essentially, in compensatory hypertrophy secondary to hypertension, systolic force at lower frequencies rise, whereas in aging there is a flattening of the ascending limb of the FFR. Potential Mechanisms of Enhanced ICa In the current study, I show hypertrophy secondary to hypertension is accompanied by an increase in L-type inward Ca2+ current at all frequencies. Slowed inactivation of Ca2+ channels may partially explain both this and the overall prolonged APD. Depression of the transient outward potassium current (ITO) has been described in models of hypertrophy.47;164 By prolonging the APD, there may be a tendency towards increased ICa. Another possible mechanism is that cellular hypertrophy itself may cause a geometric rearrangement between the Ca2+ channel and ryanodine receptor in hypertension, thus reducing Ca2+ channel inactivation by SR Ca2+ release.20;156 Consequences for SERCA Gene Therapy Although investigators have shown improvement in contractile function by SERCA overexpression in animal models of aging71 and systolic failure,87 this study calls into question its value for hypertrophy due to peripheral hypertension. Because hypertrophy is linked both to increased risk for morbidity and mortality39 and to reduced SERCA expression/activity,86 there exists a current impetus to overexpress SERCA in pressure-overload animal models.165 The FFR is flattened, usually viewed as a pathological symptom, but my data indicate that systolic force is actually enhanced, so cardiac output is maintained. Also, during the electrical remodeling stage that occurs in this form of hypertrophy, SERCA expression is not depressed, and activity seems higher. The SR

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84 appears loaded even at low frequencies. At this stage, SERCA overexpression would probably be of little functional value. Conclusions In hypertrophy secondary to peripheral hypertension, systolic force increases at lower rates yielding a flattened FFR, likely due to an early electrical remodeling that is evidenced by a prolonged APD and enhanced ICa. This is paralleled by an increase in basal SERCA activity that likely captures the increased Ca2+ influx and allows sustained increases in force by increasing SR loading.

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85 Table 6-1. Cardiovascular and hypertrophy parameters of 2-month and 9-month WKY and SHR rats. n=6-13. p<0.05 from same-strain 2-Month; * same-age WKY. 2-WKY 2-SHR 9-WKY 9-SHR Ejection Fraction (%) 81 1 78 2 71 2 68 2 Systolic Blood Pressure (mmHg) 125 3 123 2 132 3 182 3 * Heart Weight (mg) 972 106 883 64 1658 80 1891 76 * Body Weight (g) 184 18 181 16 397 10 388 5 Heart/Body Ratio (mg/g) 5.4 0.4 5.0 0.3 4.1 0.1 4.9 0.2 * LV Wall Thickness (cm) 0.139 0.004 0.138 0.003 0.182 0.008 0.268 0.008 * Mean Cardiomyocyte Cross-Sectional Area (m2) 197 16 225 12 299 15 489 41 *

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86 01234562.02.53.03.54.04.55.05.56.06.57.07.5 ASystolic Force (mN/mm2)Frequency (Hz) WKY SHR 01234562.02.53.03.54.04.55.05.56.06.57.07.5 BSystolic Force (mN/mm2)Frequency (Hz) Figure 6-1. Systolic force-frequency relationships in isolated rat papillary muscle.(A) Pre-hypertensive (2-Month) SHR (n=6) and control WKY (n=7). (B) Hypertrophied (9-Month) SHR (n=9) and control WKY (n=10).

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87 1 mN/mm2 A 0.2 Hz 9-M WKY 9-M SHR 0.5 Hz 1 Hz 2 Hz 3 Hz 4 Hz 5 Hz 6 Hz Figure 6-2. Representative forceand calcium-frequency relationships in 9-month-old WKY (thick line) and SHR (thin line) LV muscle from 0.2 to 6 Hz over 300 ms from stimulation pulse. (A) Developed force. (B) Cytosolic calcium. Like the means for the two strains, systolic force for this individual SHR was greater than this individual WKY at stimulation rates below 3 Hz and less than WKY beyond 3 Hz. At 3 Hz, systolic forces and cytosolic Ca2+ transient peaks were similar (systolic force<10% mN/mm2; [Ca2+]i<4% AU). SHR contraction was delayed at the higher frequencies; slowed contraction is a common characteristic of hypertrophy.

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88 400 AU 9-M WKY 9-M SHRB 0.2 Hz 0.5 Hz 1 Hz 2 Hz 3 Hz 4 Hz 5 Hz 6 Hz Figure 6-2. Continued.

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89 0123456405060708090100110 ********A 2-Month WKY 2-Month SHRAPD80 (ms)Frequency (Hz) 0123456405060708090100110 ** 9-Month WKY 9-Month SHR****BAPD80 (ms)Frequency (Hz) 0123456913172125293337 ************CICa(L) (-nA x ms)Frequency (Hz) 9-Month WKY 9-Month SHR 0123456100110120130140150160170 ************DICa (Integral, % 0.5 Hz)Frequency (Hz) 9-Month WKY 9-Month SHR Figure 6-3. Action potential duration (to 80% repolarization) and integral of Ca2+ current in rat ventricular myocytes as a function of stimulation frequency. APD-FR in (A) Pre-hypertensive (2-Month) SHR (n=4) and (B) hypertrophied (9-Month) SHR (n=10) and control WKY (2 month n=4, 9 month n=10). ICa (C) values and (D) percentage of 0.5 Hz value in hypertrophied SHR (n=5) and control WKY (n=6). * p<0.05 greater than 0.2 value by posttest. SHR p<0.05 from WKY.

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90 012345630405060708090 A WKY SHRForce Relaxation Velocity (mN/sec)Frequency (Hz) 012345680110140170200230260 *B 2-Month WKY 2-Month SHR 9-Month WKY 9-Month SHR% 1 Hz ValueFrequency (Hz) 0 10 20 30 40 50 601.5 Hz 3 Hz ** 2-WKY 2-SHR9-WKY9-SHRTime to Maximum PRP(sec)*CSee Legend Note Figure 6-4. Measures of relative SERCA activity in hypertrophied and control in rat LV muscle. (A) Maximum velocities of relaxation of the systolic force transients. SHR n=6; WKY n=9. (B) Maximum velocities of systolic [Ca2+]i removal during relaxation; percentage of 1 Hz value. Original units were AU/sec. 2-Month SHR n=4; WKY n=5. 9-Month SHR n=6; WKY n=6. p<0.05 from prehypertensive SHR; * 2-Month WKY. (C) Rest duration required to obtain maximum relative systolic force increase. n=4-8. * p<0.05 from duration required at 1.5 Hz. Note: Duration required for 2-month SHR and WKY to reach maximum PRP at 1.5 Hz was greater than 60 s, the longest examined period.

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91 2-Month WKYSHRWKYSHR 0 100000 200000SERCA PLB ASERCAPLBDensitometry Units 9-Month WKYSHRWKYSHR 0 50000 100000 150000BSERCAPLBDensitometry Units WKYSHR 0.0 0.5 1.0 1.5 2.0 2.5C2-MonthPLB:SERCA Ratio WKYSHR 9-Month Figure 6-5. Expression of SERCA and pentameric PLB protein. (A) Pre-hypertensive (2-Month) SHR (SERCA n=5, PLB n=6)and control WKY (n=5). (B) Hypertrophied (9-Month) SHR (n=6) and control WKY (SERCA n=6, PLB n=5). (C) PLB:SERCA ratio.

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CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS Conclusions These studies examined the fundamental basis of the cardiac force-frequency relationship (FFR) and its alterations with aging and adaptation to peripheral hypertension. In the process of developing an experimental protocol that would allow hypertensive rats to be used as a model of human hypertension, the role of the sarcoplasmic reticular (SR) Ca2+ pump (SERCA) in the FFR has been more fully explored, and an explanation for the inconsistency of earlier rat studies was presented. The view of SERCA as the cornerstone of contractility and the positive FFR was supported by the consequences of its over-activation and inhibition. However, the SR (i.e., SERCA) is only one of two sources of contractile Ca2+; the inward Ca2+ current (ICa) serves as both the instrument of SR Ca2+ release and a contributor to force development. The new finding of the importance of the action potential and ICa explains the changes in the heart that occur with hypertrophy, which could not be fully explained by previous investigations. The primary experiment for the study of cardiac function and the interaction of the molecular components is the FFR, the effect of stimulation rate on systolic force between ~1 Hz and ~3 Hz24 (positive in healthy larger mammals). Most pathological conditions flatten or even invert the FFR, providing both a diagnostic tool26 and causing the common symptom of exhaustion upon exertion.25 Most evidence for the cause of the 92

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93 positive FFR has pointed to an upregulation of SERCA activity within that frequency range,31-34 increasing recycling of systolic Ca2+ for subsequent contractions. However, full acceptance of this theory has been hindered by the fact that most in vitro myocardial preparations from small rodents have displayed a negative FFR,108;109 despite a great similarity between Ca2+ handling proteins between rats and larger mammals (Table 3-2). Many researchers have thus sought another origin of the FFR, such as intracellular Na+ and its known concentration differences in smaller species.17 I observed that with essentially the same experimental protocol, the FFR of the rat is very similar to that of the human in shape and magnitude (systolic force ~doubling between 1 and 3 Hz) (Figure 3-1). Additionally, sizes of intracellular systolic Ca2+ transients and peak systolic force are linearly related (Figure 3-2), as they have been shown to be in healthy and failing human cardiac tissue.119;120 This further supports the presumption that the cardiomyocytes are behaving normally. Thus I conclude that the rat is an acceptable experimental model for cardiac studies. A remaining question is why rat heart tissue has traditionally displayed a negative FFR. I believe the answer lies in the assumed role of SERCA in the FFR and its ability to strongly shift the FFR if its activity is altered. The key of my experimental protocol was the loading, by passive diffusion, of the intracellular Ca2+ indicator dye Fura-2. This process involves allowing the tissue to remain unmolested for up to 4 hours before performing the FFR experiment. Prior to loading, the myocardium displays a traditionally negative rat FFR but after loading displays a typically mammalian positive FFR, which also occurs if the papillary remains for 4 hours without Fura-2 loading. The pre-Fura

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94 loading FFR is not negative because of diminished force at the peak range, but increased force at the subphysiological range (Figure 3-1). Hypothesizing this was due to enhanced SERCA activity immediately after dissection, I performed post-rest potentiations (PRPs) on experimental preparations both before and after Fura-2 loading. The PRPs indicated that SERCA activity could increase with frequency after Fura-2 loading, but was already maximized prior to loading (Figure 3-5). Treatment with the -agonist (and SERCA activator) isoproterenol resulted in elevated sub-physiological forces and a flattened FFR similar to pre-Fura-loading. Treatment with the SERCA inhibitor cyclopiazonic acid resulted in an FFR with low systolic force and reduced positive FFR (Figure 3-4). Also, all these results are similar to that seen in human myocardium exposed to these drugs,125;166 giving further evidence that the rat tissue is behaving like normal human tissue. I hypothesize that excision, transport, and dissection of the rat heart activates a stress response that increases SERCA activity. Time seems to allow SERCA activity to return to its normal levels. Human and dog preparations are performed very carefully, the heart cooled and removed slowly in situ prior to resection; rodent preparations are notoriously quickly excised at body temperature. Another possibility is that because SERCA contributes more contractile Ca2+ in rodents than larger mammals,17 the SERCA stress response may be more intense and longer in duration. I further hypothesize the particular stress is ischemia, although further experiments must be performed to examine this. These data support my first conclusion: The rat is an appropriate model for cardiac function and disease in the human.

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95 I then continued my investigations into basic cardiac function using the rat as my choice of model. The above data support a largely SERCA-based view of the positive mammalian FFR. SR Ca2+ content increases proportionally to systolic force. SERCA activity shapes the normal mammalian FFR. If SERCA Ca2+ pumping activity is greatly elevated, the FFR shifts upward at low stimulation rates. If reduced, the FFR loses magnitude and slope. Although peak systolic force and Ca2+ transients maintain a close relationship, there is a disconnect between SERCA activity and systolic force and between force and Ca2+ transient decline at stimulation rates above 4 Hz. Developed force declines (Figure 3-1), but SERCA activity, by PRP (Figure 3-5) and velocity of cytosolic Ca2+ removal (Figure 3-3), does not. This suggests an effect of SR Ca2+ release, a necessary link between SERCA and force development. SR Ca2+ release is activated by extracellular Ca2+ entering the cell (ICa) via the voltage-gated Ca2+ channel during Phase 2 of the action potential. This “trigger Ca2+” opens the Ca2+-sensitive SR Ca2+ channel, the ryanodine receptor. The following experiments examined the source (action potential) and characteristics (ICa) of Ca2+ entry in the frequency range of the FFR in isolated LV myocytes. The action potential duration (APD) increases over the same stimulation range as systolic force. From 0.5 to 4 Hz the action potential lengthens from ~55 ms to ~90 ms and shortens at faster rates. The key question, though, was whether APD would translate to greater ICa. The data curves practically overlap; ICa integral increases ~30% over the same positive FFR range and declines thereafter (Figure 4-2). Increased ICa may enhance

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96 contractile force by multiple mechanisms. Foremost, as the “trigger Ca2+,” enhanced ICa may increase SERCA-sequestered SR Ca2+ release with every contraction. It would also serve as a source for more Ca2+ ions for SERCA to sequester with its rate-enhanced activity. Finally, ICa Ca2+ itself may add to force development; in addition to its role as “trigger,” ICa contributes to SR Ca2+ in the pool of contractile Ca2+. Although the SR, charged by SERCA, is the largest and necessary contributor of contractile Ca2+ in the normal cardiomyocyte, its release requires and is supplemented by the entry of extracellular Ca2+. The observed correlation between ICa and systolic force is evidence that SERCA activity is not the only factor in the FFR. This leads to my second conclusion: The action potential duration contributes to the normal mammalian force-frequency relationship. This finding also opens a new avenue of investigation into pathologies in which the FFR is altered. The previous data demonstrated the role of SERCA and the potential contribution of APD and ICa in the FFR. It is known from the literature that APD lengthens with senescence; in the WKY rat this process begins at about 18 months of age.80 There also exists strong evidence that SERCA expression declines beginning at that time,70;70;71;74 but this has never been examined in a longitudinal fashion. This seemed counter-intuitive, as the SR Ca2+ release mechanism increases as the SR Ca2+ loading mechanism declines. I next investigated the effects of the seemingly-conflicting age-related processes of SERCA decline and APD prolongation on contractile force during aging, in the process performing the first longitudinal study of function and Ca2+-handling molecular characteristics.

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97 SERCA mRNA message and protein expression decline along with the flattening of the FFR. This appears to be a normal consequence of aging. Activation of the PKC and calcineurin pathways has been shown to lead to reduce SERCA expression;53 58 circulating norepinephrine67 and LV local renin-angiotension-system activity are elevated in senescence.167 As an enzyme, SERCA total activity is more relevant than expression to function. Post-rest potentiation (PRP) data indicates SERCA loses frequency augmentation at 16 months of age in the rat. The ratio of the inhibitor of SERCA, phospholamban (PLB), to SERCA rises at both the mRNA and protein levels constantly throughout adulthood as SERCA falls (Figure 5-2). The reduced SERCA expression combined with its proportional inhibition may be a cause of the reduced positive FFR. Although the FFR shifts from positive to flattened at 16 months, intrinsic force is maintained with further aging, which is unexpected in light of a continued reduction in SERCA activity. The APD is known to lengthen in the WKY rat between 18 and 24 months, prolonging (to 80% repolarization) from ~55 ms to ~100 ms (Table 5-1). My data showing the correlation between APD and systolic force demonstrates a possible link in the normal FFR. Janczewski et al.149 demonstrated that the prolonged APD with aging maintains normal calcium cycling in senescent isolated myocytes; they hypothesized that APD prolongation is an adaptation to reduced SERCA expression. My data support this hypothesis at the tissue and functional levels. APD prolongation occurs at the same age as intrinsic developed force would otherwise be expected to decline (16-24 months). Normalized Ca2+ handling, as shown by Janczewski, might allow for sustained cardiac output, but as rate-induced SERCA activity is negligible and the APD is prolonged even at low frequencies, systolic force cannot be enhanced with rate. This data

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98 unites previous studies to create an unprecedented overview of cardiac molecular and functional aging. I also present the hypothesis that cardiac hypertrophy with aging is, unlike pressure-overload, largely due to biochemical rather than mechanical stimuli. In hypertension, cardiomyocytes enlarge to increase number of myofilaments for contractile power and to minimize wall stress. Although cardiomyocytes enlarge with aging, the LV wall does not thicken (Table 5-1), probably due to apoptosis of the non-hypertrophied cells,64 negating hypertrophy as an adaptation for muscular strength or stability. However, angiotensin II,53;157 norepinephrine,52 and elevated diastolic Ca2+ 56 (a potential consequence of reduced SERCA Ca2+ sequestration) are linked to the PKC, JNK, and ERK pathways, which are linked to cellular growth. A reduction in SERCA is a characteristic of aging observed by many investigators, but these data provide the first indication of the time course of the decline and allow correlation to function. The ratio of SERCA to its inhibitor falls with the positivity of the FFR. SERCA expression itself significantly declines thereafter, and the positive FFR becomes flattened. Another known change with aging is a prolongation of the APD, which I have observed occurs soon after SERCA reduction severely affects normal cardiac function. The studies of Janczewski et al. support the hypothesis that APD prolongation is an adaptation to SERCA decline. Thus, SERCA reduction probably induces many of the alterations in cardiac tissue with age. Together, this supports my third conclusion: Reduced cardiac performance with adult aging is a result of a decline in SERCA expression.

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99 Cardiomyocytes are known to enlarge with age, but the degree of cellular hypertrophy was surprising. Cellular cross-sectional area (CSA) became significantly different from young adulthood (age 4 months) at 24 months of age and continued to grow. At 24 months CSA also became insignificantly different from the SHR with hypertrophy and no evidence of the decline into systolic heart failure (normal ejection fraction and maintained basal force development, age 9 months). Due to similar cellular hypertrophy and functional data, such as a less positive FFR158 and prolonged APD (when observed at lower rates),47 some investigators have hypothesized that the events follow the same sequence whether the impetus of the hypertrophy is age or hypertension.85 I decided to next examine this model in detail for functional and molecular comparisons with the normotensive aging model. Prior to the appearance of peripheral hypertension, the SHR and WKY rat strains exhibited identical cardiovascular characteristics. At 9 months of age, the hypertensive SHR rat was hypertrophied at every level (organ, tissue, and cell) compared to the normotensive age-matched WKY. Its cell thickness was similar to the senescent WKY ages at which the FFR was depressed (Table 6-1). Within the range of the positive mammalian FFR (1-3 Hz) and beyond, there were no differences in developed force between the normotensive SHR and WKY rats. The FFR was flattened in hypertrophied SHR because systolic force was elevated ~equal to the 3 Hz value at the lower (ascending limb) frequencies, unlike the depressed augmented force in aging. At higher rates, there was no alteration in systolic force (Figure 6-1). Systolic force and peak cytosolic Ca2+ levels are linearly related in normal cardiac muscle (Figure 3-2). I next examined if this held true during hypertrophy. Although I was

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100 unable to quantify absolute cytosolic Ca2+ concentrations due to technical limitations, I was able to examine similar representative WKY and SHR experiments. Just as developed force was higher at lower frequencies in the hypertrophy SHR, Ca2+ transients were higher and did not rise further with increasing stimulation rates (Figure 6-2). Because systolic force and Ca2+ within the normal ascending range was no different from the values at 3 Hz, the FFR peak, I concluded that during the stages of compensatory hypertrophy, the hypertensive SHR increases developed force to the maximum the myocardium can produce. There are two major sources of contractile Ca2+ for the cardiomyocyte—extracellular influx and SR intracellular stores—that seem to work together to increase developed force (Chapter 4). Ca2+ influx during Phase 2 is the largest contributor to the length of the action potential, and the APD prolongs with frequency during the positive FFR in normotensive SHR, young WKY, and adult WKY. However, APD in the hypertrophied SHR is prolonged at low frequencies and does not lengthen with an increase in rate (Figure 6-3A,B). Inward Ca2+ current is greater at all frequencies in the hypertrophied SHR than the age-matched WKY and does not increase as greatly as rate increases (Figure 6-3C,D). I theorize that APD and ICa augment contractile Ca2+ largely indirectly, by increasing SR “trigger” Ca2+ and providing substrate for elevated SERCA SR-filling activity. Measures of SERCA activity display elevated but not maximized activity. Whereas isolated papillary muscles from normotensive WKY relax more quickly with frequency to 3 Hz, muscle from hypertrophied SHR relax more quickly at lower frequencies and do not increase speed. This is an accepted indirect measure of SERCA

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101 that I previously used in Figure 3-5, but when comparing different strains it is confounded by tissue stiffness and other Ca2+ removal systems such as Na+/Ca2+ exchanger and the plasma membrane Ca2+-ATPase. The rate of removal of cytosolic Ca2+ during relaxation (CaRV) is a better measure of SERCA activity because SERCA is the largest component (92% in rat17 under normal conditions), and I used it as such in Figure 3-3. CaRV in the hypertrophied SHR was no different from the age-matched WKY. Thus, it is likely the frequency-potentiation of SERCA, controlled by CamKII directly and indirectly through PLB, was maintained, but CaRV does not indicate basal levels of Ca2+ sequestration. To investigate this, I examined by PRP the length of rest required to practically fill the SR. Unlike nonhypertrophied animals, there was no change in time required as frequency rises from 1.5 to 3 Hz. From these three measures, I concluded that SERCA activity was higher at low frequencies, but not because its control proteins were hyperactive. The rate-limiting step of SERCA activity has been calculated to be ATP availability in high Ca2+ states, but the stoichiometric binding of two Ca2+ ions during low Ca2+ states.146 In the higher Ca2+ state of the SHR, it is possible SERCA activity is elevated. My next step was to determine if SERCA protein itself was elevated because an overabundance of Ca2+ pumps would probably fill the SR more quickly regardless of basal activity. However, immunoblotting revealed no difference in SERCA or PLB expression or ratio in SHR and WKY at either age (Figure 6-5). These data indicate that prior to the change in SERCA expression seen in systolic heart failure, the myocardium increases systolic force through a prolonged APD and elevated ICa. This supports my final conclusion:

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102 Electrical remodeling is an early adaptation to cardiac hypertrophy secondary to peripheral hypertension. These data also suggest that SERCA overexpressing gene therapy to return normal FFR function or reverse decompensatory adaptation to hypertension would be of little or no value at or prior to this stage. Future Directions These results indicate that the FFR, a fundamental property of cardiac function, is due to at least two components working synergistically—increased ICa releasing stored SR Ca2+ and adding to cytosolic Ca2+, and elevated SERCA sequestering that Ca2+ for recycling into subsequent contractions. The effects of hypertrophy, long believed to be detrimental to cardiac function, depend upon the stimulus of the hypertrophy and the duration of the stimulus. My studies in young rats were limited to the goal of establishing the model’s value to other studies. Also, investigations in hypertension ceased at the compensatory stage, when developed force is maximized. Thus I propose future experiments to answer the following questions raised by these studies: 1. What is the stress that elevates SERCA activity immediately after heart excision from the rat? Is it ischemia, rapid temperature flux, or another factor? 2. What are the roles of SERCA and APD in very early compensatory (between 2 and 9 months of age) and decompensatory hypertrophy secondary to peripheral hypertension? 3. What are the roles of SERCA and APD at the transition from compensatory to decompensatory hypertrophy and from decompensatory hypertrophy to systolic failure? 4. What effects do extracellular collagen deposits have on developed force and diastolic tension? 5. Does cellular hypertrophy itself play a role in altered force development by distancing the L-type Ca2+ channel from the ryanodine receptor?

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103 6. What is the link between altered Ca2+ cycling and changes in gene expression (ICa, IK, SERCA, PLB)? 7. What are the alterations in diastolic tension in aging and hypertensive hypertrophy?

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BIOGRAPHICAL SKETCH David Glenn Taylor was luckily born to Glenn and Vicki Taylor on February 3, 1977, in central Mississippi, joining his older brother, Matthew. In 1999 he graduated summa cum laude from the Honors College at the University of Southern Mississippi, where he received a BS in biology with minors in chemistry and English and had had many adventures. That fall David entered the graduate Interdisciplinary Program for Biomedical Sciences at the University of Florida, College of Medicine. Once in Florida, he set out to explore all the state had to offer, and found an extraordinary wife, Michelle. Since joining the Department of Pharmacology and Therapeutics under the guidance of Dr. Harm Knot and with the support and aid of many other wonderful scientists and friends, he has been investigating the basic physiology of cardiac contraction and the functional and molecular changes that accompany cardiac hypertrophy. He will earn his doctorate in August, 2004, and then probably take a day off. 118