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Mechanisms of Protection against Myocardial Ischemia-Reperfusion Injury

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Review of related literature
 Methods
 Results
 Discussion
 References
 Biographical sketch
 

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1 MECHANISMS OF PROTECTION AGAINST M YOCARDIAL ISCHEMIA-REPERFUSION INJURY By JOEL P. FRENCH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 By Joel P. French

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3 To my family: Asimina, Janet, John and Jenat heir unwavering love and support have enabled every major accomplishment in my life.

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4 ACKNOWLEDGMENTS This work would not have been possible without the guidance and support of several important people. First, I would like to expr ess my appreciation to my mentor and committee chairp, Dr. Scott Powers. You have taught me everything I know about research, grant and manuscript writing, teaching and presenting. Through example, you have also instilled in me a drive to succeed. I believe that you have given me all of the skills I need to be successful in this field and I hope that my future accomplishments will reflect the extremely high quality of your mentoring. I would also like to thank my doctoral committee, Dr. Stephen Dodd, Dr. David Criswell and Dr. Nihal Tumer for their patienc e and expertise throughout this project. Importantly, thank you to Dr. John Quindry for his guidance and friendship. I appreciate everything that you taught me and more importa ntly, your ability to ma ke the lab such an enjoyable environment to work in. Additionally, thanks to everyone in the lab who contributed to this project: Dr. Karyn Hamilton, Patrick Upchurch, Dr. Jessica Staib and Da rin Falk. Thanks also to the rest of the team in the lab: Youngil Lee, Joe McClung, Zsol t Murlasits, Melissa Deer ing, Keith Deruisseau, and Darin Van Gammeren. Finally, and most importantly I would like to th ank my family; Mina, Janet, John and Jena. Without your love and support I never could ha ve come this far. I love you all.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Rationale...................................................................................................................... ...........12 Specific Aims.................................................................................................................. ........15 2 REVIEW OF RELATED LITERATURE..............................................................................17 Introduction................................................................................................................... ..........17 Myocardial Ischemia-Reperfusion Inju ry: Characteristics and Mechanisms.........................18 The Oxyradical Hypothesis.............................................................................................19 ROS related damage.................................................................................................20 Sources of myocardial ROS.....................................................................................21 Antioxidant defenses agains t IR-induced ROS production......................................22 The Calcium-Overload Hypothesis.................................................................................25 Calcium induced ROS production............................................................................26 E-C uncoupling and decreased calcium sensitivity..................................................26 Calcium-activated proteases.....................................................................................27 Calpain and IR Injury.......................................................................................................... ...29 Calpain: Linking the Oxyradical and Calcium-Overload Theories........................................30 Calpain and Calcium-Handling Proteins................................................................................30 Regulation of Free Cytosolic Calcium............................................................................30 Oxidative Modification and Degradat ion of Calcium-Handling Proteins.......................31 SERCA and PLB......................................................................................................32 NCX.........................................................................................................................32 L-type calcium channels and the DHPR..................................................................32 Summary: Calpain and Ca lcium-Handling Proteins...............................................................33 Antioxidants and SR Dysfunction..........................................................................................33 Exercise-Induced Cardio-Protection Against IR Injury.........................................................34 Increased Myocardial Heat Shock Proteins.....................................................................35 Increased Myocardial Antioxidant Capacity...................................................................35 Exercise-Induced Regulation of Calpain Activation..............................................................36

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6 Summary........................................................................................................................ .........37 3 METHODS........................................................................................................................ .....38 Experimental Animals........................................................................................................... .38 Animal Model Justification.............................................................................................38 Animal Housing and Diet................................................................................................38 Experimental Design............................................................................................................ ..38 Experimental Design: Hypothesis One...........................................................................38 Experimental Design: Hypothesis Two...........................................................................39 General Methods................................................................................................................ .....40 The Isolated Working Heart Preparat ion/IR Protocol (Hypothesis One)........................40 In Vitro Ischemia-Reperfusion Protocol (Hypothesis One)............................................41 Cardiac Performance Measurements (Hypothesis One)..................................................41 Calpain Inhibition (Hypothesis One)...............................................................................42 Exercise Training Protoc ol (Both Hypotheses)...............................................................42 In Vivo Ischemia-Reperfusion Pr otocol (Hypothesis Two).............................................42 Inhibition of MnSOD Protein Translation (Hypothesis Two).........................................43 Dependant Measures (Both Hypotheses)................................................................................44 Measurement of Calpain Activation................................................................................44 Western Blots for Calc ium-Handling Proteins................................................................44 Immunoprecipitation of Calc ium-Handling Proteins......................................................45 Measurement of Protein Carbonyl Form ation on Calcium-Handling Proteins...............45 Measurement of HNE Formation on Calcium-Handling Proteins..................................46 Data Analysis.................................................................................................................. ........46 4 RESULTS........................................................................................................................ .......47 Hypothesis One................................................................................................................. ......47 Animal Characteristics....................................................................................................47 Cardiac Performance Measures.......................................................................................48 Percent recovery of left ventri cular developed pressure (LVDP)............................48 Percent of pre-ischemic +dp/dt and dp/dt..............................................................48 Oxidative Modification of Critic al Calcium-Handling Proteins.....................................49 Carbonyl formation on calci um-handling proteins..................................................51 HNE formation on calcium-handling proteins.........................................................51 Calpain-Mediated Degradation of Calcium-Handling Proteins......................................52 Hypothesis Two................................................................................................................. .....53 Calpain Activation (Calpain-Cleaved II-Spectrin)........................................................53 Oxidative Modification of Critic al Calcium-Handling Proteins.....................................55 Carbonyl formation on calci um-handling proteins..................................................56 HNE formation on calcium-handling proteins.........................................................57 Calpain-Mediated Degradation of Calcium-Handling Proteins......................................58 5 DISCUSSION..................................................................................................................... ....60 Overview of Principal Findings..............................................................................................60

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7 Hypothesis One: The Effects of IR and Calpain Inhibition on Myocardial CalciumHandling Proteins.............................................................................................................. ..61 Calpain Inhibition Protects Against IR-i nduced Damage and Removal of CalciumHandling Proteins.........................................................................................................62 Calpain Degrades Critical Calcium-Handling Proteins...................................................62 Hypothesis Two: IR, Exercise, MnS OD and Calcium-Handling Proteins.............................65 Exercise Training Provides Cardio-Protection................................................................65 Exercise-Induced Over-Expression of MnSOD Prevents the Oxidation of CalciumHandling Proteins.........................................................................................................66 Exercise-Induced Over-Expression of MnSOD Attenuates Calpain Activation.............68 Exercise-Induced Over-Expression of MnSOD Prevents the Degradation of Calcium-Handling Proteins..........................................................................................68 Degradation of Calcium-Handling Protei ns is Associated with Oxidation.....................68 Summary and Future Directions.............................................................................................69 LIST OF REFERENCES............................................................................................................. ..71 BIOGRAPHICAL SKETCH.........................................................................................................82

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8 LIST OF TABLES Table page 4-1 Animal body and heart weights.........................................................................................47 4-2 Correlations Between the Oxidative Modification of Ca2+-handling Proteins and Their Degradation Following IR........................................................................................53 4-3 Correlations between the oxidative modificat ion of calcium-handling proteins and their degradation following IR...........................................................................................58

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9 LIST OF FIGURES Figure page 1-1 Proposed mechanisms underlying IR-induced calpain activation and myocardial dysfunction.................................................................................................................... .....16 3-1 Experimental design for Hypothesis One..........................................................................39 3-2 Experimental design for Hypothesis Two..........................................................................40 4-1 % Recovery of left ventricula r developed pressure (LVDP).............................................47 4-2 Rate of systolic pressure increase (+dp/dt)........................................................................48 4-3 Rate of systolic pressure decline (-dp/dt)...........................................................................49 4-4 Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reti culum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX).......................................50 4-5 HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), P hospholamban (PLB) and Sodium/calcium exchanger (NCX)...............................................................................................................51 4-6 Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reticulum calci um ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX)..................................................................52 4-7 Western blotting for calpain-cleaved II-Spectrin.............................................................54 4-8 Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reti culum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX).......................................55 4-9 HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), P hospholamban (PLB) and Sodium/calcium exchanger (NCX)...............................................................................................................56 4-10 Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reticulum calci um ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX)..................................................................57 5-1 Proposed mechanisms underlying the IR-induc ed increase in calpain activity and myocardial dysfunction......................................................................................................70

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISMS OF PROTECTION AGAINST M YOCARDIAL ISCHEMIA-REPERFUSION INJURY By Joel P. French December 2006 Chair: Scott K. Powers Major: Health and Human Performance Myocardial ischemia-reperfusion (IR) is char acterized by an increase in reactive oxygen species (ROS) generation as well as increased free cytosolic Ca2+ (Ca2+-overload) resulting in myocardial contractile dy sfunction and injury. We have previously demonstrated that both exercise training a nd inhibition of the Ca2+activated protease calpain, protect the heart against IR injury. A dditionally, we have shown that exercise regulates IR-induced calpain activa tion. However, the mechanisms involved in exercise-induced calpain regula tion and calpain-mediated injury are not completely understood. We hypothesized that the oxida tion and calpain-mediated de gradation of critical Ca2+-handling proteins was an important mechanism of IR in jury and exercise-induced cardio-protection. Therefore, we conducted two sepa rate experiments to examine the relationships between IR, calpain activation, exercise tr aining and the oxidation and de gradation of myocardial Ca2+handling proteins. Our first set of experiments looked at the effects of calpain in hibition on myocardial function, Ca2+-handling protein oxidation and degradation following IR. We found that IR resulted in impaired LVDP, +dp/dt and dp/dt and increased oxidati ve modification and

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11 degradation of several Ca2+-handling proteins (LTCC, SERCA2 a, PLB, NCX). In addition, we found that pharmacological inhibition of calpain prevented contractile dy sfunction as well as the degradation of these Ca2+-handling proteins. Our second set of experiments looked at th e mechanisms of exercise-induced cardioprotection against IR injury. We found that short-term exercise tr aining attenuated calpain activation as well as the oxidative modification and degradation of Ca2+-handling proteins. In addition, when the exercise-induced over-expre ssion of the endogenous antioxidant enzyme MnSOD was prevented, using an antisense oligonuc leotide, the protective effects of exercise training were lost. Therefore, we propose a series of events dur ing IR, which are initiated by ROS-mediated oxidative modification of critical Ca2+-handling proteins, resulting in increased free cytosolic Ca2+ and calpain activation. Once active, calpain can cleave Ca2+-handling proteins, facilitating their degradation, exacerbating Ca2+-overload, ROS generation and IR injury. Exercise appears to provide protection against these events by over-expressing MnSOD, which attenuates IR-induced Ca2+-handling protein oxidation, calpain activation and Ca2+handling protein degradation.

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12 CHAPTER 1 INTRODUCTION Rationale Myocardial ischemia-reperfusion (IR) injury is a prevalent conseque nce of cardiovascular disease. Although there are many factors lead ing to myocardial dysfunction following IR, growing evidence suggests that increased reactive oxygen species (ROS) production and cytosolic free Ca2+-overload, either independently or coope ratively, are major contributors to IRinduced injury (10, 80, 89). ROS generated during IR can have several targets within the m yocardium (including proteins, lipids, and DNA), impairing their functi on and/or promoting their degradation, leading to contractile dysfunction, cellular damage and ce ll death. Additionally, ROS can interfere with intracellular Ca2+ homeostasis, further exacerbating th e deleterious effects of IR (9, 64). Increases in myocardial cytosolic Ca2+ levels have been observed during both ischemia and reperfusion. In this regard, it has been hypothesized that one role of cytosolic Ca2+ in the pathogenesis of IR-induced myocardial in jury is through the activation of the Ca2+-dependent protease, calpain. Calpain exists in myocytes in two primary isoforms micro (calpain I) and milli (calpain II), named for the respective amounts of Ca2+ required for their activation in vitro Both calpain isoforms are activated by prolonged exposure to elevated cytosolic Ca2+ and it is well documented that calpain activ ation occurs in the heart during IR (55, 120, 122, 128). This is significant because calpain can injure cardiac myocytes via se veral different pathways. For example, calpains cleave several structural prot eins leading to the re lease of myofilaments, facilitating their degradation by the proteoso me. In addition, calpains may contribute to apoptosis, through cleavage of Bid, mediating cytochrome c release from the mitochondria. Also, calpains increase the expression of cell ad hesion molecules, leading to an increase in

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13 neutrophil-mediated oxidative damage. Each of these pathways has been shown to significantly contribute to IR-associated injury. Moreover, calpains deleterious role in IR injury is supported by strong evidence indicating that calpain inhibition significa ntly attenuates myocardial contractile dysfunction, cellular inju ry, and cell death (55, 120, 122, 128). Recent evidence suggests an additional role fo r calpain as a link between the oxyradical and Ca2+-overload theories of IR-induced myocardial injury. This research suggests that ROS may oxidatively modify Ca2+-handling proteins, impairing thei r function and possibly leading to their cleavage by calpain (64, 126, 129, 130). De gradation and/or functional impairment of these proteins would lead to an increase in free cytosolic Ca2+, further exacerbating IR-induced calpain activation, ROS production a nd myocardial dysfunction (Figure 1-1). In support of this postulate, work from our laboratory has descri bed an IR-induced increase in oxidative stress (protein carbonyls) as well as an increas e in calpain-mediate d cleavage of the Ca2+-handling protein, SERCA2a. Indeed, th is suggests that some Ca2+-handling proteins are cleaved by calpain, and degraded during IR. Therefore, the first goal of thes e experiments was to determine the effects of IR on the oxidative modifi cation and degradatio n of several key Ca2+-handling proteins. The proteins studied were: the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a), phospholamban (PLB), the Na+/Ca2+ exchanger (NCX) and L-type calcium channels (LCC). We also determined if t hose proteins/protein complexes, which were oxidatively modified, had a higher incidence of degradation. Additionally, we determined the effects of in-vitro calpain inhibition on the degradation of Ca2+-handling proteins and myocardial function following IR. Endurance exercise training is an established means of i nducing cardioprotection against IR-induced injury (71, 72, 93, 96, 113, 114). Alt hough the mechanisms of exercise-induced

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14 cardioprotection are unknown, work from our labor atory indicates that exercise may provide cardioprotection against IR injur y, at least in part, th rough the regulation of calpain (30). This work reveals that exercise training completely attenuated the IR-induced increase in calpain activation and was associated w ith improved myocardial contra ctile function following IR. Additionally, exercise trained hearts showed an increase in the endogenous antioxidant MnSOD and a decrease in oxidative stress (protein car bonyl formation). We hypothesize that endurance exercise may regulate calpain activation through an up-regulation of endogenous antioxidants, such as Mn-SOD. Superoxide production is believed to be a major source of oxidative stress during IR. Mitochondrial produced superoxide is thought to play an important role in myocardial IR injury due to the aerobic nature of th e heart. Superoxide production has been shown to dramatically increase following ischemia and the use of supe roxide scavengers has been shown to improve contractile function following IR. Because of th e detrimental impact of superoxide generation during IR, we hypothesize that an exercise-induced increase in MnSOD may provide protection against oxidative modification of key ce llular proteins, including critical Ca2+-handling proteins associated with both the SR and plasma memb rane. Reducing oxidative damage to calcium handling proteins may result in improved Ca2+-handling and reduced calp ain activation during IR and, therefore reduce myocardial dysfunction and injury. Therefore, the second goal of these experiments was to investigate the effects of exercise training on oxi dative modification and degradation of Ca2+-handling proteins following IR. Additionally, we determined if the exerciseinduced reduction in calpain activity and preservation of Ca2+-handling proteins during IR was dependant on the exercise-induced ove r-expression of MnSOD in the heart.

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15 Specific Aims The goal of these experiments was to addr ess two separate bu t related hypotheses involving myocardial IR injury, oxid ative stress, and calpain activation. Hypothesis one: IR will result in increase d oxidative modification (carbonyl and HNE formation) and/or calpain-mediated degradation of myocardial calcium handling proteins. Further, calpai n inhibition will a ttenuate the IRinduced degradation of Ca2+-handling proteins. Th is hypothesis will be tested by achieving the following specific aims. Aim (A): To determine if IR results in increased carbonyl and/or HNE formation to Ca2+-handling proteins within the heart. Aim (B): To ascertain if oxidative modification to calci um handling proteins is associated with an increase in the degradation of these proteins. Aim (C): To discern if calpain inhib ition attenuates the degradation of key Ca2+handling proteins in th e heart following IR. Hypothesis two: Exercise training will provi de protection against IR-induced oxidative modification (carbonyl and HNE fo rmation) and degradation of myocardial Ca2+-handling proteins via an increase in the endogenous antioxidant, MnSOD. This hypothesi s will be rigorously tested by achieving the following specific aims. Aim (A): To determine if exercise trai ning attenuates IR-indu ced carbonyl and/or HNE formation on key Ca2+-handling proteins and/or degradation of these proteins within the heart. Aim (B): To ascertain if exercise-induced protection against IR-induced oxidative modification and degradation of Ca2+-handling proteins is dependent on an increase in myocardial MnSOD.

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16 Ischemia-Reperfusion ROS Production Calcium Handling Protein Damage / Cleavage Cytosolic Ca2+ Calpain Activation IR Injury Cytosolic Ca2+ Hypothesis Two MnSOD Hypothesis One Calpain Inhibition Ischemia-Reperfusion ROS Production Calcium Handling Protein Damage / Cleavage Cytosolic Ca2+ Calpain Activation IR Injury Cytosolic Ca2+ Hypothesis Two MnSOD Hypothesis One Calpain Inhibition Figure 1-1. Proposed mechanisms underlying IR -induced calpain activation and myocardial dysfunction.

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17 CHAPTER 2 REVIEW OF RELATED LITERATURE Introduction Coronary heart disease (CHD) is the number one cause of death for both men and women in the United States, as well as most other i ndustrialized nations. In 2002, CHD was responsible for 927,448 deaths in the U.S., roughly one in ever y three deaths (34.2%). Additionally, health care costs related to CHD in 2005 were estimated at $393.5 billion w ithin the U.S. alone (6). Because CHD typically results in periods of myocardial ischem ia, often leading to terminal infarction, understanding the m echanisms of myocardial IR injury as well as possible mechanisms of protection against IR injury is important in the treatment and management of patients with CHD. Many factors can contribute to IR-induced myocardial IR injury. The first goal of this review will be to provide an overview of IR-induced cellula r injury and to discuss the mechanisms responsible for IR-i nduced cellular injury. Although se veral factors contribute to IR injury, this review will focus prim arily on the two dominant theories of myocardial IR injury, the oxyradical theory and the Ca2+ overload theory, as well as possi ble interaction between the two. The second goal of this review will be to di scuss calpain and its role in IR injury. Increases in both free Ca2+and ROS have been shown to increase the cellular activity of the Ca2+activated protease calpain (8, 12, 19). Further, calpain activa tion has been shown to play a deleterious role in the heart. Calpain may play a critical role in IR injury by linking the theories of Ca2+ overload and ROS production. Calpain may cleave Ca2+-handling proteins, which have been oxidatively modified by ROS, leading to a further increase in the levels of free Ca2+ and active calpain, exacerbating myocar dial damage and dysfunction.

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18 The third goal of this review will be to de scribe exercise-induced cardioprotection and the role of the antioxidant, manganese superoxide dismutase (MnSOD), as a potential mediator of exercise-induced cardioprotec tion. Endurance exercise traini ng has been shown to provide protection against IR-induced elev ations in calpain activity, myo cardial injury and contractile dysfunction, however, the precise m echanisms of this cardio-protec tion have not been elucidated (95, 96). One possible explanation is that exercise provides cardiop rotection through an increase in the endogenous antioxidant MnSOD. Increas ing MnSOD protein and/or activity would result in a reduction in IR-induced RO S damage. This could lead to a reduction in the oxidative modification of Ca2+-handling proteins, as well as their degradation by calpain, reducing not only ROS-related injury but disturbances in Ca2+ homeostasis and calpain -related pathology as well. Myocardial Ischemia-Reperfusion Injury: Characteristics and Mechanisms Myocardial ischemia is defined as the reduc tion or cessation of blood flow to myocardial tissue, below the metabolic requirements of that tissue. In addition to being a clinical manifestation of coronary artery disease, the la test clinical treatments for this disease (i.e. coronary bypass surgery, balloon angioplasty) subj ect the heart to episod es of ischemia and subsequent restoration of bl ood flow (reperfusion). Following a period of ischemia and reperfusion, the heart can undergo temporary or permanent injur y, depending on the duration of the ischemia (10). Brief periods of ischemia, less than 5 minutes, will generally result in arrhythmias. However this length of ischemia is not associated with any long-term loss in heart function or cell death. A period of ischemia lasting approximately 5-20 minutes results in ventricular contract ile dysfunction and is referred to as myocardial stunning. This ischemic duration will typically result in temporary cont ractile function without causing any permanent damage (necrosis or apoptosis) to the heart (10). The most severe form of IR injury, myocardial

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19 infarction, occurs when the duration of ischem ia exceeds 20 minutes. Infarction results in loss of contractile function as well as cellular death, through pathways of both necrosis and apoptosis (7). There are many mechanistic factors that may contribute to myocar dial dysfunction and cellular death (7). These factors include, but are not limited to: ROS production, Ca2+-overload, increased proteolytic activity, a nd platelet / inflammatory cell infiltration. Although there are many factors leading to myocar dial dysfunction and cell death following IR, growing evidence suggests that increased free cytosolic Ca2+ and/or ROS production, e ither independently or cooperatively, are two of the major contributors to IR-induced injury (9). A brief discussion of the roles of both ROS and free cytosolic Ca2+ in IR-induced injury follows. The Oxyradical Hypothesis The oxyradical hypothesis of IR injury was first proposed in 1985 when it was postulated that the generation of reactive oxygen and free ra dical species such as the superoxide anion (O2 -), hydrogen peroxide (H2O2), and the hydroxyl radical (OH) during reperfusion contributed to myocardial injury (86). Reactive oxygen speci es (ROS) are derived fr om the reduction of molecular oxygen (78). Some ROS (such as O2 and OH) are known as free radicals because they contain one or more unpair ed electrons in their outer mo st orbital, making them highly unstable and reactive (42). For example, when an O2 molecule accepts a single free electron, the product is superoxide (O2 -). When a second free electron is accepted, hydrogen peroxide (H2O2) is formed. In the presence of an iron (ferrous) salt, the O_O bond can be broken resulting in the formation of two hydroxyl radicals (OH). Importantly, the OH produced is one of the most highly reactive free radicals, capable of reacting with almost ever y component of the cell (76). Free radicals produced via these pathways have been implicated in damage to several structures

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20 within the cell, includ ing cellular proteins, lip ids, and DNA (104). Supporting the deleterious role of ROS, ischemia-reperfus ion experiments using iron chelat ors and/or various antioxidants (free radical scavengers) have de monstrated a reduction in free radical generation and protection against IR-induced injury (4, 9, 28, 86, 106). ROS related damage Damage caused by free radicals has been iden tified during both periods of ischemia and reperfusion in the heart, although it is currently believed that the major ity of the radicals are produced during the first few minutes of reperfusion (4, 11, 13, 28, 57, 74, 119). Once generated, free radicals have seve ral targets within the cell, wh ich will be discussed in the following sections. Myocardial protein oxidation. Many cardiac proteins can be oxidatively modified by free radicals in the heart during IR. Included in the list of target pr oteins are: enzymes, structural proteins, contract ile proteins, and membrane-bound proteins (41, 43, 49, 100, 101). Often, the damage caused by free radical interacti on is irreparable. In addition, many proteins, which are oxidatively modified, become more su sceptible to proteolytic cleavage by calpain and degradation by the proteosome. One example of oxi dative modification to ce llular proteins is the formation of carbonyl groups. This carbonyl fo rmation can then be measured, providing an indirect indication of oxidative stress within the cell (15). Myocardial lipid peroxidation. Polyunsaturated fatty ac ids are highly susceptible to free radical modification at thei r unsaturated sites (3). Once ROS extract electrons, or hydrogen atoms, from the methylene groups of fatty aci ds, a chain reaction is initiated where one modified fatty acid chain reacts with a neighbor ing chain, and so on. This eventually leads to the damage of the lipid membrane, resulting in altered cell membrane permeability and fluidity. In many cases, this damage to the cell membrane leads to cellular death through necrosis and / or

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21 apoptosis. There are examples of lipid peroxida tion by-products which can be measured in order to indirectly quantify oxidative st ress within the cell (43). One such by-product is the formation of the reactive aldehyde, HNE (4-hydroxy-2-nonenal ). The w-6-family (linoleic and arachidonic acids) of polyunsaturated fatty acids produce HNE as a result of free radical attack. HNE is a highly reactive compound and it can react with seve ral functional groups on biological material, particularly sulfydryl groups, to form thioester adduct and then he miacetals. HNE may also react with histidine and lysine residues of proteins to form stable Mich ael addition-type of adducts. In addition, HNE-modification of proteins may impair biological functions. DNA damage. ROS have also been reported to da mage DNA, preventing the translation and transcription of new cellular proteins by stimulating the degradation of DNA and oligonucleosomal fragments (50). More specif ically, ROS can cause permanent or transient damage to nucleic acids within the cells, lead ing to such events as DNA strand breakage and disruption of Ca2+ metabolism. Additionally, a high rate of oxidative damage to mammalian DNA has been demonstrated by measuring oxidi zed DNA bases excreted in urine following DNA repair. Further, the rate of oxidative DNA damage has been found to be directly related to metabolic rate and inversely related to life span. Sources of myocardial ROS There are several potential sources of free ra dical production in the heart during ischemiareperfusion. Major sources include: electron leak from the mitochondrial respiratory complexes, xanthine oxidase, enzymatic arachadonic acid oxyge nation, the synthesis of nitric oxide, catecholamine oxidation and oxidative burst from neutrophils (9, 17, 19, 27). Nonetheless, a large volume of evidence implicates mitochondr ial production of radicals as the primary source of oxidants during both ischemia and reperf usion. Therefore, a detailed discussion of mitochondrial ROS production follows.

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22 Mitochondrial ROS production during IR. Again, the mitochondrial respiratory chain has been identified as one of the largest contributors of IR-induc ed free radical production (9). Mitochondrial oxidative phosphoryla tion is the major ATP synthetic pathway in eukaryotes. During this process, electrons liberated from re ducing substrates are delivered to oxygen via a chain of respiratory H+ pumps. These pumps (complexes I-IV) establish a H+ gradient across the inner mitochondrial membrane. The electrochemical energy of this gr adient is then used to drive ATP synthesis by complex V (ATP synthase). Duri ng this process, it has been estimated that approximately 1-2% of the oxygen present is reduced to form some sort of ROS or free radical (36). The primary radical made by the mitochondria is superoxide (O2 ). It is believed that the majority of the superoxide generated origin ates from electrons l eaked from ubisemiquinone, located at complex III. Once generated, superoxi de can be converted to a less reactive oxygen species, hydrogen peroxide (H2O2) by the antioxidant enzyme s uperoxide dismutase (SOD), or converted to a more reactive hydroxyl (OH) radical, in the presence of iron. Confirming the importance of mitochondrial free radical production, studies using antioxidants targeted specifically to the mitoc hondria have demonstrated a significant reduction in oxidant-related damage within the myocardi um, as well as improved myocardial function (4, 11, 28, 86). Antioxidant defenses against IR-induced ROS production Fortunately, the cell has several antioxidant defense mechanisms against the increase in free radicals typically seen during IR. An antioxi dant has been defined as any substance that significantly delays or prevents th e oxidation of that substrate ( 42). The cell contains a variety of enzymatic and non-enzymatic antioxidants, located in vari ous strategic locations and specifically targeted to different ROS.

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23 Superoxide dismutase (SOD). Superoxide dismutase (SOD) was discovered in 1969 by McCord and Fridovich (81). SOD is an enzyme that catalyzes the reduction of superoxide to hydrogen peroxide, thus forming a le ss reactive, non-radical species. O2 + O2 + 2H+ H2O2 + O2 SOD exists in three isoforms: manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD) and extracellu lar superoxide dismutaes (ECSOD). Although each isoform catalyzes the reacti on of superoxide to hydrogen pe roxide, they each reside in different locations within the cell. MnSOD is located exclusively within the mitochondria, CuZnSOD is found predominantly within the cyto sol and, as its name implies, ECSOD is found in extracellular fluids such as plasma, as well as in the extracellular matrix of tissues. Because superoxide generation has been identified as a major contributing factor to IR injury, the regulation of SOD plays a critical role in the heart. Catalase (CAT). The enzyme catalase (CAT) catalyzes the breakdown of hydrogen peroxide to water and oxygen. H2O2 2H2O + O2 Catalase is found predominantly within the pe roxisome along with several other enzymes, which can generate hydrogen peroxide such as ur ate oxidase, glycolate oxidase, and flavenoid dehydrogenases, involved in beta -oxidation of fatty acids (81). Glutathione peroxidase (GPx). The antioxidant enzyme glutathione peroxidase (GPx) also catalyzes the breakdow n hydrogen peroxide to two mol ecules of water. However, this reaction depends on the concomitant oxidat ion of reduced glutathi one (GSH) to oxidized glutathione (GSSG).

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24 H2O2 + 2GSH GSSG + 2H2O Because of the dependant relationship betw een the oxidation of glutathione and the reduction of H2O2, the measurement of the ratio of GSH to GSSG is often used to assess the amount of hydrogen peroxide produc tion, as well as GPx activity within the cell. Both GPx and CAT work in tandem to remove hydrogen peroxide from the cell. CAT has a much higher Km than GPx, responding very quickly to increases in hydrogen peroxide. GPx has a much lower Km but is more ubiquitous, breaking down the majo rity of hydrogen peroxide generated from the mitochondria and sarcoplasmic reticulum (81). Glutathione (GSH). Glutathione (GSH) is an intr acellular thiol-containing tripeptide, which is produced inside cells. Importantl y, since very little CAT resides within the mitochondria, GSH, in conjunction with Gpx, is the major means of hydrogen peroxide breakdown (82). The importance of GSH to the mito chondria is illustrated by the fact that when cytosolic GSH levels begin to fall, the mitoc hondria reduce GSH release in order to conserve their own reserves. Other Antioxidants. The cell contains several ot her non-enzymatic antioxidants that contribute to maintaining re dox balance. Many of these non-enzymatic antioxidants are consumed in the diet, such as vitamin E, vitami n C, lipoic acid, carotenoids, and flavenoids to name a few. In addition other endogenous anti oxidants such as heat shock proteins and ubiquinones also play a role in maintaining cellular redox balance. Because of the extensive number of antioxidants, complexity of their func tion, as well as relevance to these experiments, these antioxidants will not be discussed in this proposal.

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25 The Calcium-Overload Hypothesis The Ca2+ overload hypothesis predicts that myocardi al IR injury results from a disturbance in cellular Ca2+ homeostasis. Intracellular cytosolic Ca2+ levels are typically maintained at a low level (approximately 0.1 M) while total cellular Ca2+ is considerably higher. Much of the cellular Ca2+ is stored within the sarcoplasmic reticu lum (SR) and the mitochondria. Numerous studies have demonstrated a dramatic increase in cytosolic Ca2+ levels during ischemia (60, 68, 78, 110). This elevation in free Ca2+ persists during the early st ages of reperfusion, finally returning to normal levels during late reperfusion (65, 78). The Ca2+-overload hypothesis was first described in detail by Grinwald (37), who proposed the following mechanisms to attempt to explain this increase in free Ca2+, or Ca2+-overload. During ischemia, intracellular sodium accumulates due to energy depletion, and Na+/Ca2+ exchange is inhibited by the concomitant acidosis. Upon reperfusi on, the rapid reversal of acidosis reactivates Na+/Ca2+ exchange at a time when sodium overload has not yet been resolved, driving Ca2+ into the cells. The damaging effects of the Ca2+-overload were later documented by Kus uoka (66) who discovered that hearts which were reperfused with a low Ca2+ solution showed a marked decr ease in IR-induc ed injury. In fact, a transient Ca2+-overload, even in the absence of ischemia, has been shown to cause myocardial dysfuncti on and injury (63). Increased free Ca2+ can contribute to the pathology of the heart cell through several mechanistic pathways. In fact, since the earl y work of Grinwald and Kusuoka (38, 67), the calcium hypothesis has evolved to incorporate several distinct mechanisms, which attempt to explain the means through which Ca2+ may lead to myocardial dysfunction and/or injury. The proposed mechanisms include: increased RO S production, excitation-contraction (E-C) uncoupling due to decreased Ca2+ responsiveness and increased protease activity (12).

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26 Calcium induced ROS production Several researchers have suggested that IR-induced increases in free Ca2+ may lead to increased ROS generation. For example, Seno et al. found that Ca2+ stimulated increased radical production from NADPH oxidase (103). Furthe r, Gottlieb et al. proposed a model through which Ca2+ could increase mitochondrial ROS production via several pathways (36). First, Ca2+ stimulates KREBS cycle activation which results in increased electron flow into the respiratory chain and therefore, in creased leaking of electrons from the respiratory complexes to oxygen, forming the superoxide radical. In addition, Ca2+ stimulates nitric oxi de production, which can inhibit electron flow into the m itochondria through complex IV as well as complex I, resulting in increased ROS production. E-C uncoupling and decreased calcium sensitivity It is well accepted that IR results in a decr ease in myocardial contractile function. One possible explanation is a decrea se in E-C coupling. Early work by Kusuoka et al. described a decline in maximal Ca2+-activated force production in the heart following IR (65). Because electrical activation was not impa ired in the heart following IR ( 47), the explanation for the IRinduced reduction in E-C uncoupling must lie in either of two mechanisms: a reduction in Ca2+ availability within the cell or a decrease in calcium responsivene ss of the contract ile machinery. As discussed earlier, several groups have shown an increase in intracellular Ca2+ following IR (38, 67). Therefore, a reduction in calcium availability is not a likely explanation for the observed decrease in E-C coupling. This leaves the possibility of a reduced Ca2+ sensitivity of the contractile machinery within the cell. The idea that myocardial Ca2+ sensitivity is reduced followi ng IR originated from the observation that although contra ctile function was significant ly impaired following IR, Ca2+ levels were actually elevated. Since Ca2+ stimulates muscular contra ction, it was postulated that

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27 the Ca2+ sensitivity of the contractile proteins must be reduced. Although several studies have confirmed these findings, the exact mechanisms responsible for the IR-i nduced reduction in Ca2+ responsiveness are not complete ly understood. Most of the work completed to date has implicated the structural modification of one or more of the myofibrillar proteins (65). Studies using skinned fibers have suggest ed that IR results in modifica tion of the myofilaments (51). This may be an additional point of interaction between the Ca2+ overload and oxyradical theories because ROS have been shown to modify ce llular proteins, impairing their function. Among their many possible targets, radi cals may modify myofibrillar pr oteins, by oxidizing thiol groups, resulting in impaired Ca2+ responsiveness (150, 92). Using immunohistochemistry, Matsumura observed degradation of the myofilame nt-associated scaffolding protein -actin following IR (79). In addition, Gao documented a decrease in the thin-filament re gulatory protein troponin-I following IR (32, 33). Further, Gao prevente d the degradation of troponin-I by altering the reperfusate in such a way to mitigate Ca2+-overload in the heart following ischemia. These observations are particularly impor tant given the crucial role of troponin-I as an intermediary between Ca2+ activation and cross-bridge cycling. Th is degradation of troponin-I may explain much of the depression in myocardial contractile function following IR. This idea becomes even more pertinent to the experiments proposed in this manuscript when considering the fact that troponin-I is also cleaved by the Ca2+-activated protease calpain. Calcium-activated proteases Cathepsins. The cathepsins are a group of lysoso mal proteases, which are found in innate immune cells such as neutrophils and macrophages. Th erefore these lysosomal enzymes are frequently found in areas of inflammation a nd injury. For example, increased levels of cathepsin B and D are frequently observed in patients with heart disease and other chronic

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28 inflammatory conditions (84). Although cathepsins clearly play a role in chronic inflammatory conditions, their contribution to acu te IR injury is unlikely to be important given the fact that reperfusion times of almost two hours are require d for significant neutrophil infiltration into myocardial cells. Calpain. Increases in free Ca2+ during IR can lead to increased activation of Ca2+activated cysteine proteases, such as calpain. Calpain exists in myocytes in two primary isoforms, micro (calpain I) a nd milli (calpain II), named fo r the respective amounts of Ca2+ required for their activation in vitro Both calpain isoforms are he terodimers made up of a large (80 kDa) and small (28-30 kDa) regulatory subunit (35). Calpain has several Ca2+ binding domains similar to calmodulin. Ca2+ binding causes a shift in th e structure of the protein, exposing a site for interaction with various subs trates (35). Although the two calpain isoforms are named for Ca2+ concentrations n eeded for activation in vitro there is evidence that calpain II can be activated by far less than millimolar Ca2+ concentrations in vivo increasing its relevance to myocardial IR injury (35). Regardless, once activated, calpain migrates in the cytosol toward the SR and/or plasma membrane where the majo rity of its substrates are located. These substrates include struct ural and contractile pr oteins as well as Ca2+ handling proteins, to name a few. The exact number of calpain-targeted pr oteins is currently unknown, however, a recent review by Goll et al. (35) reported over 100 differe nt proteins that serve as calpain substrates. Note that this review only disc ussed cytoskeletal prot eins, kinases and phosphateses, just a few categories of potential calpain substrates (35). Hen ce, it is likely that calpain cleaves many more than 100 proteins in cells. Although both calpain I and II have similar subs trates, there is some evidence to suggest slightly different role s for the two isoforms in vivo For example, work in skeletal and cardiac

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29 tissues have documented an initial increase in both calpain I and II activ ities in response to various modes of tissue injury followed by a late r, second increase in cal pain I activity. This evidence has led some to believe that while both isoforms clearly contribute to cellular injury in response to various stressors (IR, hypoxemia, hydrogen peroxide calcium overload) calpain I may also play a more productive role in th e removal of damaged proteins, etc. following injury. In addition, long-term i nhibition of both isoforms typically results in the death of the animal suggesting that calpain plays a role in normal homeostatic cellular pathways. However, in the current review, the primary interest in calpain is dire cted toward its contributing role in cellular injury following a stressor, such as IR. Calpain and IR Injury The IR-induced increase in calpain activati on has been well documented in the heart (9, 19, 35, 55, 115, 122, 128). This increase in calpai n activity has long been known to play a deleterious role in myocardial IR-induced inju ry. Once activated through binding with calcium, calpain can injure cardiac myocytes via severa l different pathways. Calpain cleaves several structural proteins leading to the release of myofilaments, facilitating their degradation by the proteosome (35, 83, 99, 124, 125). Moreover, calp ains may contribute to apoptosis, through cleavage of Bid, mediating cyto chrome c release from the mitochondria (18, 19, 35). Also, calpains increase the expression of cell adhesion molecules, leading to an increase in neutrophilmediated oxidative damage (91, 108). Each of these pathways has been shown to significantly contribute to IR-associated injury. In suppor t of this postulate, several studies have demonstrated cardioprotection through the use of calpain inhibitors pr ior to IR (55, 120, 122, 128). Recent work from our laboratory supports th ese findings, demonstrating almost complete cardioprotection against IR-induced contractile function and injury using the calp ain inhibitor MDL-28170 (30). These results provide physiolo gical support to the notion that calpain

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30 activation plays a significant role in IR-induced m yocardial injury. In fa ct, calpain may play a unique role in linking the two predominant mechan istic theories of myo cardial IR injury, the Ca2+-overload hypothesis and the oxyradical hypothesis. Calpain: Linking the Oxyradical and Calcium-Overload Theories Although several independent theo ries have been proposed in order to explain myocardial injury and dysfunction following IR, the pathogen esis of IR most likely involves a complex interaction between the oxyradical and Ca2+-overload hypotheses. Several mechanisms have been proposed linking the two theories. One possibi lity is that free radica ls generated during IR could interact with various SR proteins, causing SR dysfunc tion and damage (58, 61, 64, 102). The Impairment in SR function would likely result in increased free cytosolic Ca2+, and calpain activation leading to further ce llular damage. Supporting this postulate, cell culture studies administering antioxidants prio r to reoxygenation have observed a significant attenuation in Ca2+-overload (85). In addi tion studies using both in vitro working heart and langendorf IR models have described an attenuation in IR-indu ced calpain activation th rough the use of various antioxidants (107). Because calpain is activated by Ca2+ this indirectly indicates a reduction in IR-induced Ca2+-overload (35). When also taking in to consideration the possibility that oxidative modification of SR proteins by free radicals may in crease the likelihood of their cleavage by calpain, it appears that calpain may play a critical role in IR-induced injury, linking the oxyradical and Ca2+-overload theories. Calpain and Calcium-Handling Proteins. Regulation of Free Cytosolic Calcium The bulk of the Ca2+ released within the cell co mes from calsequestrin-bound Ca2+ stores within the sarcoplasmic reticulum (SR). Ca2+ is normally released by the process of Ca2+induced Ca2+ release in which the entry of a small amount of Ca2+ across the plasma membrane

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31 (sarcolemma) triggers the release of much more from the SR. This mechanism depends on the fact that Ca2+ entry from the extracellular fluid, vi a the dihydropyridine receptor (DHPR) and Ltype Ca2+ channels, and to a le sser extent the sodium calcium exchanger (NCX), increases the probability that the SR Ca2+ release channel (ryanodine receptor, RyR) is open. The greater the probability that the RyR is open the greater the release of Ca2+ from the SR. Once cyto s olic calcium levels are elevated, the ca lcium must either be moved back into the SR, or removed from the cell completely via the plasma membrane. There are two primary means of calci um removal from the cytosol: the sarcoplasmic/endoplasmic reticulum calci um ATPase (SERCA), and the sodium calcium transporter (NCX). The SERCA is an ATP-de pendant pump located in the SR, which removes calcium from the cytosol, re turning to the SR where it is stored bound to the protein calsequestrin for later release. SERCA ac tivity is regulated by the protein phospholamban (PLB), as well as ATP levels (77). Calcium can also be rem oved via the plasma membrane by the NCX. The NCX couples the transport of three Na+ molecules to one Ca2+ molecule in the opposite direction in two consecutive steps. Toge ther, these proteins/protein complexes play a critical role in the regulation of free cytosolic Ca2+ levels and therefore, calpain regulation, during IR. Importantly, all of these proteins/protein comp lexes are targets for oxidative modification by ROS during IR as well (64). Oxidative Modification and Degradat ion of Calcium-Handling Proteins There is a significant body of work deta iling the oxidative modification of Ca2+-handling proteins within the cell (64). Following is a very brief review of pertinent studies, which deal with oxidative stress and the Ca2+-handling proteins mentioned earlier.

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32 SERCA and PLB SERCA, and its regulatory prot ein PLB, play a critical role in the removal of Ca2+ from the cytosol, returning it to the SR. Any impairment in function could lead to elevations in free cytosolic Ca2+. Importantly, Favero et al. (29) found that hydrogen peroxide inhibited SERCA activity within the myocardium. In addition, Grov er et al. (39) studied the interaction of ROS with smooth muscle SERCA and found that hyd rogen peroxide-induced damage to SERCA diminished the SR Ca2+ pool as well as the smooth muscle response to Angiotensin II. Grover also reported similar results us ing superoxide (39). In additio n, Suzuki and Ford (111) reported that ROS induced concentration-dependant in hibition of SERCA. Collectively, these studies indicate that ROS can impair SERCA function and impair Ca2+ uptake into the SR. NCX The NCX is the primary means of removing Ca2+ from the cell via the plasma membrane. A reduction in NCX function could elevate levels of free cytosolic calcium. There is evidence suggesting that this exchanger is a t e tramer linked by disulfide bonds, and therefore, is susceptible to modification by ROS (16, 59, 90). Supporting this theor y, Coetzee et al. (21) reported NCX inhibition in guin ea pig cardiac myocytes follow ing a hypoxanthine / xanthine oxidase treatment (i.e., superoxide ge nerating system). Kato et al (59) also observed similar results in isolated SR vesicles from bovine hearts. In a ddition, DiPolo and Beauge (26) proposed that NCX inhibition is due to a ROS-indu ced reduction in the calcium sensitivity of the exchanger. Hence, similar to ROS damage to SERCA, it also appears th at ROS can modify and damage membrane Ca2+ transport as well. L-type calcium channels and the DHPR Several groups have documented a decrease in L-type Ca2+ channel current by ROS. For example, Tokube et al. (118) found that hypoxanthine / xanthine oxidase treatments, as well as

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33 hydrogen peroxide treatments inhibited the L-type Ca2+ channel current in cardiac myocytes. Further, this inhibition was prevented by SOD. Guerra et al. (40) observed almost identical findings, using dihydroxyfumaric acid (DHF). In addition, Coeteez et al. (22) described a decrease in the peak Ca2+ current of L-type Ca2+ channels following hypoxanthine / xanthine oxidase treatments. Summary: Calpain and Calcium-Handling Proteins There is recent evidence suggesti ng that calpain may cleave key SR Ca2+-handling proteins during IR (64). Degradation of these pr oteins would lead to a disruption in Ca2+ transport within the cell, further exacerba ting the IR-induced increase in free cytosolic Ca2+. Importantly, there is reason to believe that Ca2+-handling proteins within the SR may become targets for calpain cleavage after they are oxidatively modified by ROS. This may provide a very important link between the two predominant mechanis tic theories of IR injury, the Ca2+-overload theory and the oxyradical theory. Additionally, th is would present an interesting scenario in which calpain can could regulate its own activ ation in a feed-forward mechanism. Therefore, the first goal of this these experiments will be determine the relati onships between IR-induced ROS production, the oxidative modification of Ca2+-handling proteins, calpain activ ation, and the calpain-mediated cleavage of Ca2+-handling proteins. Antioxidants and SR Dysfunction Because of the possible link between the oxidation of Ca2+-handling proteins and their subsequent degradation by calpain, alterations in the antioxidant st atus within the cell may play a critical role in reduc ing calpain activation, Ca2+-overload, and the associated deleterious effects within the myocardium. As discussed earlier, the addition of antioxidants has been shown to significantly reduce Ca2+-overload in cardiac myocyte cultures and the intact heart following IR (58, 61, 64, 102). Additionally, antioxidant treatments can also preserve Ca2+-handling protein

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34 function in cells following oxidative stress. Howe ver, which antioxidant(s) are most effective in providing protection to Ca2+-handling proteins in vivo are cu rrently unknown. Nonetheless, an antioxidant that is thought to play a critical role in pr otecting the myocardium against IR injury is MnSOD (45, 93, 127). As discussed earlier, Mn SOD may play a very important role in protecting the heart against ROS because of its location within the mitochondria, a major source of myocardial radical production. It is possible that an increase in myocardial MnSOD activity may reduce the oxidative modification of critical SR and plasma membrane Ca2+-handling proteins, maintaining their functi on and reducing the likelihood of their degradation by calpain. This would in turn serve to attenuate any furt her IR-induced increase in free cytosolic calcium and calpain activation. One well established mo del of increasing MnSOD protein content and enzyme activity is endurance ex ercise training (55, 120, 122, 128). Exercise-Induced Cardio-Prot ection Against IR Injury Regular bouts of muscular ex ercise (e.g., 60 minutes of endurance exercise) is a wellestablished means of inducing cardio-protection against IR-i nduced injury (71, 72, 93, 113, 114). Work from numerous laboratories has consiste ntly demonstrated exercise-induced cardioprotection against IR insults of varying severities, ranging from minor injury to infarction. Additionally, work from our labor atory has determined that three days of exercise training provides the same degree of car dio-protection as long-term (w eeks) training (14, 25, 44, 93). Although there is little debate concerning the protective eff ects of exercise training, the mechanisms through which it provides cardio-protec tion are not completely understood. Several potential mechanisms to explain exercise-induced cardio-protection exist, including increases in myocardial heat shock proteins (HSPs), increased antioxidant capacity, and reduced calpain activation. The following sections will address each of these possibilitie s in greater detail.

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35 Increased Myocardial He at Shock Proteins Heat shock proteins are a multifunctional gr oup of proteins, which are stimulated by a variety of stimuli such as heat, oxidative stress, Ca2+-overload, exercise training, and low pH. Once active, these proteins serve several function s within the cell, including: chaperoning and / or translocating proteins, fold ing and refolding proteins, scav enging free radicals, and even facilitating protein synthesis. However, alt hough the cardio-protectiv e properties of various HSPs have been demonstrated, the importance of HSPs to exercise-induced cardio-protection is somewhat controversial. For example, work from our laboratory has suggested that an elevation in HSPs is not essential for exer cise induced cardio-prot ection (46). In these studies, hearts from animals, which were exercise trained in a cold environment (4 C), were compared to the hearts from animals that were trained at room temper ature. Hearts from the cold-trained animals demonstrated a similar level of cardio-protection against IR injury compared to the warm-trained animals, even without the exercise -induced increase in HSP protein content. These results have been confirmed by other groups as well (114). Therefore, it appears that an elevation in myocardial HSPs is not essent ial for exercise-induced cardi o-protection. Another possible mechanism of exercise-induced cardio-protection is through an up-regulation of endogenous antioxidants. Increased Myocardial Antioxidant Capacity As discussed earlier, the cell c ontains several antioxidant de fenses against IR-induced ROS production. The primary antioxida nt defenses are t hought to include GSH, GPX, CAT, and SOD. Although there is an abunda nce of research demonstrating the cytoprotective effects of these antioxidants during IR, the question of whic h antioxidants may play a critical role in exercise-induced cardio-protecti on is yet unanswered. Importantl y, protein levels and activities of only a few antioxidant enzymes have been s hown to increase consistently following exercise

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36 training (95, 96). For example, GSH content has been shown to be elevated following long-term (8-10 weeks) exercise traini ng ((52, 53, 56, 98) whereas short-te rm training has been found to increase GSH protein content (75) decrease it (69), or result in no change (105). Therefore it can be concluded that long-term exercise training elevates GS H but the conflicting evidence in the literature does not pe rmit a firm conclusion about the impa ct of short-term training on cardiac levels of GSH. Moreover, most studies have concluded that exercise training does not elevate GPX levels in the heart (25, 92, 94). The effect of exercise on CAT activ ity is also somewhat unclear with some studies repor ting increases and others reporti ng no change following training (25, 44, 46, 109). In contrast, it is widely agr eed that exercise elev ates myocardial MnSOD protein content and activity (25, 44, 45). Increased MnSOD activity Growing evidence suggests that endurance exercise may provide protection, at least in part, by up-regulating th e endogenous antioxidant Mn SOD. As discussed earlier, MnSOD may play an impor tant cardio-protective role in the heart during IR due to its localization in the mitochondria and ability to prevent oxidative stress induced by mitochondrial superoxide production. Se veral studies have documented the protective effects of MnSOD. For example, Chen et al. (20) demonstrated that MnSOD over-expression reduced infarct size following IR injury. Further, A bunasra et al. (1) observed cytoprot ection against IR injury using adenoviral gene transfer of MnSOD. In addi tion, recent studies using a MnSOD mimetic, which was directed almost exclusively into the mitoc hondria, observed that hearts from animals which were given the mimetic showed a significan t improvement in myocardial function and a reduction in myocardial injury following IR (2). Exercise-Induced Regulation of Calpain Activation Recent work from our laboratory has revealed an exercise-induced decrease in calpain activation following IR (30, 97). This work also demonstrated a decrease in calpain-mediated

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37 cleavage and degradation of the Ca2+-handling protein SERCA2a ( 30). This suggests that exercise may provide cardio-prot ection, at least in part, throu gh the regulation of IR-induced calpain activation. However, the mechanisms of exercise-induced calp ain regulation are not currently known. One possibili ty is that exercise-induced increases in MnSOD may reduce oxidative modification to Ca2+-handling proteins, attenuating the IR-induced increase in free Ca2+ and calpain activation. Therefore, the s econd goal of these experiments will be to determine the relationships between MnSOD, oxidative modification of Ca2+-handling proteins, calpain activation, and calpainmediated degradation of Ca2+-handling proteins. Summary Myocardial IR injury is a complex problem involving both the generation of free radical species (the oxyradical theory), as well as increases in free cytosolic Ca2+ (the Ca2+-overload theory), resulting in loss of m yocardial function, damage and degr adation of cellular proteins and lipids, and cell death. Incr eased activation of the Ca2+-dependant protease, calpain during IR may provide an important link between these two theories by preferentially cleaving Ca2+handling proteins which have b een modified by free radicals, t hus exacerbating the problem of Ca2+-overload and calpain-mediated injury within the myocardium. It is believed that much of the ROS producti on during IR originates from the mitochondrial respiratory chain. The antioxida nt MnSOD is localized in the mitochondria and can reduce the generation of the free radical supe roxide. Additionally, exercise training ha s been consistently shown to provide cardio-p rotection against myocardial IR injury and is also associated with an increase in MnSOD and a decrease in calpain activation. This increase in MnSOD may reduce the oxidative modification of cal cium handling proteins thus reducing calpain activation and maintaining Ca2+ homeostasis.

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38 CHAPTER 3 METHODS The methods segment of this proposal will be divided into five sections with the first providing details about the experimental animals, the second outlining the experimental designs, the third and fourth detailing the general methods and dependant measurements used in these experiments and the fifth desc ribing statistical analyses. Experimental Animals Animal Model Justification Adult (3 5 month old) male Sprague-Dawley (SD) rats were used for these experiments. The animals were 3 5 months of age (young adult) at the time of sacrifice. The SD rat was chosen for several reasons: first, the invasive nature of these experiments precludes the use of human subjects. Second, the SD model is a we ll accepted model for the study of myocardial ischemia reperfusion injury (14, 56, 70, 88, 92). Third, the SD rat does not display large interanimal variation in measures of cardiac contractil ity and/or collateral circulation. In addition, we chose to study male rats to avoid the possibl y confounding effects of varying estrogen levels across the estrus cycle (116). Animal Housing and Diet All animals were housed at the University of Florida Animal Care Services Center. Animals was maintained on a 12:12 hour light-d ark cycle and provided food (AIN93 diet) and water ad libitum throughout the experimental protocol. Experimental Design Experimental Design: Hypothesis One Animals were randomly assigned to one of five experimental groups (Figure 3-1). The Control group (hearts were quickly remove d from anesthetized animals, i.e. no in-vitro

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39 perfusion) served as a control for all dependant measures. A Control-perfused group was also included to control for any dependa nt variable changes that result from perfusion on the isolated working heart preparation. To i nvestigate the effects of IR, th ree experimental groups (ControlIR, Calpain-Inhibited-IR, and Vehi cle-IR) were subjected to an in -vitro IR stress (i.e., no flow ischemia, followed by reperfusion). At the conclu sion of each experiment, segments of the left ventricle were rapidly frozen in li quid nitrogen and stored at -80oC until assay. Samples were subsequently assayed to determine the levels of selected biochemical dependent measures. Figure 3-1. Experimental design for Hypothesis One. Experimental Design: Hypothesis Two Animals were randomly assigned to one of si x experimental groups. Four groups were exercise trained, as detailed belo w, while the other two groups re mained sedentary (Figure 3-2). To elucidate the role that MnSOD plays in ex ercise-induced cardio-pro tection, one exercise trained experimental group rece ived an antisense oligonucleotid e against MnSOD following each exercise training session. Importantly, our experi ence with this antisense oligonucleotide is that this treatment consistently attenuates the ex ercise-induced increase in myocardial MnSOD Control Control Perfused Control IR Calpain Inh IR Perfusion Only Calpain Inhibition In-vitro DEPENDANT MEASURES Contractile function / Ca2+-handling LVDP, +dp/dt, -dp/dt Oxidation of critical Ca2+-handling proteinsWB for carbonyl & HNE formation Degradation of critical Ca2+-handling proteinsWB Vehicle IR Ischemia-reperfusion In-vitro Control Control Perfused Control IR Calpain Inh IR Perfusion Only Calpain Inhibition In-vitro DEPENDANT MEASURES Contractile function / Ca2+-handling LVDP, +dp/dt, -dp/dt Oxidation of critical Ca2+-handling proteinsWB for carbonyl & HNE formation Degradation of critical Ca2+-handling proteinsWB Vehicle IR Ischemia-reperfusion In-vitro

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40 activity associated with exercise training without reduc ing MnSOD activity and/ or protein levels below those of control animals (45, 73). In addition, an oligonucleot ide mismatch group of trained animals was included to account for any extraneous effects of the oligonucleotide. All experimental groups underwent either a sham surgery or an in vivo IR surgery, also described below. The Control-Sham group served as a control for all dependant measures. At the conclusion of each experiment, segments of the left ventricle were assayed to assess the levels of numerous biochemical dependant measures. Figure 3-2. Experimental design for Hypothesis Two. General Methods The Isolated Working Heart Preparatio n/IR Protocol (Hypothesis One) To investigate myocardial function before and after an IR insult, we selected the in vitro working heart model. This model is a highly repr oducible preparation for examination of cardiac performance, as cardiac preload and after-load pr essures are maintained constant. Further, an DEPENDANT MEASURES Calpain activityWB for II-spectrin Oxidation of critical Ca2+-handling proteinsWB for carbonyl & HNE formation Degradation of critical Ca2+-handling proteinsWB Control Sham Trained IR Trained IR MnSODAS Trained IR Mismatch Exercise Training Trained Sham Control IR Sham Surgery IR In-vivo DEPENDANT MEASURES Calpain activityWB for II-spectrin Oxidation of critical Ca2+-handling proteinsWB for carbonyl & HNE formation Degradation of critical Ca2+-handling proteinsWB Control Sham Trained IR Trained IR MnSODAS Trained IR Mismatch Exercise Training Trained Sham Control IR Sham Surgery IR In-vivo Control Sham Trained IR Trained IR MnSODAS Trained IR Mismatch Exercise Training Trained Sham Control IR Sham Surgery IR In-vivo

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41 advantage of the working heart model versus an in vivo IR model is the elimination of the confounding influence of other organ system s, systemic circulation, and peripheral complications. This preparation has been success fully used by our laboratory for over five years (30, 71-73, 92, 97) In Vitro Ischemia-Reperfusion Protoc ol (Hypothesis One) Simultaneous clamping of aortic and atrial lin es was used to induce global, normothermic, no flow ischemia. Following 30 minutes of perfus ion during the pre-ischemic protocol, ischemia was maintained for 25 minutes followed by 45 mi nutes of reperfusion. During ischemia, the heart was enclosed in a sealed, wate r-jacketed chamber maintained at 37 C. Following the ischemic period, the heart was switched to th e retrograde perfusion mode for 10 minutes followed by 10 minutes of assist mo de (retrograde perfus ed with the atrial cannula open) and 25 minutes of normal reperfusion. Upon the c onclusion of non-perfusi on, perfusion, or IR treatments, the left ventricular free wall was immedi ately sectioned into four strips cut from base to apex. Prior to storage, heart sections we re rinsed in a cold antioxidant buffer (50mM NaHPO4, 0.1mM butylated hydroxytoluene, and 0.1mM EDTA). These tissue sections were then rapidly frozen in liquid nitrog en, and stored at -80 C until subsequent biochemical analysis. Cardiac Performance Measurem ents (Hypothesis One) Cardiac performance measurements were reco red every 5 minutes prior to ischemia and during reperfusion. Measurements included: left ventricular de veloped pressure (LVDP), the rates of pressure development (+ dp/dt) and decline (-dp/dt), and h eart rate. These variables were measured via a calibrated pressure transducer (Harvard Instrume nts) connected to the aortic cannula. Data was recorded and stored using a customized computer data-acquisition system.

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42 Calpain Inhibition (Hypothesis One) To determine the effects of IR-induced calpain activation on oxidation and degradation of myocardial calcium handling prot eins, calpain was inhibited ( in vitro ) using the selective inhibitor MDL 28170, also known as calpain inhib itor three or (CI3) (EMD Biosciences, La Jolla, CA). The efficacy of CI3 as a selectiv e inhibition of calpain I and II has been well established (18, 19, 30, 122, 125). Th e inhibitor was dissolved in Dimethyl Sulfoxide (DMSO) and added to the perfusion buffer prior to heart perfusion at a concentration of 10 M. In preliminary experiments, this co ncentration of CI3 was shown to inhibit calpain I and II without inhibiting the proteosome. Exercise Training Protocol (Both Hypotheses) Exercise trained animals began by performing 5 consecutive days of gradual habituation to treadmill running. Treadmill habitua tion initiated with 10 minutes of training the first day and was increased by 10 minutes each day, ending in 50 minutes of runni ng on the fifth day. Following habituation, exercise trained animals performed 3 consecutive days of treadmill running (60 minutes/day) at an intens ity of approximately 60-70% of VO2max. In Vivo Ischemia-Reperfusion Prot ocol (Hypothesis Two) The in vivo model of coronary artery ligation has been used succe ssfully by our laboratory for over 12 years. In our hands, ligation of the le ft main coronary artery (close to its origin) using this in vivo preparation consistently results in ischem ia in 60% of the ventricular free wall. Rats were anesthetized (80 mg/kg sodium pentobarb ital i.p.) and ve ntilated (Harvard Apparatus, Holliston, MA) with room air via a tracheostomy tube. A saline-filled catheter attached to a pressure transducer was placed in the carotid ar tery and interfaced with a computerized heart performance analyzer for continuous monitori ng of arterial blood pressures (Digi-Med, Louisville, KY). Arterial blood (<100 l) samples were obtained prior to ischemia to assess

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43 blood gases and pH using a calibrated elec tronic blood gas analyz er (Instrumentation Laboratories, Lexington, MA). When indicated, th e tidal volume delivered by the ventilator was adjusted to correct blood gas abnormalities. An additional catheter was placed in the jugular vein for delivery of sodium pentobarbital (20 mgkg-1) as needed. Following a left thoracotomy, a ligature was placed around the left anterior de scending coronary artery (LCA), close to its origin. In sham surgery, the hearts were rem oved without occlusion of the LCA. For the IR surgery, a soft piece of polyethylene tubing was threaded through the ligature, pressed on the surface of the LCA, and secured with a small he mostat. Coronary occlusion was maintained for 50 min followed by 120 min of reperfusion. Elect rocardiographic activity was continuously monitored and recorded via an interfaced custom ized data acquisition program with data points recorded every millisecond. Following reperfusion, hearts were removed, rinsed in a cold antioxidant buffer (50 mM NaHPO4, 0.1 mM BH T, 0.1 mM diethylenetriaminepentaacetic acid, pH 7.4), and quickly frozen in li quid nitrogen for later analysis. Inhibition of MnSOD Protein Translation (Hypothesis Two) Antisense oligonucleotides (AS-ODN) are sing le-stranded synthetic DNA that typically contain a backbone with modifica tion to a specific se quence to hybridize to a specific messenger RNA. Hybridization of the ODN to mRNA inhibits the mRNA fr om initiating translation. To block the translation of MnSOD protein, an imals were injected (i.p.) with a 22-mer phosphorothioate derivative of the AS-ODN (5-CACGCCGCCCGACACAACATTG-3) immediately post-exercise at a dose of 10 mg/kg. The injection time and dose of this specific AS-ODN have been shown to provide optimal e xperimental conditions to inhibit the exerciseinduced increase of MnSOD activity in myocardial tissue (45, 73). This has been confirmed by previous experiments from our laboratory. In addition, a mismatch control group (MM-ODN (CAC TCC TCC CAG CAC AAC AGTC)) was included in these experiments to verify that the

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44 inhibition of MnSOD translation was due to the specific AS-ODN sequence, as well as accounting for any extraneous factors re sulting from the delivery of the ODN. Dependant Measures (Both Hypotheses) Measurement of Calpain Activation To assess calpain activity, calpain-sp ecific cleavage product of the protein II-spectrin was analyzed. Briefly, proteins were separated us ing standard SDS-PAGE techniques on a 4%-20% polyacrylamide gel. Proteins were then transferred to nitrocellu lose membranes and exposed to a mouse monoclonal primary antibody to II-spectrin (SIGMA, St. Louise, MO). Following washing, an anti-mouse IgG-HRP-conjugate d secondary antibody was applied for chemiluminescence detection (Amersham, Piscataway, NJ). Both II-spectrin intact and calpaincleaved fragments were analyzed using a Kodak imaging system. The cleaved band was expressed as a percentage of the intact band and finally expressed as a percentage of the Perfused Control group. Western Blots for Calcium-Handling Proteins Western blots were used to determin e protein levels of the following Ca2+-handling proteins: SERCA2a, phospholamban, L-type Ca2+ channels and the Na+/Ca2+ exchanger. These measurements were used to determine which Ca2+-handling proteins were degraded following IR, as well which proteins were degraded spec ifically by calpain during IR (Hypothesis one). Briefly, proteins were separated using st andard SDS-PAGE techniques on 4%-20% polyacrylamide gels. Proteins were then tran sferred to Polyvinylidene Difluoride (PVDF) membranes and exposed to a monoclonal primary antibody. Following primary antibody exposure, an anti-mouse, or an ti-rabbit 800 (green) or 680 (red) infared secondary antibody (LiCor, Lincoln, Nebraska) was app lied for infared detection. Each blot was then analyzed using an Odyssey infared imaging system (Li-Cor, Lincoln, Nebraska) and normalized to a commassie

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45 blue protein stain in order to ad just for protein loading. Results were then expressed as a percent of either Control-Perfused (Hypothesis one), or Cont rol-Sham (Hypothesis two). Immunoprecipitation of Calcium-Handling Proteins To determine the effects of exercise, IR and calpain inhibition on the oxidation of Ca2+handling proteins, each of the following pr oteins (SERCA2a, phospholamban, L-type Ca2+ channels and the Na+/Ca2+ exchanger) were first isolated by immunoprecipitation. Briefly, heart tissue was homogenized at a 1:10 dilution factor in a 100mM KPO4 buffer containing 1 m lactacystine, 1 m MG-132 (SIGMA, St. Louise, MO), pH 7.4. The homogenate was then centrifuged at 1000 g for 20 minutes to clear cellul ar debris. Approximately 1000 g of protein was then transferred to a new tube and exposed to 10 l of primary antibody to the protein of interest. Following an overnigh t incubation on a Fisher rocker, 40 l of protein A/G PLUSagrose (Santa Cruz Biotechnology, Santa Cruz, CA ) were added and incubated overnight. Four centrifugations (2500 rpm for 10 minutes) were then used to separa te the agrose-bound antibody/protein complex. Following each spi n, the pelleted complex was suspended in 118 l of KPO4 buffer. Finally, the Bradford protein assay was run to determine final protein concentration and the samples were normalized to approximately 2 mg of protein / ml. Measurement of Protein Carbonyl Form ation on Calcium-Handling Proteins Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive indices of oxidative injury (15) Proteins, isolated via immun oprecipitation, were examined for carbonyl formation using a commercially ava ilable Western Blot kit from Chemicon International (Chemicon Internati onal, Temecula, CA). This allowed for the determination of the level of oxidative modification to specific calcium-handling proteins.

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46 Measurement of HNE Formation on Ca lcium-Handling Proteins HNE (4-hydroxy-2-nonenal) formation is a major product of endogenous lipid peroxidation. The w-6-family (linoleic and ar achidonic acids) of polyuns aturated fatty acids produce HNE as a result of free radical-induced lipid peroxidation. HNE is a highly reactive aldehyde and can react with several functiona l groups on biological material, particularly sulfydryl groups to form thioes ter adduct and then hemiacetals. HNE may also react with histidine and lysine residues of proteins to form stable Michael addition-type of adducts. In addition, HNE-modification of proteins may impair biological functions. Pr oteins, isolated via immunoprecipitation, were examined for HNE fo rmation using a commercially available Western Blot kit from Calbiochem (SanDiego, Ca). This allowed for the determination of the level of oxidative modification to specific Ca2+ handling proteins. Data Analysis To test our hypotheses, one-way ANOVAs were performed to assess IR, calpain inhibition, and exercise training differences for the primary dependent measures. A Tukey post hoc test was used to determine group differences when indicated. Significance was established a priori at P < 0.05. The relationship between the oxi dative modificati on (carbonyl and HNE formation) of Ca2+-handling proteins and the degradation of Ca2+-handling proteins was assessed using a Pearsons simple correlation.

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47 CHAPTER 4 RESULTS Hypothesis One Animal Characteristics The physical characteristics for the animals in all experimental groups are presented in Table 4-1. Although body mass differed between the experimental groups, heart weights and heart-to-body weight ratios were similar. Table 4-1. Animal body and heart weights. Group Number Body Weight (g) Heart Weight (g) Heart / Body Weight Ratio (mg/g) Non-perfused Control 11 360 4.2 1.20 .02 3.34 .07 Non-perfused Trained 9 339 5.8 1.21 .03 3.57 .10 Perfused Control 11 354 4.4 1.16 .03 3.28 .09 Control-IR 11 347 8.4 1.22 .03 3.53 .11 Trained-IR 12 322 4.2 1.17 .02 3.65 .08 Calpain inhibited-IR 6 374 7.9 1.28 .08 3.41 .20 Vehicle-IR 6 401 8.0 1.25 .04 3.13 .06 Values are means SE. Significantly different from Trained-IR, Significantly different from VehicleIR, P < 0.05. Note that Non-perfused Trained and vehicle-IR groups had significantly different body weights and heart / body weight ratios compared to Non-perfused Controls, although heart weight did not differ. 0 20 40 60 80 100 120 Control-IRTrained-IRInhibited-IR % of pre-ischemic LVDP 0 20 40 60 80 100 120 Control-IRTrained-IRInhibited-IR % of pre-ischemic LVDP Figure 4-1. % Recovery of left ventricular de veloped pressure (LVDP). Values are means SE. Significantly different from Control-IR, P < 0.05. Note that inhibition denotes the inhibition of calpain using the inhibitor CI3. % of pre-ischemic LVDP

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48 Cardiac Performance Measures Percent recovery of left ventricular developed pressure (LVDP) LVDP is commonly used as an index of myo cardial function. By comparing LVDP prior to ischemia with post-ischemic LVDP we can quantify myocardial dysf unction. As expected, % recovery of LVDP was significan tly depressed in the Control-IR group (Figure 4-1). However, both exercise training and calpain inhibition at tenuated the loss of LVDP following IR. This demonstrates the cardio-protectiv e effects of exercise as well as the deleterious effects of the Ca2+-activated protease calpain. Figure 4-2. Rate of systolic pressure increase (+dp/dt). Values are means SE. Significantly different from Control-IR, P < 0.05. Note that inhibition denotes the inhibition of calpain using the inhibitor CI3 and vehic le denotes vehicle treatment only without CI3. Percent of pre-ischemic +dp/dt and dp/dt The measurements of +dp/dt and -dp/dt are in dicative of the rate of systolic pressure development (i.e., rate of ventricular contraction) and the rate of systolic pressure decline (i.e., rate of ventricular relaxation), respectively. Because myocardial contraction is regulated by the % of pre-ischemic +dp/dt 0 20 40 60 80 100 120Control-IRTrained-IRVehicle-IRInhibited-IR 0 20 40 60 80 100 120Control-IRTrained-IRVehicle-IRInhibited-IR % of pre-ischemic +dp/dt

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49 release and re-sequestering of Ca2+ within the sarcoplasmic reticulum, these measurements also reflect myocardial Ca2+ release and uptake kinetics. Furt her, by comparing +dp/dt and -dp/dt prior to and following ischemia, we can quantify the changes in myocardial contraction/relaxation rates and/or Ca2+-handling kinetics. As illustrated in Figures 4-2 and 4-3, both +dp/dt and dp/dt decreased significantly following IR in both the control-IR and vehicle-IR experimental groups, suggesting a decrease in Ca2+-handling efficiency. However, both exerci se training and calpain inhibition nearly completely prevent this IR-induced dysfunction. Figure 4-3. Rate of systolic pressure decline (-dp/dt). Values are means SE. Significantly different from Control-IR, P < 0.05. Note that inhibition denotes the inhibition of calpain using the inhibitor CI3 and vehic le denotes vehicle treatment only without CI3. Oxidative Modification of Critical Calcium-Handling Proteins The increase in free cytosolic Ca2+ and corresponding increase in calpain activity have been shown to play a deleterious role in car diac myocytes following IR Because the oxidative modification of Ca2+-handling proteins has been shown to lead to impaired Ca2+-handling, we postulated that the oxidation of Ca2+-handling proteins within the cardiac myocytes may impair 0 20 40 60 80 100 120 Control-IR Trained-IR Vehicle-IR Inhibited-IR % of pre-ischemic dp/dt % of pre-ischemic dp/dt

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50 function of the proteins and thus le ad to increases in free cytosolic Ca2+, the activation of calpain, myocardial damage and contractile dysfunction. Figure 4-4. Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reti culum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium ex changer (NCX). Representative blots are displayed above. Values, below, are means SE. Significantly different from respective Control-Perfused group, P < 0.05. Note that inhibition denotes the inhibition of calpain using the inhibitor CI 3 and vehicle denotes vehicle treatment only without CI3. We assessed oxidative modification of cal cium handling proteins by measuring both carbonyl and HNE formation via Wester n Blotting of each individual Ca2+-handling protein. Protein carbonyl levels of prot eins are indicative of the magni tude of oxidative modification of proteins whereas HNE-protein inte raction is taken as an indicati on of protein reactions with the reactive aldehyde, HNE. The measurement of both carbonyl and HNE fo rmation are commonly used to assess oxidative stress to proteins and lipids within the cell. 0 50 100 150 200 250 300 350 400 450 500 ControlTrainedControlPerfused Control-IRTrained-IRVehicle-IRInhibited-IR LTCC SERCA2a PLB NCX % of Control-Perfused % of ControlControl Trained Control Control Trained Vehicle Inhibited Perfused IR IR IR IR LTCC SERCA2 PLB NCX

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51 Figure 4-5. HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), P hospholamban (PLB) and Sodium/calcium exchanger (NCX). Representative blots are displayed above. Values below are means SE. Significantly different from re spective ControlPerfused group, P < 0.05. Note that inhibition denotes the inhi bition of calpain us ing the inhibitor CI3 and vehicle denotes vehicl e treatment only without CI3. Carbonyl formation on calcium-handling proteins IR resulted in an increase in carbonyl formation to all four Ca2+-handling proteins measured (LTCC, SERCA2a, PLB, and NCX). Im portantly, exercise trai ning attenuated the IRassociated increase in carbonyl form ation in all four proteins (Figur e 4-4). This suggests that all four proteins are subject to oxidative modification following IR. HNE formation on calcium-handling proteins An increase in HNE formation on SERCA2a and PLB was also observed following IR. However, the oxidative modification of these proteins was attenuate d by exercise training % of Control-PerfusedSERCA2a PLB NCX Control Trained Control Control Trained Vehicle Inhibited Perfused IR IR IR IR 0 50 100 150 200 250 300 350 400 450 ControlTrainedControlPerfused Control-IRTrained-IRVehicle-IRInhibited-IR SERCA2a PLB NCX % of Control

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52 (Figures 4-5). Note that no si gnificant changes in HNE forma tion were detected on the NCX, and there were not detectable leve ls of HNE formation on the LTCC. 0 50 100 150 200 250 ControlTrainedControlPerfused Control-IRTrained-IRVehicle-IRInhibited-IR LTCC SERCA2a PLB NCX Figure 4-6. Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reticulum calci um ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX). Representative blots are displayed above. Values below are means SE. Significantly different from respective Control-Perfused group, P < 0.05. Note that inhibition denotes the inhibition of calpain using the inhibitor CI3 and vehic le denotes vehicle treatment without CI3. Calpain-Mediated Degradation of Calcium-Handling Proteins We have previously demonstrated that IR re sults in increased calpain activation. Once active, calpain degrades many proteins/protein co mplexes within the cell. We hypothesized that calpain may cleave critical Ca2+-handling proteins, which have been oxidatively modified, LTCC SERCA2a PLB NCX Control Trained Control Co ntrol Trained Vehicle Inhibited Perfused IR IR IR IR % of Control

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53 exacerbating IR-induced Ca2+-overload, calpain activation and m yocardial dysfunction. To test this hypothesis we performed Western Blot analyses to determine protein content of each of the four Ca2+-handling proteins with and without calpai n inhibition. Our results demonstrated a decrease in intact LTCC, SERCA2a, PLB, a nd NCX following IR in both control-IR and vehicle-IR groups. However, bot h exercise training and calpain inhibition prevented this IRinduced degradation in all four proteins. Table 4-2. Correlations Between the Oxidative Modification of Ca2+-handling Proteins and Their Degr adation Following IR Carbonyl formation / Intact protein (R2) HNE formation / Intact protein (R2) LTCC -.901 SERCA2a -.939 -.939 PLB -.827 -.955 NCX -.511 -.263 Pearson correlation R2 values depicting the rela tionship between oxidative modification (carbonyl formation and HNE formation) to critical Ca2+-handli ng proteins and the total amount of intact protein, determined by Western Blot. All experiment al groups were pooled for this analysis. Our results indicate that all four of the measured Ca2+-handling proteins, LTCC, SERCA2a, PLB and NCX, are degraded by calpain during IR (Figure 4-6). In addition, exercise training attenuated the degrada tion of these proteins, most likely through a re duction in IRinduced calpain activ ation. Consistent with this postulate, a strong negative correlation exists between the oxidative modificati on (carbonyl and HNE formation) and amount of intact protein of each of the Ca2+-handling proteins (Table 4-2). This may suggest that oxidative modification makes these proteins more susceptible to calpain-mediated degradation. Hypothesis Two Calpain Activation (Calpain-Cleaved II-Spectrin) The deleterious effects of IR -induced calpain activation have been well documented (55, 120, 122, 128). Additionally, we have previously dem onstrated that exercise training attenuates

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54 calpain activation and provides cardioprotection agai nst IR injury (71, 72, 93, 96, 113, 114). The mechanism(s) of this protection, however, are currently unknown. 0 50 100 150 200 250 300 350 400 Figure 4-7. Western blo tting for calpain-cleaved II-Spectrin (an in-vivo calpain substrate). A representative blot, above, displaying intact (250 kD) and calpain-cleaved IISpectrin (145 kD). Values below are means SE. Significantly different from Control-Sham group, P < 0.05. We hypothesized that exercise may provide pr otection through up-regul ation of myocardial MnSOD resulting in protection against IR-induced oxidative stress to Ca2+-handling proteins, Ca2+-overload and calpain activation. Theref ore, we performed Western Blotting for IISpectrin, a well-characterized in-vivo calpain subs trate, in order to determine the effects of MnSOD on IR-induced calpain activation. As expected, IR resulted in an increase in ca lpain activation in control (sedentary animals), which was attenuated by exercise training (Figur e 4-7). Importantly, exercise trained animals treated with the antisense o ligonucleotide against MnSOD had similar levels of calpain Control Trained Control Trained Trained-IR Trained-IR Sham Sham IR IR Antisense Mismatch 250 kD 145 kD Control Trained Control Trained Trained-IR Trained-IR Sham Sham IR IR Antisense Mismatch % of Control Sham

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55 activation to control-IR animals. This finding is consis tent with the hypothesis that MnSOD plays a critical role in regula ting IR-induced calpain activation. Oxidative Modification of Critical Calcium-Handling Proteins The increase in free cytosolic Ca2+ and corresponding increase in calpain activity have been shown to play a deleterious role in the heart following IR. Because the oxidative modification of Ca2+-handling proteins has been shown to lead to impaired Ca2+-handling, we have hypothesized that the oxidation of Ca2+-handling proteins within the myocardium may lead to increases in free cytosolic Ca2+, the activation of calpain, myocardial damage and contractile dysfunction. Figure 4-8. Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel (LTCC), Sarcoplasmic/endoplasmic reti culum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium ex changer (NCX). Representative blots are displayed above. Values below are means SE. Significantly different from respective Control-Sham group, P < 0.05. % of Control-Sham Control Control Trained-IR Trained-IR Trained-IR Sham IR Mismatch Antisense LTCC SERCA2a PLB NCX 0 50 100 150 200 250 300 350 400 450 Control-ShamControl-IRTrained-MM-IRTrained-IRTrained-AS-IR LTCC SERCA2a PLB NCX % of Control Sham Control Control Tr ained-IR Trained-IR Trained-IR Sham IR Mismatch Antisense

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56 Carbonyl formation on calcium-handling proteins IR resulted in an increase in carbonyl formation on all four Ca2+-handling proteins measured in hearts from control (untrain ed animals): LTCC, SERCA2a, PLB, and NCX (illustrated in Figure 4-8). Ex ercise training attenuated the IR -associated increase in carbonyl formation in three of the four measured Ca2+-handling proteins: LTCC, SERCA2a, and PLB. Importantly, exercise-induced protection agai nst IR-induced carbonyl formation was abolished by the antisense oligonucleotide against MnS OD in both the LTCC and SERCA2a, but not in PLB. In addition, neither exercise training nor antisense treatment a ffected IR-induced carbonyl formation on the NCX. Figure 4-9. HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), P hospholamban (PLB) and Sodium/calcium exchanger (NCX). Representative blots are displayed above. Values below are means SE. Significantly different from respective Control-Sham group, P < 0.05. Control Control Trained-IR Trained-IR Trained-IR Sham IR Mismatch Antisense % of Control-Sham 0 50 100 150 200 250 300 Control-ShamControl-IRTrained-MM-IRTrained-IRTrained-AS-IR SERCA2a PLB NCX SERC A PLB NCX Control Control Tr ained-IR Trained-IR Trained-IR Sham IR Mismatch Antisense % of Control Sham

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57 HNE formation on calcium-handling proteins An increase in HNE formation on SERCA2a and PLB in the heart was also observed following IR, which was attenuated by exercise tr aining (Figures 4-9). In addition, exerciseinduced protection against IR-induced HNE fo rmation on SERCA2a and PLB was abolished by the antisense oligonucleotide against MnSOD. However, no significant changes in HNE formation were detected on the NCX, and ther e was no detectable amount of HNE formation on the LTCC. Figure 4-10. Western blotting for intact Ca2+-handling proteins: Ltype calcium channel (LTCC), Sarcoplasmic/endoplasmic reti culum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium exchanger (NCX). Values are means SE. Significantly different from respective Cont rol-IR group, P < 0.05. Control Trained Traine d Control Trained-IR Trained-IR Sham Sham IR IR Mismatch Antisense 0 20 40 60 80 100 120 140 160 ControlSham TrainedSham Trained-IRControl-IRTrained-MMIR Trained-ASIR LTCC SERCA2a PLB NCX % of Control-IR SERCA2a PLB NCX LTCC Control Trained Trained Control Trained-IR Trained-IR Sham Sham IR IR Mismatch Antisense % of Control Sham

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58 Calpain-Mediated Degradation of Calcium-Handling Proteins We and others (18, 19, 31, 97) ha ve previously demonstrated th at IR results in increased calpain activation in the heart. Once active, calpain degrades calpain substrates (proteins / protein complexes) within the cell. We hypothesized that calpain cleaves important Ca2+handling proteins, which have been oxidat ively modified, exacerbating IR-induced Ca2+overload, calpain activation and myocardial injury. To test this hypothesis we performed Western Blot analyses to determine protein content of each of the four Ca2+-handling proteins. Our results demonstrated a decrease in myocar dial levels of intact LTCC, SERCA2a, PLB, and NCX following IR, which was attenuated by ex ercise training. Moreover, MnSOD antisense oligonucleotide treatment abolished the ex ercise-induced cardiopr otection against Ca2+-handling protein degradation in all four proteins (Figure 4-10). These re sults are consistent with the notion that MnSOD plays a critical role in both the regulation of calpain activation as well as the preservation of Ca2+-handling proteins during IR. Add itionally, these results suggest that oxidative modification of these prot eins leads to their degradation. Table 4-3. Correlations between the oxidative modification of calcium-handling proteins and their degradation following IR Carbonyl formation / Intact protein (R2) HNE formation / Intact protein (R2) LTCC -.809 SERCA2a -.802 -.789 PLB -.722 -.823 NCX -.810 -.792 Pearson correlation R2 values depicting the rela tionship between oxidative modification (carbonyl formation and HNE formation) to critical Ca2+-handli ng proteins and the total amount of intact protein, determined by Western Blot. All experiment al groups were pooled for this analysis. Finally, there was a strong ne gative correlation between the oxidative modification and amount of intact protein of each of the Ca2+-handling proteins (Table 4-3). This finding is

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59 consistent with the concept that oxidative modifi cation makes these proteins more susceptible to calpain-mediated degradation.

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60 CHAPTER 5 DISCUSSION Overview of Principal Findings These experiments examined both the mechanis tic role that calpain plays in IR-induced cardiac injury as well as the mechanism res ponsible for exercise-induced cardioprotection against IR injury vi a two separate groups of experiments. Experiments conducted to determine the mechanism(s) responsible for calpain-induced cardiac injury (Hypothesis One) tested the fo llowing separate hypotheses: (1) IR promotes increased oxidative modificati on of important myocardial Ca2+-handling proteins; (2) oxidative modification of Ca2+-handling proteins is associated w ith increased degradation of these proteins; and (3) inhibition of calpain will attenu ate the IR-induced degradation of myocardial Ca2+-handling proteins. Our data confirm the de leterious effects of IR on the myocardium; including impaired Ca2+-handling and contractile function, ox idation and degrad ation of critical Ca2+-handling proteins, and increased calpain activation. In addition, we observed strong negative correlations between the degree of Ca2+-handling protein oxidative modification and the amount of intact protein suggesting a link between oxidative stress and protein degradation during IR. Importantly, our experi ments established that inhibiti on of calpain protects the heart against IR-induced contract ile dysfunction as well as the degradation of Ca2+-handling proteins. Our results provide the first evidence th at calpain cleaves the following critical Ca2+-handling proteins (LTCC, NCX and PLB) in the intact he art during IR. In additi on, our data confirms previous work describing the calpainmediated cleavage of SERCA2a during IR. Experiments completed to examine the m echanism responsible for exercise-induced cardioprotection against IR injury (Hypothesis Two) tested the follo wing postulates: (1) exercise training attenuates IR-induced oxidation and degradation of critical myocardial Ca2+-handling

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61 proteins; and (2) exercise-induced cardioprotec tion is dependent on an exercise-induced overexpression of cardiac levels of MnSOD. Our resu lts reveal that exerci se attenuated IR-induced calpain activation, ox idative modification of critical Ca2+-handling proteins and the degradation of these proteins. Importantly, when the exercise-induced over-expression of MnSOD was attenuated via gene silencing (i.e., antisense oligonucleotide against MnSOD), cardioprotection against Ca2+-handling protein oxidation and degrada tion, as well as calp ain activation was abolished. These results suppor t the concept that MnSOD play s a critical role in exerciseinduced cardio-protection against IR injury, at least in part, by preventing the IR-induced oxidation and degradation of Ca2+-handling proteins and calpai n activation. Moreover, these findings represent the first in-vivo data de monstrating that oxidatively modified Ca2+-handling proteins in the heart are more su sceptible to degradation during IR. In combination, the results of experiments s uggest a chain of events during IR beginning with an increase in oxidative damage of Ca2+-handling proteins, leading to impaired Ca2+handling and calpain activation, in turn resulting in the calpain-me diated degradation of critical Ca2+-handling proteins by calpain, exacerbating Ca2+-overload, and myocardial injury. In contrast, exercise attenuates these events, due in a large part, to the upregulation of MnSOD in the heart. Increased myocardial MnSOD reduces the oxidation of Ca2+-handling proteins, and the deleterious chain of events that follow by dismutating superoxide produced in the mitochondria during IR. Hypothesis One: The Effects of IR and Ca lpain Inhibition on Myocardial CalciumHandling Proteins The following paragraphs provide a detailed discussion of the findings of experiments designed to determine the role that calpain plays in IR-induced damage and removal of Ca2+handling proteins in the heart.

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62 Calpain Inhibition Protects Against IR-i nduced Damage and Removal of CalciumHandling Proteins. Calpain can modify and damage the myocardi um during IR via multiple pathways. For example, calpains cleave several structural prot eins leading to the re lease of myofilaments, facilitating their degradation by the proteoso me. In addition, calpains can also promote apoptosis, through cleavage of Bid, mediating cytochrome c release from the mitochondria. Moreover, calpains increase the expression of cel l adhesion molecules, leading to an increase in neutrophil-mediated oxidative damage. Each of these pathways has been shown to significantly contribute to IR-induced injury (55, 120, 122, 128). Further suppor ting the deleteri ous role of calpain during IR, inhibition of calpain has been previously re ported, by our group and others, to reduce many of the deleterious effects of IR including contractile dysfunction, infarct area and apoptosis (18, 19, 122, 123). Our experiments contribu te to previous studies, demonstrating that calpain inhibition attenuates the decline in LVDP, +dp/dt and dp/dt typically observed following IR (Figures 4-1, 4-2, 4-3). Decreased peak pr essures (LVDP), as well as decreased rates of pressure development (+dp/dt) and relaxation (dp/dt) typically occu r following myocardial ischemia and are indicative of impaired Ca2+ transport within the myocardium. Therefore, our data suggests that calpain impairs myocardial Ca2 +handling following ischem ia. To elucidate a potential mechanism of calpain-induced impairments in Ca2 +handling we measured the protein content of several cr itical myocardial Ca2 +handling proteins following IR, with or without calpain inhibition. Calpain Degrades Critical Calc ium-Handling Proteins We postulated that calpain degrades myocardial Ca2+-handling proteins during IR, exacerbating Ca2+-overload and IR injury. Further, we hypothesized that IR-induced oxidation

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63 of Ca2+-handling proteins would be associated with calpain-mediated degrad ation. We were lead to these hypotheses by three key observations. First, calpain cleaves Ca2+-handling proteins. There is growing evidence from in-vitro studies suggesting that calpain can cleave Ca2+-handling proteins. For example, Belles et al. (8) and De Jongh et al. (24) have report ed that calpain degrades the LTCC in-vitro Other in-vitro experiments have demonstrated calpain-cl eavage of the ryanodine receptor and the sodium/potassium ATPase (126, 130). Our re sults demonstrated a decrease in intact Ca2+handling proteins following IR, which was attenuat ed by calpain inhibition (Figure 4-6). These findings indicate that calpain mediates the de gradation of several cr itical myocardial Ca2+handling proteins, including the LTCC, SERCA2 a, PLB and NCX, during IR. Although calpainmediated cleavage of various Ca2+-handling proteins has been demonstrated in-vitro to our knowledge, the present study is the first to invest igate calpain-mediated degradation of these Ca2+-handling proteins in intact hearts. Second, Ca2+-handling proteins lose function when oxidized during IR. There is a strong body of in-vitro evidence examin ing the effects of ROS on Ca2+-handling protein function. This work is summarized in review papers detailing the ROS-mediated loss of function in all of the Ca2+-handling proteins examined in these expe riments (LTCC, NCX, SERCA, and PLB) (64, 129). In addition, several studies have demonstrated a loss in Ca2+-handling protein function following IR (48, 62). In combination with th e knowledge that widesp read protein damage occurs during IR, we postulated that Ca2+-handling proteins might al so be oxidized during IR, accounting for their loss of func tion. This postulate was supported by our findings that IR resulted in increased oxidativ e modification of critical Ca2+-handling proteins (F igures 4-4, 4-5).

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64 In addition, physiological m easures of myocardial Ca2+-handling (+dp/dt, -dp/dt and LVDP) were depressed following isch emia (Figures 4-1, 4-2, 4-3). Third, oxidative modification of proteins can le ad to degradation. Several studies have demonstrated that oxidative modification predispo ses cellular proteins for degradation. Recent work by Zolotarjova et al. (130) demonstrated that when oxidized, the sodium/potassium exchanger is increasingly prone to calpain-medi ated degradation. Wu et al also found that oxidation of the RYR leads to an increased incidence of degrada tion by calpain (126). Supporting this work, our results demonstrated strong negative co rrelations between the amount of oxidized calcium handling pr oteins (i.e., LTCC, NCX, SERCA and PLB) and the level of intact protein (Table 4-2), suggesting a li nk between the oxidative modification of Ca2+-handling proteins and their degradation. Nonetheless, this finding should be viewed with caution because, although strong correlations exist between the degree of Ca2+-handling protein oxidation and degradation, a strong positive correlation al one does not confirm a causal relationship. In combination, we feel that these data supports the idea that ROS produced during IR oxidatively modify cr itical myocardial Ca2+-handling proteins, resul ting in both a loss in Ca2+handling ability, as well as calpa in-mediated degradation of Ca2+-handling proteins, exacerbating IR injury. If this line of reasoning proves correct, a reduction in IR -induced oxidative stress would attenuate the oxidation and degradation of myocardial Ca2+-handling proteins, thereby decreasing IR-induced Ca2+ overload and calpain activation, attenuating m yocardial injury. Exercise training has been shown to reduce oxi dative stress within the myocardium during IR, possibly through the over-expre ssion of endogenous antioxidant enzymes such as MnSOD. We have also previously demonstrated that ex ercise reduces IR-induced calpain activation. We postulate that exercise regul ates IR-induced calpain activa tion by reducing the oxidative

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65 modification of myocardial Ca2+-handling proteins. This postula te was tested by a second series of experiments and these results will discussed in the subsequent sections. Hypothesis Two: IR, Exercise, MnSOD and Calcium-Handling Proteins These experiments tested the hypothesis that exercise would provide cardio-protection against the IR-induced oxidative modifi cation and degrada tion of critical Ca2+-handling proteins as well as calpain activation. In addition, we pos tulated that exercise-i nduced over-expression of MnSOD is a critical component of this exercise-induced cardio-protection. Exercise Training Provides Cardio-Protection We and others have previously demonstrated that exercise protects the heart against IR injury (71, 72, 93, 96, 113, 114). In addition, we have reported that exercise training prevents IR-induced calpain activation (31) However, the mechanism(s) responsible for this protection have not been determined. Expanding on our pr evious work, the current experiments identified one possible mechanism through which exercise may regulate calpain activation and provide cardioprotection by the pr eservation of critical Ca2+-handling proteins within the myocardium. Our results reveal that exercise provides car dioprotection against both the IR-induced oxidation and degradation of Ca2+-handling proteins (Fi gures 4-8, 4-9, 4-10). In addition, exercise attenuates IR-induced calpain act ivation (Figure 4-7), potentially through an improved regulation of free cytosolic Ca2+ in cardiac myocytes. Although the mechanism(s) responsible for these protective effects are not comple tely understood, we hypothesize that one possibility is that an exercise-induced over-expression of the anti oxidant MnSOD may provide protection against ROS-mediated Ca2+-handling protein degradatio n, calpain activation and Ca2+-handling protein degradation. To further inves tigate this possibility the exercise-induced over-expression of MnSOD was prevented via a gene si lencing using an antisense oli gonucleotide against MnSOD.

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66 Exercise-Induced Over-Expression of MnSO D Prevents the Oxidation of CalciumHandling Proteins. As discussed previously, ther e is strong evidence that bo th ROS and free cytosolic Ca2+ increase dramatically during IR Moreover, there is a biologi cal rationale to support a causal relationship between the IR-induced increase in ROS production and Ca2+-overload. In regard to the source of radical production during both ischemia and reperfusion, the mitochondrial respiratory chain has been identified as a majo r contributor to IR-indu ced free radical production (9). Further supporting the importance of mito chondrial free radical production, studies using antioxidants targeted specifically to the mitoc hondria have demonstrated a significant reduction in oxidant-related damage within the myocardi um, as well as improved myocardial function (4, 11, 28, 86). Results from our experiments demonstrated a reduction in the IR -induced oxidation of Ca2+-handling proteins following exer cise training (Figures 4-8, 4-9). Importa ntly, the exerciseinduced reduction in Ca2+-handling protein oxidation wa s abolished by the antisense oligonucleotide against MnSOD. This suggests th at mitochondrial super oxide production during IR contributes to the oxidati on of critical myocardial Ca2+-handling proteins. The mechanism through which mitochondr ia-produced superoxide affects Ca2+-handling proteins located in the SR and/or plasma me mbrane is currently not understood. Mitochondriaproduced superoxide has several potential fate s including conversion to a less reactive oxygen species, such as hydrogen peroxide (H2O2) by the antioxidant enzyme superoxide dismutase (SOD), or conversion to a more reactive species such as the hydroxyl radical (OH), in the presence of iron, or peroxynitrite (ONOO), through reaction with ni tric oxide. Although highly reactive, peroxynitrite an d the hydroxyl radical have very s hort half-lives a nd therefore would not be likely to oxidize Ca2+-handling proteins located in th e SR and/or plasma membrane.

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67 Hydrogen peroxide, however, has a longer half-lif e and could leave the m itochondria to directly interact with Ca2+-handling proteins. If hydrogen peroxide is responsible for oxida tion of cytosolic calcium handling proteins during an IR insult, an important question emerge s. That is, what is the fate of the excess hydrogen peroxide produced due to the exerci se-induced over-expression of MnSOD? As discussed earlier, MnSOD convert s superoxide to hydrogen peroxide Therefore, it is probable that an exercise-induced over-e xpression of MnSOD would resu lt in a marked increase in hydrogen peroxide, which has also been shown to exacerbate myocardial injury. Since we have consistently observed a decrease, not an incr ease, in IR-induced oxida tive modification of proteins with exercise training, we reason that exercise must al so over-express or up-regulated the activity of one or more hydrogen peroxide s cavenging systems within the myocardium. One possibility is an elevation in either protein content and/or activity of the hydrogen peroxide scavenger, catalase (12, 31). A nother possibility is that exerci se training increases the hydrogen peroxide buffering capacity of the glutathione system through an increas e in the amount of glutathione protein and/or an increase in the amount of glutat hione reductase protein or activity. Any of these alterations would allow the cell to more effectiv ely manage increased amounts of hydrogen peroxide. Nonetheless, previous experi ments have failed to confirm that exercise training results in an increase in myocardial le vels of catalase, glut athione peroxidase, or glutathione (44, 72, 87, 96, 117). Hence, it seems likely that another mechanism exists in cardiac myocytes to remove hydrogen peroxide. Two rece ntly discovered molecules involved in the removal of hydrogen peroxide from the m itochondria include both periredoxin III and thioredoxin (5, 23, 34, 54, 112, 121). However, at presen t, it is unclear if one or both of these

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68 antioxidants are exercise-induced in the heart. Cl early, this is an important topic for future research. Exercise-Induced Over-Expression of M nSOD Attenuates Calpain Activation In addition to protecting the heart against ROS-mediated damage to Ca2+-handling proteins, exercise-induced over-e xpression of MnSOD also attenua ted the IR-induced increase in calpain activation (F igure 4-7). This supports the idea that IR-induced calpain activation, is at least in part, due to ROS-mediated damage to Ca2+-handling proteins. It is possible that the oxidative modification of Ca2+-handling proteins cont ributes to IR-induced Ca2+-overload, resulting in greater calpain activation. Exercise-Induced Over-Expression of MnSOD Prevents the Degradation of CalciumHandling Proteins MnSOD also appears to play a role in the exercise-induced reduction of Ca2+-handling protein degradation during IR (Figure 4-10). Ind eed, prevention of exercise-induced increases in myocardial MnSOD via antisense oligonucleotides eliminated the exercise-induced protection against IR-induced degradation of these important proteins. This fi nding is consistent with the concept that the oxi dation of these Ca2+-handling proteins may make them more susceptible to cleavage by calpain. Degradation of Calcium-Handling Protei ns is Associated with Oxidation As previously discussed, research links the IR-induced oxidation of Ca2+-handling proteins with a loss of function both in-v itro as well as in the intact heart (62, 64, 129). Additionally there is evidence supporti ng the idea that oxidatio n of these proteins may facilitate their degradation by calpain (126, 130). Our experi ments provide two lines of evidence to support these ideas. First, strong correlations were observed between the amount of protein oxidation and degradation of Ca2+-handling proteins following IR suggesting that oxidized Ca2+-handling

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69 proteins are more likely to be degraded (Table s 4-2, 4-3). Second, exercise-induced protection against both the oxidation and degradation of Ca2+-handling proteins wa s almost completely prevented by MnSOD antisense oligonucleotide treatm ent. This finding is consistent with the notion that a causal relationship exists between the oxidative modification of Ca2+-handling proteins and their degradation. Summary and Future Directions This project utilized two separate experime nts to investigate the relationships between ROS, Ca2+-handling protein oxidation and degradation as well as calpain activation during IR. Major findings include the follo wing: (1) IR results in cont ractile dysfunction and impaired Ca2+-handling, calpain activation an d the oxidation and degradati on of critical myocardial Ca2+handling proteins; (2) calpain degrades several important Ca2+-handling proteins during IR including LTCC, SERCA2a, PLB, and NCX; (3 ) exercise training attenuates IR-induced oxidation of these calcium handling proteins and preserves their leve ls in the heart; (4) exerciseinduced cardioprotection is critically dependant on an up-regu lation of MnSOD. In combination, these findings are consistent with the mechanistic series of events, which is depicted in Figure 51. We postulate that IR results in an incr ease in mitochondrial s uperoxide production, which leads to the oxidation of critical Ca2+-handling proteins, resulting in increased free cytosolic Ca2+ and calpain activation and fi nally the calpain-mediated degradation of critical Ca2+-handling proteins. The results of these experiments provide a unique contribution to the existing research describing the mechanisms of IR injury and the mechanisms of exercise-induced cardioprotection against IR injur y. Indeed, these new data provide a mechanistic link connecting the relationship between IR-induced ROS and Ca2+-overload. This work is also the first to demonstrate calpain-cleavage of several critical Ca2+-handling proteins. Finally, our work

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70 provides additional insight into the mechanisms of exercise-induced cardioprotection as well as the cardioprotective effects of MnSOD. In fact, these experiments may suggest a possible beneficial clinical role for the acute use of calpain inhibitors and/ or mitochondria-targeted superoxide scavengers. Future research is needed to determine if exercise training elevates mitochondrial or cytosolic antioxidants capable of removing hydrogen peroxide. Moreover, more work is required to clarify the oxidation pathway connecting mitoc hondrial superoxide produc tion to the oxidative damage of Ca2+-handling proteins. Finally, the sequen ce of events involving the oxidation and calpain-mediated degradation of Ca2+-handling proteins is not yet completely understood and warrants additional research. Figure 5-1. Proposed mechanisms underlying the IR-induced increase in calpain activity and myocardial dysfunction.

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76 66. Kusuoka H, Porterfield JK, Weisma n HF, Weisfeldt ML, and Marban E. Pathophysiology and pathogenesis of stunned myo cardium. Depressed Ca2+ activation of contraction as a consequence of reperfusioninduced cellular calcium overload in ferret hearts. J Clin Invest 79: 950-961, 1987. 67. Kusuoka H, Weisfeldt ML, Zweier JL, Jacobus WE, and Marban E. Mechanism of early contractile failure duri ng hypoxia in intact ferret hear t: evidence for modulation of maximal Ca2+-activated force by inorganic phosphate. Circ Res 59: 270-282, 1986. 68. Lee HC, Mohabir R, Smith N, Franz MR, and Clusin WT. Effect of ischemia on calcium-dependent fluorescence transients in rabbit hearts containi ng indo 1. Correlation with monophasic action pot entials and contraction. Circulation 78: 1047-1059, 1988. 69. Leeuwenburgh C, Leichtweis S, Hollander J, Fiebig R, Gore M, and Ji LL. Effect of acute exercise on glutathione deficient heart. Mol Cell Biochem 156: 17-24, 1996. 70. Leichtweis SB, Leeuwenburgh C, Chan dwaney R, Parmelee DJ, and Ji LL. Ischaemia-reperfusion induced alterations of mitochondrial function in hypertrophied rat heart. Acta Physiol Scand 156: 51-60, 1996. 71. Lennon SL, Quindry J, Hamilton KL, French J, Staib J, Mehta JL, and Powers SK. Loss of exercise-induced cardioprote ction after cessation of exercise. J Appl Physiol 96: 1299-1305, 2004. 72. Lennon SL, Quindry JC, French JP, Kim S, Mehta JL, and Powers SK. Exercise and myocardial tolerance to ischaemia-reperfusion. Acta Physiol Scand 182: 161-169, 2004. 73. Lennon SL, Quindry JC, Hamilton KL, French JP, Hughes J, Mehta JL, and Powers SK. Elevated MnSOD is not required for exercise-induced cardi oprotection against myocardial stunning. Am J Physiol Heart Circ Physiol 287: H975-980, 2004. 74. Li XY, McCay PB, Zughaib M, Jeroudi MO, Triana JF, and Bolli R. Demonstration of free radical generation in the "stunned" myocardium in the conscious dog and identification of major differences be tween conscious and open-chest dogs. J Clin Invest 92: 1025-1041, 1993. 75. Liu J, Yeo HC, Overvik-Douki E, Hagen T, Doniger SJ, Chyu DW, Brooks GA, and Ames BN. Chronically and acutely exercised ra ts: biomarkers of oxidative stress and endogenous antioxidants. J Appl Physiol 89: 21-28, 2000. 76. Lloyd RV, Hanna PM, and Mason RP. The origin of the hydroxyl radical oxygen in the Fenton reaction. Free Radic Biol Med 22: 885-888, 1997. 77. MacLennan DH, Asahi M, and Tupling AR. The regulation of SERCA-type pumps by phospholamban and sarcolipin. Ann N Y Acad Sci 986: 472-480, 2003.

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79 104. Serbinova E, Khwaja S, Reznick AZ, and Packer L. Thioctic acid protects against ischemia-reperfusion injury in the is olated perfused Langendorff heart. Free Radic Res Commun 17: 49-58, 1992. 105. Seward SW, Seiler KS, and Starnes JW. Intrinsic myocardial function and oxidative stress after exhaustive exercise. J Appl Physiol 79: 251-255, 1995. 106. Shea MJ, Murtagh JJ, Jolly SR, Abrams GD, Pitt B, and Lucchesi BR. Beneficial effects of nafazatrom on ischem ic reperfused myocardium. Eur J Pharmacol 102: 63-70, 1984. 107. Singh RB, Chohan PK, Dhalla NS, and Netticadan T. The sarcoplasmic reticulum proteins are targets for calpain actio n in the ischemic-reperfused heart. J Mol Cell Cardiol 37: 101-110, 2004. 108. Squier MK, Sehnert AJ, Sellins KS, Malk inson AM, Takano E, and Cohen JJ. Calpain and calpastatin re gulate neutrophil apoptosis. J Cell Physiol 178: 311-319, 1999. 109. Starnes JW, Taylor RP, and Park Y. Exercise improves postischemic function in aging hearts. Am J Physiol Heart Circ Physiol 285: H347-351, 2003. 110. Steenbergen C, Murphy E, Levy L, and London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 60: 700-707, 1987. 111. Suzuki YJ and Ford GD. Superoxide stimulates IP3-indu ced Ca2+ release from vascular smooth muscle sarcoplasmic reticulum. Am J Physiol 262: H114-116, 1992. 112. Tao L, Gao E, Hu A, Coletti C, Wang Y, Christopher TA, Lopez BL, Koch W, and Ma XL. Thioredoxin reduces post-ischemic myocardial apoptosis by reducing oxidative/nitrative stress. Br J Pharmacol 2006. 113. Taylor RP, Ciccolo JT, and Starnes JW. Effect of exercise training on the ability of the rat heart to tolerate hydrogen peroxide. Cardiovasc Res 58: 575-581, 2003. 114. Taylor RP, Harris MB, and Starnes JW. Acute exercise can improve cardioprotection without increasing heat shock protein content. Am J Physiol 276: H1098-1102, 1999. 115. Temsah RM, Netticadan T, Chapman D, Takeda S, Mochizuki S, and Dhalla NS. Alterations in sarcoplasmic re ticulum function and gene expr ession in ischemic-reperfused rat heart. Am J Physiol 277: H584-594, 1999. 116. Thibodeau PA, Kachadourian R, Lemay R, Bisson M, Day BJ, and Paquette B. In vitro proand antioxidant properties of estrogens. J Steroid Biochem Mol Biol 81: 227-236, 2002. 117. Tiidus PM, Pushkarenko J, and Houston ME. Lack of antioxidant adaptation to shortterm aerobic training in human muscle. Am J Physiol 271: R832-836, 1996.

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80 118. Tokube K, Kiyosue T, and Arita M. Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes. Pflugers Arch 437: 155-157, 1998. 119. Triana JF, Li XY, Jamaluddin U, Thornby JI, and Bolli R. Postischemic myocardial "stunning". Identification of major differences between the open-chest and the conscious dog and evaluation of the oxygen radical hypothesis in the conscious dog. Circ Res 69: 731-747, 1991. 120. Trumbeckaite S, Neuhof C, Zierz S, and Gellerich FN. Calpain inhibitor (BSF 409425) diminishes ischemia/reperfusion-induced damage of rabbit heart mitochondria. Biochem Pharmacol 65: 911-916, 2003. 121. Turoczi T, Chang VW, Engelman RM, Maulik N, Ho YS, and Das DK. Thioredoxin redox signaling in the ischemic heart: an insight with tran sgenic mice overexpressing Trx1. J Mol Cell Cardiol 35: 695-704, 2003. 122. Urthaler F, Wolkowicz PE, Digerness SB, Harris KD, and Walker AA. MDL-28170, a membrane-permeant calpain inhibitor, attenua tes stunning and PKC epsilon proteolysis in reperfused ferret hearts. Cardiovasc Res 35: 60-67, 1997. 123. Wang CH, Chen YJ, Lee TH, Chen YS, Ja wan B, Hung KS, Lu CN, and Liu JK. Protective effect of MDL28170 ag ainst thioacetamide-induced acu te liver failure in mice. J Biomed Sci 11: 571-578, 2004. 124. Wang KK, Roufogalis BD, and Villalobo A. Further characterization of calpain-mediated proteolysis of the human erythrocyt e plasma membrane Ca2+-ATPase. Arch Biochem Biophys 267: 317-327, 1988. 125. Wang KK and Yuen PW. Development and therapeutic po tential of calpain inhibitors. Adv Pharmacol 37: 117-152, 1997. 126. Wu Y and Hamilton SL. Functional interactions of cyt oplasmic domains of the skeletal muscle Ca2+ release channel. Trends Cardiovasc Med 8: 312-319, 1998. 127. Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, and Hori M. Exercise provides direct biphasic cardioprotection via manga nese superoxide dismutase activation. J Exp Med 189: 1699-1706, 1999. 128. Yoshikawa Y, Hagihara H, Ohga Y, Naka jima-Takenaka C, Murata KY, Taniguchi S, and Takaki M. Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics. Am J Physiol Heart Circ Physiol 288: H1690-1698, 2005.

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81 129. Zima AV and Blatter LA. Redox regulation of cardiac calciu m channels and transporters. Cardiovasc Res 71: 310-321, 2006. 130. Zolotarjova N, Ho C, Mellgren RL, Askari A, and Huang WH. Different sensitivities of native and oxidized forms of Na+/K(+ )-ATPase to intracellular proteinases. Biochim Biophys Acta 1192: 125-131, 1994.

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82 BIOGRAPHICAL SKETCH Joel French was born in Sioux Fa lls, South Dakota. He attained two bachelor degrees from the University of Sioux Falls (Exercise Scie nce and Psychology) along with two minors in Computer Science and English Literature. Duri ng this time, Joel also taught Tae-kwon-do, started a personal training business and worked as a youth director at the Sioux Falls YMCA. Following graduation, he then worked for two y ears as a rehabilitation specialist at Central Plains Clinic and McKennan hospital in Sioux Falls. Joel graduated with his masters in Exercise Physiology from St. Cloud State University (St. Cloud, MN) in 1998. He then worked for a year at the US Olympic Training Center in Lake Placid, NY, returning to Minneapolis, MN for another two years working in cardiac and orthop edic rehabilitation. Finally, deciding to focus his career in basic scienc e, Joel began his doctoral work at the University of Florida in 2001, studying the mechanisms of heart disease and pr otection against myocardial infarction.


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Table of Contents
    Title Page
        Page 1
        Page 2
    Dedication
        Page 3
    Acknowledgement
        Page 4
    Table of Contents
        Page 5
        Page 6
        Page 7
    List of Tables
        Page 8
    List of Figures
        Page 9
    Abstract
        Page 10
        Page 11
    Introduction
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Review of related literature
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
    Methods
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
    Results
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
    Discussion
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
    References
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
    Biographical sketch
        Page 82
Full Text





MECHANISMS OF PROTECTION AGAINST MYOCARDIAL ISCHEMIA-REPERFUSION
INJURY




















By

JOEL P. FRENCH


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

2006



































Copyright 2006

By

Joel P. French
































To my family: Asimina, Janet, John and Jena-their unwavering love and support have enabled
every major accomplishment in my life.










ACKNOWLEDGMENTS

This work would not have been possible without the guidance and support of several

important people. First, I would like to express my appreciation to my mentor and committee

chairp, Dr. Scott Powers. You have taught me everything I know about research, grant and

manuscript writing, teaching and presenting. Through example, you have also instilled in me a

drive to succeed. I believe that you have given me all of the skills I need to be successful in this

field and I hope that my future accomplishments will reflect the extremely high quality of your

mentoring. I would also like to thank my doctoral committee, Dr. Stephen Dodd, Dr. David

Criswell and Dr. Nihal Tumer for their patience and expertise throughout this project.

Importantly, thank you to Dr. John Quindry for his guidance and friendship. I appreciate

everything that you taught me and more importantly, your ability to make the lab such an

enjoyable environment to work in.

Additionally, thanks to everyone in the lab who contributed to this project: Dr. Karyn

Hamilton, Patrick Upchurch, Dr. Jessica Staib and Darin Falk. Thanks also to the rest of the

team in the lab: Youngil Lee, Joe McClung, Zsolt Murlasits, Melissa Deering, Keith Deruisseau,

and Darin Van Gammeren.

Finally, and most importantly I would like to thank my family; Mina, Janet, John and Jena.

Without your love and support I never could have come this far. I love you all.











TABLE OF CONTENTS




A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF TABLES ................. ........................ .. ........... ...................................... .. 8

L IST O F F IG U R E S ......................................................................... ................................... . 9

A B S T R A C T ............... ................................................................ .......................................... 10

CHAPTER

1 INTRODUCTION ............................................... ..................................... 12

R rationale ............................................................................................. ......... 12
Specific Aims .................................................... .................. 15

2 REVIEW OF RELATED LITERA TURE ............................................................ ............... 17

In tro d u ctio n 1.................. .. ..... .... ... ....................................................... 1 7
Myocardial Ischemia-Reperfusion Injury: Characteristics and Mechanisms...................... 18
The O xyradical H ypothesis .................................................................... ............... 19
R O S related dam age ..... .................................................................. ............... 20
Sources of m yocardial R O S ................................................................ ................ 2 1
Antioxidant defenses against IR-induced ROS production.................................22
The Calcium-Overload Hypothesis ........................................................ 25
C alcium induced R O S production....................................................... ................ 26
E-C uncoupling and decreased calcium sensitivity............................. ................ 26
C alcium -activated proteases....................................... ...................... ................ 27
Calpain and IR Injury ............... .. .............. .............. ...... ................. 29
Calpain: Linking the Oxyradical and Calcium-Overload Theories..................................30
Calpain and Calcium -H handling Proteins .................................... .................... ................ 30
R regulation of Free Cytosolic Calcium ....................................................................... 30
Oxidative Modification and Degradation of Calcium-Handling Proteins....................31
SERCA and PLB ...................................... .............................32
N C X .............. .. .. .............................. .. ..................... 3 2
L-type calcium channels and the DHPR ............................................. ................ 32
Summary: Calpain and Calcium -Handling Proteins.......................................... ................ 33
Antioxidants and SR Dysfunction ................................................... ........................... 33
Exercise-Induced Cardio-Protection Against IR Injury .................................... ................ 34
Increased M yocardial H eat Shock Proteins................................................ ................ 35
Increased M yocardial A ntioxidant Capacity .............................................. ................ 35
Exercise-Induced Regulation of Calpain Activation......................................... ................ 36









S u m m a ry ................................................................................................................................. 3 7

3 M E T H O D S .............................................................................................................................3 8

Experim mental A nim als .............. ................................................ ............... .. .... .. .... 38
A nim al M odel Justification .................................................................... ................ 38
A nim al H housing and D iet. ....................................................................... ................ 38
E xperim mental D design ............... .. .................... ................ .................. ......... ....... . ........... 38
Experim ental D esign: H ypothesis O ne ...................................................... ................ 38
Experim ental D esign: H ypothesis Tw o...................................................... ................ 39
General M methods ................... ... ............ ...................... ... ................... 40
The Isolated Working Heart Preparation/IR Protocol (Hypothesis One).....................40
In Vitro Ischemia-Reperfusion Protocol (Hypothesis One) ......................................41
Cardiac Performance Measurements (Hypothesis One).............................................41
C alpain Inhibition (H ypothesis O ne).......................................................... ................ 42
Exercise Training Protocol (Both Hypotheses) ................ ....................................42
In Vivo Ischemia-Reperfusion Protocol (Hypothesis Two).......................................42
Inhibition of MnSOD Protein Translation (Hypothesis Two)....................................43
D ependant M measures (B oth H ypotheses)........................................................... ................ 44
M easurem ent of C alpain A ctivation........................................................... ................ 44
Western Blots for Calcium-Handling Proteins...........................................................44
Immunoprecipitation of Calcium-Handling Proteins .................................................45
Measurement of Protein Carbonyl Formation on Calcium-Handling Proteins ............45
Measurement of HNE Formation on Calcium-Handling Proteins ................................46
D ata A n aly sis ......................................................................................................... ........ .. 4 6

4 R E SU L T S ..................................................................................................... ............ 4 7

H hypothesis O ne.................................................................................................... ....... .. 47
Animal Characteristics .......................................... ......... ...... ...............47
C ardiac Perform ance M easures.................................................................. ................ 48
Percent recovery of left ventricular developed pressure (LVDP) ............................48
Percent of pre-ischem ic +dp/dt and -dp/dt ................................... ..................... 48
Oxidative Modification of Critical Calcium-Handling Proteins ...............................49
Carbonyl formation on calcium-handling proteins .............................................51
HNE formation on calcium-handling proteins....................................................51
Calpain-Mediated Degradation of Calcium-Handling Proteins ................................ 52
Hypothesis Two .................... .. ........ .......... ..................... 53
Calpain Activation (Calpain-Cleaved all-Spectrin)...................................................53
Oxidative Modification of Critical Calcium-Handling Proteins ............................... 55
Carbonyl formation on calcium-handling proteins .............................................56
HNE formation on calcium-handling proteins....................................................57
Calpain-Mediated Degradation of Calcium-Handling Proteins .................................58

5 D ISC U S SIO N ....................................................................................................... ....... .. 60

O verview of Principal Findings ...................................................................... ................ 60









Hypothesis One: The Effects of IR and Calpain Inhibition on Myocardial Calcium-
H an d lin g P protein s ..................... .... ... ..................................................................... 6 1
Calpain Inhibition Protects Against IR-induced Damage and Removal of Calcium-
Handling Proteins.................. ... .. ................ ............................. 62
Calpain Degrades Critical Calcium-Handling Proteins.............................. ................ 62
Hypothesis Two: IR, Exercise, MnSOD and Calcium-Handling Proteins..........................65
Exercise Training Provides Cardio-Protection.................................... ....................... 65
Exercise-Induced Over-Expression of MnSOD Prevents the Oxidation of Calcium-
Handling Proteins................. ...... .... ........ ..... .. ........ ................... 66
Exercise-Induced Over-Expression of MnSOD Attenuates Calpain Activation ..........68
Exercise-Induced Over-Expression of MnSOD Prevents the Degradation of
C alcium -H handling P roteins..................................................................... ................ 68
Degradation of Calcium-Handling Proteins is Associated with Oxidation.................. 68
Sum m ary and Future D directions ..................................................................... ................ 69

L IST O F R EFE R E N C E S ............................................................................................. 71

B IO G R A PH IC A L SK E T C H .............. ....................................................................... 82










LIST OF TABLES


Table page
4-1 A nim al body and heart w eights ......................................... ........................ ................ 47

4-2 Correlations Between the Oxidative Modification of Ca2+-handling Proteins and
T heir D egradation F ollow ing IR ...................................................................... ................ 53

4-3 Correlations between the oxidative modification of calcium-handling proteins and
their degradation follow ing IR ......................................................................... ................ 58










LIST OF FIGURES


Figure page

1-1 Proposed mechanisms underlying IR-induced calpain activation and myocardial
dy sfu n action ..................................................................................................... ......... .. 16

3-1 Experim ental design for H ypothesis One ..................................................... ................ 39

3-2 Experim ental design for H ypothesis Tw o ........................ ..................... ......................40

4-1 % Recovery of left ventricular developed pressure (LVDP)........................................47

4-2 R ate of systolic pressure increase (+dp/dt) ........................................................................48

4-3 R ate of systolic pressure decline (-dp/dt)...................................................... ................ 49

4-4 Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel
(LTCC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a),
Phospholamban (PLB) and Sodium/calcium exchanger (NCX) .. .................................. 50

4-5 HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium
ex ch an g er (N C X ) .............................................................................................................. 5 1

4-6 Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC),
Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Phospholamban
(PLB) and Sodium/calcium exchanger (N CX) ..................................... ...................... 52

4-7 W western blotting for calpain-cleaved all-Spectrin........................................ ................ 54

4-8 Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel
(LTCC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a),
Phospholamban (PLB) and Sodium/calcium exchanger (NCX). .................................... 55

4-9 HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium
exchanger (NCX)............................... ............ ............................... 56

4-10 Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC),
Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Phospholamban
(PLB) and Sodium/calcium exchanger (NCX) ..................................................... 57

5-1 Proposed mechanisms underlying the IR-induced increase in calpain activity and
myocardial dysfunction .................. ............ ............................... 70










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

MECHANISMS OF PROTECTION AGAINST MYOCARDIAL ISCHEMIA-REPERFUSION
INJURY

By

Joel P. French

December 2006

Chair: Scott K. Powers
Major: Health and Human Performance

Myocardial ischemia-reperfusion (IR) is characterized by an increase in reactive oxygen

species (ROS) generation as well as increased free cytosolic Ca2 (Ca2+-overload) resulting in

myocardial contractile dysfunction and injury.

We have previously demonstrated that both exercise training and inhibition of the Ca2+-

activated protease calpain, protect the heart against IR injury. Additionally, we have shown that

exercise regulates IR-induced calpain activation. However, the mechanisms involved in

exercise-induced calpain regulation and calpain-mediated injury are not completely understood.

We hypothesized that the oxidation and calpain-mediated degradation of critical Ca2+-handling

proteins was an important mechanism of IR injury and exercise-induced cardio-protection.

Therefore, we conducted two separate experiments to examine the relationships between IR,

calpain activation, exercise training and the oxidation and degradation of myocardial Ca2+-

handling proteins.

Our first set of experiments looked at the effects of calpain inhibition on myocardial

function, Ca2+-handling protein oxidation and degradation following IR. We found that IR

resulted in impaired LVDP, +dp/dt and -dp/dt and increased oxidative modification and









degradation of several Ca2+-handling proteins (LTCC, SERCA2a, PLB, NCX). In addition, we

found that pharmacological inhibition of calpain prevented contractile dysfunction as well as the

degradation of these Ca2+-handling proteins.

Our second set of experiments looked at the mechanisms of exercise-induced cardio-

protection against IR injury. We found that short-term exercise training attenuated calpain

activation as well as the oxidative modification and degradation of Ca2+-handling proteins. In

addition, when the exercise-induced over-expression of the endogenous antioxidant enzyme

MnSOD was prevented, using an antisense oligonucleotide, the protective effects of exercise

training were lost.

Therefore, we propose a series of events during IR, which are initiated by ROS-mediated

oxidative modification of critical Ca2+-handling proteins, resulting in increased free cytosolic

Ca2+ and calpain activation. Once active, calpain can cleave Ca2+-handling proteins, facilitating

their degradation, exacerbating Ca2+-overload, ROS generation and IR injury.

Exercise appears to provide protection against these events by over-expressing MnSOD,

which attenuates IR-induced Ca2+-handling protein oxidation, calpain activation and Ca2+-

handling protein degradation.









CHAPTER 1
INTRODUCTION

Rationale

Myocardial ischemia-reperfusion (IR) injury is a prevalent consequence of cardiovascular

disease. Although there are many factors leading to myocardial dysfunction following IR,

growing evidence suggests that increased reactive oxygen species (ROS) production and

cytosolic free Ca2+-overload, either independently or cooperatively, are major contributors to IR-

induced injury (10, 80, 89).

ROS generated during IR can have several targets within the myocardium (including

proteins, lipids, and DNA), impairing their function and/or promoting their degradation, leading

to contractile dysfunction, cellular damage and cell death. Additionally, ROS can interfere with

intracellular Ca2+ homeostasis, further exacerbating the deleterious effects of IR (9, 64).

Increases in myocardial cytosolic Ca2+ levels have been observed during both ischemia and

reperfusion. In this regard, it has been hypothesized that one role of cytosolic Ca2+ in the

pathogenesis of IR-induced myocardial injury is through the activation of the Ca2+-dependent

protease, calpain. Calpain exists in myocytes in two primary isoforms, micro (calpain I) and

milli (calpain II), named for the respective amounts of Ca2+ required for their activation in vitro.

Both calpain isoforms are activated by prolonged exposure to elevated cytosolic Ca2+ and it is

well documented that calpain activation occurs in the heart during IR (55, 120, 122, 128). This

is significant because calpain can injure cardiac myocytes via several different pathways. For

example, calpains cleave several structural proteins leading to the release of myofilaments,

facilitating their degradation by the proteosome. In addition, calpains may contribute to

apoptosis, through cleavage of Bid, mediating cytochrome c release from the mitochondria.

Also, calpains increase the expression of cell adhesion molecules, leading to an increase in









neutrophil-mediated oxidative damage. Each of these pathways has been shown to significantly

contribute to IR-associated injury. Moreover, calpains' deleterious role in IR injury is supported

by strong evidence indicating that calpain inhibition significantly attenuates myocardial

contractile dysfunction, cellular injury, and cell death (55, 120, 122, 128).

Recent evidence suggests an additional role for calpain as a link between the oxyradical

and Ca2+-overload theories of IR-induced myocardial injury. This research suggests that ROS

may oxidatively modify Ca2+-handling proteins, impairing their function and possibly leading to

their cleavage by calpain (64, 126, 129, 130). Degradation and/or functional impairment of

these proteins would lead to an increase in free cytosolic Ca2+, further exacerbating IR-induced

calpain activation, ROS production and myocardial dysfunction (Figure 1-1). In support of this

postulate, work from our laboratory has described an IR-induced increase in oxidative stress

(protein carbonyls) as well as an increase in calpain-mediated cleavage of the Ca2+-handling

protein, SERCA2a. Indeed, this suggests that some Ca2+-handling proteins are cleaved by

calpain, and degraded during IR. Therefore, the first goal of these experiments was to determine

the effects of IR on the oxidative modification and degradation of several key Ca2+-handling

proteins. The proteins studied were: the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

(SERCA2a), phospholamban (PLB), the Na /Ca2+ exchanger (NCX) and L-type calcium

channels (LCC). We also determined if those proteins/protein complexes, which were

oxidatively modified, had a higher incidence of degradation. Additionally, we determined the

effects of in-vitro calpain inhibition on the degradation of Ca2+-handling proteins and myocardial

function following IR.

Endurance exercise training is an established means of inducing cardioprotection against

IR-induced injury (71, 72, 93, 96, 113, 114). Although the mechanisms of exercise-induced









cardioprotection are unknown, work from our laboratory indicates that exercise may provide

cardioprotection against IR injury, at least in part, through the regulation of calpain (30). This

work reveals that exercise training completely attenuated the IR-induced increase in calpain

activation and was associated with improved myocardial contractile function following IR.

Additionally, exercise trained hearts showed an increase in the endogenous antioxidant MnSOD

and a decrease in oxidative stress (protein carbonyl formation). We hypothesize that endurance

exercise may regulate calpain activation through an up-regulation of endogenous antioxidants,

such as Mn-SOD.

Superoxide production is believed to be a major source of oxidative stress during IR.

Mitochondrial produced superoxide is thought to play an important role in myocardial IR injury

due to the aerobic nature of the heart. Superoxide production has been shown to dramatically

increase following ischemia and the use of superoxide scavengers has been shown to improve

contractile function following IR. Because of the detrimental impact of superoxide generation

during IR, we hypothesize that an exercise-induced increase in MnSOD may provide protection

against oxidative modification of key cellular proteins, including critical Ca2+-handling proteins

associated with both the SR and plasma membrane. Reducing oxidative damage to calcium

handling proteins may result in improved Ca2+-handling and reduced calpain activation during IR

and, therefore reduce myocardial dysfunction and injury. Therefore, the second goal of these

experiments was to investigate the effects of exercise training on oxidative modification and

degradation of Ca2+-handling proteins following IR. Additionally, we determined if the exercise-

induced reduction in calpain activity and preservation of Ca2+-handling proteins during IR was

dependant on the exercise-induced over-expression of MnSOD in the heart.









Specific Aims


The goal of these experiments was to address two separate but related hypotheses

involving myocardial IR injury, oxidative stress, and calpain activation.


Hypothesis one:





Aim (A):


Aim (B):


Aim (C):



Hypothesis two:


Aim (A):



Aim (B):


IR will result in increased oxidative modification carbonyll and HNE
formation) and/or calpain-mediated degradation of myocardial calcium
handling proteins. Further, calpain inhibition will attenuate the IR-
induced degradation of Ca2+-handling proteins. This hypothesis will be
tested by achieving the following specific aims.

To determine if IR results in increased carbonyl and/or HNE formation to
Ca2+-handling proteins within the heart.

To ascertain if oxidative modification to calcium handling proteins is
associated with an increase in the degradation of these proteins.

To discern if calpain inhibition attenuates the degradation of key Ca2+
handling proteins in the heart following IR.


Exercise training will provide protection against IR-induced oxidative
modification carbonyll and HNE formation) and degradation of
myocardial Ca2+-handling proteins via an increase in the endogenous
antioxidant, MnSOD. This hypothesis will be rigorously tested by
achieving the following specific aims.

To determine if exercise training attenuates IR-induced carbonyl and/or
HNE formation on key Ca2+-handling proteins and/or degradation of these
proteins within the heart.

To ascertain if exercise-induced protection against IR-induced oxidative
modification and degradation of Ca2+-handling proteins is dependent on an
increase in myocardial MnSOD.












T ROS J
Production
Hypothesis Two
MnSOD
1 Calcium I
Damage

Hypothesis One
Calpain Inhibition I


Figure 1-1. Proposed mechanisms underlying IR-induced calpain activation and myocardial
dysfunction.









CHAPTER 2
REVIEW OF RELATED LITERATURE

Introduction

Coronary heart disease (CHD) is the number one cause of death for both men and women

in the United States, as well as most other industrialized nations. In 2002, CHD was responsible

for 927,448 deaths in the U.S., roughly one in every three deaths (34.2%). Additionally, health

care costs related to CHD in 2005 were estimated at $393.5 billion within the U.S. alone (6).

Because CHD typically results in periods of myocardial ischemia, often leading to terminal

infarction, understanding the mechanisms of myocardial IR injury as well as possible

mechanisms of protection against IR injury is important in the treatment and management of

patients with CHD.

Many factors can contribute to IR-induced myocardial IR injury. The first goal of this

review will be to provide an overview of IR-induced cellular injury and to discuss the

mechanisms responsible for IR-induced cellular injury. Although several factors contribute to IR

injury, this review will focus primarily on the two dominant theories of myocardial IR injury, the

oxyradical theory and the Ca2+ overload theory, as well as possible interaction between the two.

The second goal of this review will be to discuss calpain and its role in IR injury.

Increases in both free Ca2+and ROS have been shown to increase the cellular activity of the Ca2+-

activated protease calpain (8, 12, 19). Further, calpain activation has been shown to play a

deleterious role in the heart. Calpain may play a critical role in IR injury by linking the theories

of Ca2+ overload and ROS production. Calpain may cleave Ca2+-handling proteins, which have

been oxidatively modified by ROS, leading to a further increase in the levels of free Ca2+ and

active calpain, exacerbating myocardial damage and dysfunction.









The third goal of this review will be to describe exercise-induced cardioprotection and the

role of the antioxidant, manganese superoxide dismutase (MnSOD), as a potential mediator of

exercise-induced cardioprotection. Endurance exercise training has been shown to provide

protection against IR-induced elevations in calpain activity, myocardial injury and contractile

dysfunction, however, the precise mechanisms of this cardio-protection have not been elucidated

(95, 96). One possible explanation is that exercise provides cardioprotection through an increase

in the endogenous antioxidant MnSOD. Increasing MnSOD protein and/or activity would result

in a reduction in IR-induced ROS damage. This could lead to a reduction in the oxidative

modification of Ca2+-handling proteins, as well as their degradation by calpain, reducing not only

ROS-related injury but disturbances in Ca2+ homeostasis and calpain-related pathology as well.

Myocardial Ischemia-Reperfusion Injury:
Characteristics and Mechanisms

Myocardial ischemia is defined as the reduction or cessation of blood flow to myocardial

tissue, below the metabolic requirements of that tissue. In addition to being a clinical

manifestation of coronary artery disease, the latest clinical treatments for this disease (i.e.

coronary bypass surgery, balloon angioplasty) subject the heart to episodes of ischemia and

subsequent restoration of blood flow (reperfusion). Following a period of ischemia and

reperfusion, the heart can undergo temporary or permanent injury, depending on the duration of

the ischemia (10). Brief periods of ischemia, less than 5 minutes, will generally result in

arrhythmias. However this length of ischemia is not associated with any long-term loss in heart

function or cell death. A period of ischemia lasting approximately 5-20 minutes results in

ventricular contractile dysfunction and is referred to as "myocardial stunning." This ischemic

duration will typically result in temporary contractile function without causing any permanent

damage (necrosis or apoptosis) to the heart (10). The most severe form of IR injury, "myocardial









infarction", occurs when the duration of ischemia exceeds 20 minutes. Infarction results in loss

of contractile function as well as cellular death, through pathways of both necrosis and apoptosis

(7).

There are many mechanistic factors that may contribute to myocardial dysfunction and

cellular death (7). These factors include, but are not limited to: ROS production, Ca2+-overload,

increased proteolytic activity, and platelet / inflammatory cell infiltration. Although there are

many factors leading to myocardial dysfunction and cell death following IR, growing evidence

suggests that increased free cytosolic Ca2+ and/or ROS production, either independently or

cooperatively, are two of the major contributors to IR-induced injury (9). A brief discussion of

the roles of both ROS and free cytosolic Ca2+ in IR-induced injury follows.

The Oxyradical Hypothesis

The oxyradical hypothesis of IR injury was first proposed in 1985 when it was postulated

that the generation of reactive oxygen and free radical species such as the superoxide anion (02*

), hydrogen peroxide (H202), and the hydroxyl radical (OH*) during reperfusion contributed to

myocardial injury (86). Reactive oxygen species (ROS) are derived from the reduction of

molecular oxygen (78). Some ROS (such as 02' and OH*) are known as free radicals because

they contain one or more unpaired electrons in their outer most orbital, making them highly

unstable and reactive (42). For example, when an 02 molecule accepts a single free electron, the

product is superoxide (02*'). When a second free electron is accepted, hydrogen peroxide (H202)

is formed. In the presence of an iron (ferrous) salt, the 0-0 bond can be broken resulting in the

formation of two hydroxyl radicals (OH*). Importantly, the OH* produced is one of the most

highly reactive free radicals, capable of reacting with almost every component of the cell (76).

Free radicals produced via these pathways have been implicated in damage to several structures









within the cell, including cellular proteins, lipids, and DNA (104). Supporting the deleterious

role of ROS, ischemia-reperfusion experiments using iron chelators and/or various antioxidants

(free radical scavengers) have demonstrated a reduction in free radical generation and protection

against IR-induced injury (4, 9, 28, 86, 106).

ROS related damage

Damage caused by free radicals has been identified during both periods of ischemia and

reperfusion in the heart, although it is currently believed that the majority of the radicals are

produced during the first few minutes of reperfusion (4, 11, 13, 28, 57, 74, 119). Once

generated, free radicals have several targets within the cell, which will be discussed in the

following sections.

Myocardial protein oxidation. Many cardiac proteins can be oxidatively modified by

free radicals in the heart during IR. Included in the list of "target proteins" are: enzymes,

structural proteins, contractile proteins, and membrane-bound proteins (41, 43, 49, 100, 101).

Often, the damage caused by free radical interaction is irreparable. In addition, many proteins,

which are oxidatively modified, become more susceptible to proteolytic cleavage by calpain and

degradation by the proteosome. One example of oxidative modification to cellular proteins is the

formation of carbonyl groups. This carbonyl formation can then be measured, providing an

indirect indication of oxidative stress within the cell (15).

Myocardial lipid peroxidation. Polyunsaturated fatty acids are highly susceptible to

free radical modification at their unsaturated sites (3). Once ROS extract electrons, or hydrogen

atoms, from the methylene groups of fatty acids, a chain reaction is initiated where one

"modified" fatty acid chain reacts with a neighboring chain, and so on. This eventually leads to

the damage of the lipid membrane, resulting in altered cell membrane permeability and fluidity.

In many cases, this damage to the cell membrane leads to cellular death through necrosis and / or









apoptosis. There are examples of lipid peroxidation by-products which can be measured in order

to indirectly quantify oxidative stress within the cell (43). One such by-product is the formation

of the reactive aldehyde, HNE (4-hydroxy-2-nonenal). The w-6-family (linoleic and arachidonic

acids) of polyunsaturated fatty acids produce HNE as a result of free radical attack. HNE is a

highly reactive compound and it can react with several functional groups on biological material,

particularly sulfydryl groups, to form thioester adduct and then hemiacetals. HNE may also react

with histidine and lysine residues of proteins to form stable Michael addition-type of adducts. In

addition, HNE-modification of proteins may impair biological functions.

DNA damage. ROS have also been reported to damage DNA, preventing the translation

and transcription of new cellular proteins by stimulating the degradation of DNA and

oligonucleosomal fragments (50). More specifically, ROS can cause permanent or transient

damage to nucleic acids within the cells, leading to such events as DNA strand breakage and

disruption of Ca2+ metabolism. Additionally, a high rate of oxidative damage to mammalian

DNA has been demonstrated by measuring oxidized DNA bases excreted in urine following

DNA repair. Further, the rate of oxidative DNA damage has been found to be directly related to

metabolic rate and inversely related to life span.

Sources of myocardial ROS

There are several potential sources of free radical production in the heart during ischemia-

reperfusion. Major sources include: electron leak from the mitochondrial respiratory complexes,

xanthine oxidase, enzymatic arachadonic acid oxygenation, the synthesis of nitric oxide,

catecholamine oxidation and oxidative burst from neutrophils (9, 17, 19, 27). Nonetheless, a

large volume of evidence implicates mitochondrial production of radicals as the primary source

of oxidants during both ischemia and reperfusion. Therefore, a detailed discussion of

mitochondrial ROS production follows.









Mitochondrial ROS production during IR. Again, the mitochondrial respiratory chain

has been identified as one of the largest contributors of IR-induced free radical production (9).

Mitochondrial oxidative phosphorylation is the major ATP synthetic pathway in eukaryotes.

During this process, electrons liberated from reducing substrates are delivered to oxygen via a

chain of respiratory H pumps. These pumps (complexes I-IV) establish a H gradient across the

inner mitochondrial membrane. The electrochemical energy of this gradient is then used to drive

ATP synthesis by complex V (ATP synthase). During this process, it has been estimated that

approximately 1-2% of the oxygen present is reduced to form some sort of ROS or free radical

(36). The primary radical made by the mitochondria is superoxide (02*). It is believed that the

majority of the superoxide generated originates from electrons "leaked" from ubisemiquinone,

located at complex III. Once generated, superoxide can be converted to a less reactive oxygen

species, hydrogen peroxide (H202) by the antioxidant enzyme superoxide dismutase (SOD), or

converted to a more reactive hydroxyl (OH') radical, in the presence of iron.

Confirming the importance of mitochondrial free radical production, studies using

antioxidants targeted specifically to the mitochondria have demonstrated a significant reduction

in oxidant-related damage within the myocardium, as well as improved myocardial function (4,

11,28, 86).

Antioxidant defenses against IR-induced ROS production

Fortunately, the cell has several antioxidant defense mechanisms against the increase in

free radicals typically seen during IR. An antioxidant has been defined as "any substance that

significantly delays or prevents the oxidation of that substrate" (42). The cell contains a variety

of enzymatic and non-enzymatic antioxidants, located in various strategic locations and

specifically targeted to different ROS.









Superoxide dismutase (SOD).Superoxide dismutase (SOD) was discovered in 1969 by

McCord and Fridovich (81). SOD is an enzyme that catalyzes the reduction of superoxide to

hydrogen peroxide, thus forming a less reactive, non-radical species.

02*" + 02*" + 2H+ -- H202 + 02

SOD exists in three isoforms: manganese superoxide dismutase (MnSOD), copper-zinc

superoxide dismutase (CuZnSOD) and extracellular superoxide dismutaes (ECSOD). Although

each isoform catalyzes the reaction of superoxide to hydrogen peroxide, they each reside in

different locations within the cell. MnSOD is located exclusively within the mitochondria,

CuZnSOD is found predominantly within the cytosol and, as its name implies, ECSOD is found

in extracellular fluids such as plasma, as well as in the extracellular matrix of tissues. Because

superoxide generation has been identified as a major contributing factor to IR injury, the

regulation of SOD plays a critical role in the heart.

Catalase (CAT). The enzyme catalase (CAT) catalyzes the breakdown of hydrogen

peroxide to water and oxygen.

H202 -> 2H20 + 02

Catalase is found predominantly within the peroxisome along with several other enzymes,

which can generate hydrogen peroxide such as urate oxidase, glycolate oxidase, and flavenoid

dehydrogenases, involved in beta-oxidation of fatty acids (81).

Glutathione peroxidase (GPx). The antioxidant enzyme glutathione peroxidase

(GPx) also catalyzes the breakdown hydrogen peroxide to two molecules of water. However,

this reaction depends on the concomitant oxidation of reduced glutathione (GSH) to oxidized

glutathione (GSSG).









H202 + 2GSH -> GSSG + 2H20

Because of the dependant relationship between the oxidation of glutathione and the

reduction of H202, the measurement of the ratio of GSH to GSSG is often used to assess the

amount of hydrogen peroxide production, as well as GPx activity within the cell. Both GPx and

CAT work in tandem to remove hydrogen peroxide from the cell. CAT has a much higher Km

than GPx, responding very quickly to increases in hydrogen peroxide. GPx has a much lower

Km but is more ubiquitous, breaking down the majority of hydrogen peroxide generated from the

mitochondria and sarcoplasmic reticulum (81).

Glutathione (GSH). Glutathione (GSH) is an intracellular thiol-containing tripeptide,

which is produced inside cells. Importantly, since very little CAT resides within the

mitochondria, GSH, in conjunction with Gpx, is the major means of hydrogen peroxide

breakdown (82). The importance of GSH to the mitochondria is illustrated by the fact that when

cytosolic GSH levels begin to fall, the mitochondria reduce GSH release in order to conserve

their own reserves.

Other Antioxidants. The cell contains several other non-enzymatic antioxidants that

contribute to maintaining redox balance. Many of these non-enzymatic antioxidants are

consumed in the diet, such as vitamin E, vitamin C, lipoic acid, carotenoids, and flavenoids to

name a few. In addition other endogenous antioxidants such as heat shock proteins and

ubiquinones also play a role in maintaining cellular redox balance. Because of the extensive

number of antioxidants, complexity of their function, as well as relevance to these experiments,

these antioxidants will not be discussed in this proposal.









The Calcium-Overload Hypothesis

The Ca2+ overload hypothesis predicts that myocardial IR injury results from a disturbance

in cellular Ca2+ homeostasis. Intracellular cytosolic Ca2+ levels are typically maintained at a low

level (approximately 0.1 [tM) while total cellular Ca2+ is considerably higher. Much of the

cellular Ca2+ is stored within the sarcoplasmic reticulum (SR) and the mitochondria. Numerous

studies have demonstrated a dramatic increase in cytosolic Ca2+ levels during ischemia (60, 68,

78, 110). This elevation in free Ca2+ persists during the early stages of reperfusion, finally

returning to normal levels during late reperfusion (65, 78). The Ca2+-overload hypothesis was

first described in detail by Grinwald (37), who proposed the following mechanisms to attempt to

explain this increase in free Ca2+, or Ca2+-overload. During ischemia, intracellular sodium

accumulates due to energy depletion, and Na+/Ca2+ exchange is inhibited by the concomitant

acidosis. Upon reperfusion, the rapid reversal of acidosis reactivates Na /Ca2+ exchange at a

time when sodium overload has not yet been resolved, driving Ca2+ into the cells. The damaging

effects of the Ca2+-overload were later documented by Kusuoka (66) who discovered that hearts

which were reperfused with a low Ca2+ solution showed a marked decrease in IR-induced injury.

In fact, a transient Ca2+-overload, even in the absence of ischemia, has been shown to cause

myocardial dysfunction and injury (63).

Increased free Ca2+ can contribute to the pathology of the heart cell through several

mechanistic pathways. In fact, since the early work of Grinwald and Kusuoka (38, 67), the

calcium hypothesis has evolved to incorporate several distinct mechanisms, which attempt to

explain the means through which Ca2+ may lead to myocardial dysfunction and/or injury. The

proposed mechanisms include: increased ROS production, excitation-contraction (E-C)

uncoupling due to decreased Ca2+ responsiveness and increased protease activity (12).









Calcium induced ROS production

Several researchers have suggested that IR-induced increases in free Ca2+ may lead to

increased ROS generation. For example, Seno et al. found that Ca2+ stimulated increased radical

production from NADPH oxidase (103). Further, Gottlieb et al. proposed a model through

which Ca2+ could increase mitochondrial ROS production via several pathways (36). First, Ca2+

stimulates KREBS cycle activation which results in increased electron flow into the respiratory

chain and therefore, increased "leaking" of electrons from the respiratory complexes to oxygen,

forming the superoxide radical. In addition, Ca2+ stimulates nitric oxide production, which can

inhibit electron flow into the mitochondria through complex IV as well as complex I, resulting in

increased ROS production.

E-C uncoupling and decreased calcium sensitivity

It is well accepted that IR results in a decrease in myocardial contractile function. One

possible explanation is a decrease in E-C coupling. Early work by Kusuoka et al. described a

decline in maximal Ca2+-activated force production in the heart following IR (65). Because

electrical activation was not impaired in the heart following IR (47), the explanation for the IR-

induced reduction in E-C uncoupling must lie in either of two mechanisms: a reduction in Ca2+

availability within the cell or a decrease in calcium responsiveness of the contractile machinery.

As discussed earlier, several groups have shown an increase in intracellular Ca2+ following IR

(38, 67). Therefore, a reduction in calcium availability is not a likely explanation for the

observed decrease in E-C coupling. This leaves the possibility of a reduced Ca2+ sensitivity of

the contractile machinery within the cell.

The idea that myocardial Ca2+ sensitivity is reduced following IR originated from the

observation that although contractile function was significantly impaired following IR, Ca2+

levels were actually elevated. Since Ca2+ stimulates muscular contraction, it was postulated that









the Ca2+ sensitivity of the contractile proteins must be reduced. Although several studies have

confirmed these findings, the exact mechanisms responsible for the IR-induced reduction in Ca2+

responsiveness are not completely understood. Most of the work completed to date has

implicated the structural modification of one or more of the myofibrillar proteins (65). Studies

using skinned fibers have suggested that IR results in modification of the myofilaments (51).

This may be an additional point of interaction between the Ca2+ overload and oxyradical theories

because ROS have been shown to modify cellular proteins, impairing their function. Among

their many possible targets, radicals may modify myofibrillar proteins, by oxidizing thiol groups,

resulting in impaired Ca2+ responsiveness (150, 92). Using immunohistochemistry, Matsumura

observed degradation of the myofilament-associated scaffolding protein c.-actin following IR

(79). In addition, Gao documented a decrease in the thin-filament regulatory protein troponin-I

following IR (32, 33). Further, Gao prevented the degradation of troponin-I by altering the

reperfusate in such a way to mitigate Ca2+-overload in the heart following ischemia. These

observations are particularly important given the crucial role of troponin-I as an intermediary

between Ca2+ activation and cross-bridge cycling. This degradation of troponin-I may explain

much of the depression in myocardial contractile function following IR. This idea becomes even

more pertinent to the experiments proposed in this manuscript when considering the fact that

troponin-I is also cleaved by the Ca2+-activated protease calpain.

Calcium-activated proteases

Cathepsins. The cathepsins are a group of lysosomal proteases, which are found in

innate immune cells such as neutrophils and macrophages. Therefore these lysosomal enzymes

are frequently found in areas of inflammation and injury. For example, increased levels of

cathepsin B and D are frequently observed in patients with heart disease and other chronic









inflammatory conditions (84). Although cathepsins clearly play a role in chronic inflammatory

conditions, their contribution to acute IR injury is unlikely to be important given the fact that

reperfusion times of almost two hours are required for significant neutrophil infiltration into

myocardial cells.

Calpain. Increases in free Ca2+ during IR can lead to increased activation of Ca2+-

activated cysteine proteases, such as calpain. Calpain exists in myocytes in two primary

isoforms, micro (calpain I) and milli (calpain II), named for the respective amounts of Ca2+

required for their activation in vitro. Both calpain isoforms are heterodimers made up of a large

(80 kDa) and small (28-30 kDa) regulatory subunit (35). Calpain has several Ca2+ binding

domains similar to calmodulin. Ca2+ binding causes a shift in the structure of the protein,

exposing a site for interaction with various substrates (35). Although the two calpain isoforms

are named for Ca2+ concentrations needed for activation in vitro there is evidence that calpain II

can be activated by far less than millimolar Ca2+ concentrations in vivo, increasing its relevance

to myocardial IR injury (35). Regardless, once activated, calpain migrates in the cytosol toward

the SR and/or plasma membrane where the majority of its substrates are located. These

substrates include structural and contractile proteins as well as Ca2+ handling proteins, to name a

few. The exact number of calpain-targeted proteins is currently unknown, however, a recent

review by Goll et al. (35) reported over 100 different proteins that serve as calpain substrates.

Note that this review only discussed cytoskeletal proteins, kinases and phosphateses, just a few

categories of potential calpain substrates (35). Hence, it is likely that calpain cleaves many more

than 100 proteins in cells.

Although both calpain I and II have similar substrates, there is some evidence to suggest

slightly different roles for the two isoforms in vivo. For example, work in skeletal and cardiac









tissues have documented an initial increase in both calpain I and II activities in response to

various modes of tissue injury followed by a later, second increase in calpain I activity. This

evidence has led some to believe that while both isoforms clearly contribute to cellular injury in

response to various stressors (IR, hypoxemia, hydrogen peroxide, calcium overload) calpain I

may also play a more "productive" role in the removal of damaged proteins, etc. following

injury. In addition, long-term inhibition of both isoforms typically results in the death of the

animal suggesting that calpain plays a role in normal homeostatic cellular pathways. However,

in the current review, the primary interest in calpain is directed toward its contributing role in

cellular injury following a stressor, such as IR.

Calpain and IR Injury

The IR-induced increase in calpain activation has been well documented in the heart (9,

19, 35, 55, 115, 122, 128). This increase in calpain activity has long been known to play a

deleterious role in myocardial IR-induced injury. Once activated through binding with calcium,

calpain can injure cardiac myocytes via several different pathways. Calpain cleaves several

structural proteins leading to the release of myofilaments, facilitating their degradation by the

proteosome (35, 83, 99, 124, 125). Moreover, calpains may contribute to apoptosis, through

cleavage of Bid, mediating cytochrome c release from the mitochondria (18, 19, 35). Also,

calpains increase the expression of cell adhesion molecules, leading to an increase in neutrophil-

mediated oxidative damage (91, 108). Each of these pathways has been shown to significantly

contribute to IR-associated injury. In support of this postulate, several studies have

demonstrated cardioprotection through the use of calpain inhibitors prior to IR (55, 120, 122,

128). Recent work from our laboratory supports these findings, demonstrating almost complete

cardioprotection against IR-induced contractile function and injury using the calpain inhibitor

MDL-28170 (30). These results provide physiological support to the notion that calpain









activation plays a significant role in IR-induced myocardial injury. In fact, calpain may play a

unique role in linking the two predominant mechanistic theories of myocardial IR injury, the

Ca2+-overload hypothesis and the oxyradical hypothesis.

Calpain: Linking the Oxyradical and Calcium-Overload Theories

Although several independent theories have been proposed in order to explain myocardial

injury and dysfunction following IR, the pathogenesis of IR most likely involves a complex

interaction between the oxyradical and Ca2+-overload hypotheses. Several mechanisms have

been proposed linking the two theories. One possibility is that free radicals generated during IR

could interact with various SR proteins, causing SR dysfunction and damage (58, 61, 64, 102).

The Impairment in SR function would likely result in increased free cytosolic Ca2+, and calpain

activation leading to further cellular damage. Supporting this postulate, cell culture studies

administering antioxidants prior to reoxygenation have observed a significant attenuation in

Ca2+-overload (85). In addition studies using both in vitro working heart and langendorf IR

models have described an attenuation in IR-induced calpain activation through the use of various

antioxidants (107). Because calpain is activated by Ca2+ this indirectly indicates a reduction in

IR-induced Ca2+-overload (35). When also taking into consideration the possibility that

oxidative modification of SR proteins by free radicals may increase the likelihood of their

cleavage by calpain, it appears that calpain may play a critical role in IR-induced injury, linking

the oxyradical and Ca2+-overload theories.

Calpain and Calcium-Handling Proteins.

Regulation of Free Cytosolic Calcium

The bulk of the Ca2+ released within the cell comes from calsequestrin-bound Ca2+ stores

within the sarcoplasmic reticulum (SR). Ca2 is normally released by the process of Ca2+-

induced Ca2+ release in which the entry of a small amount of Ca2+ across the plasma membrane









sarcolemmaa) triggers the release of much more from the SR. This mechanism depends on the

fact that Ca2+ entry from the extracellular fluid, via the dihydropyridine receptor (DHPR) and L-

type Ca2+ channels, and to a lesser extent the sodium-calcium exchanger (NCX), increases the

probability that the SR Ca2+ release channel (ryanodine receptor, RyR) is open. The greater the

probability that the RyR is open the greater the release of Ca2+ from the SR. Once cytosolic

calcium levels are elevated, the calcium must either be moved back into the SR, or removed from

the cell completely via the plasma membrane.

There are two primary means of calcium removal from the cytosol: the

sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and the sodium-calcium

transporter (NCX). The SERCA is an ATP-dependant pump located in the SR, which removes

calcium from the cytosol, returning to the SR where it is stored bound to the protein

calsequestrin for later release. SERCA activity is regulated by the protein phospholamban

(PLB), as well as ATP levels (77). Calcium can also be removed via the plasma membrane by

the NCX. The NCX couples the transport of three Na+ molecules to one Ca2+ molecule in the

opposite direction in two consecutive steps. Together, these proteins/protein complexes play a

critical role in the regulation of free cytosolic Ca2+ levels and therefore, calpain regulation,

during IR. Importantly, all of these proteins/protein complexes are targets for oxidative

modification by ROS during IR as well (64).

Oxidative Modification and Degradation of Calcium-Handling Proteins

There is a significant body of work detailing the oxidative modification of Ca2+-handling

proteins within the cell (64). Following is a very brief review of pertinent studies, which deal

with oxidative stress and the Ca2+-handling proteins mentioned earlier.









SERCA and PLB

SERCA, and its regulatory protein PLB, play a critical role in the removal of Ca2+ from the

cytosol, returning it to the SR. Any impairment in function could lead to elevations in free

cytosolic Ca2+. Importantly, Favero et al. (29) found that hydrogen peroxide inhibited SERCA

activity within the myocardium. In addition, Grover et al. (39) studied the interaction of ROS

with smooth muscle SERCA and found that hydrogen peroxide-induced damage to SERCA

diminished the SR Ca2+ pool as well as the smooth muscle response to Angiotensin II. Grover

also reported similar results using superoxide (39). In addition, Suzuki and Ford (111) reported

that ROS induced concentration-dependant inhibition of SERCA. Collectively, these studies

indicate that ROS can impair SERCA function and impair Ca2+ uptake into the SR.

NCX

The NCX is the primary means of removing Ca2+ from the cell via the plasma membrane.

A reduction in NCX function could elevate levels of free cytosolic calcium. There is evidence

suggesting that this exchanger is a tetramer linked by disulfide bonds, and therefore, is

susceptible to modification by ROS (16, 59, 90). Supporting this theory, Coetzee et al. (21)

reported NCX inhibition in guinea pig cardiac myocytes following a hypoxanthine / xanthine

oxidase treatment (i.e., superoxide generating system). Kato et al. (59) also observed similar

results in isolated SR vesicles from bovine hearts. In addition, DiPolo and Beauge (26)

proposed that NCX inhibition is due to a ROS-induced reduction in the calcium sensitivity of the

exchanger. Hence, similar to ROS damage to SERCA, it also appears that ROS can modify and

damage membrane Ca2+ transport as well.

L-type calcium channels and the DHPR

Several groups have documented a decrease in L-type Ca2+ channel current by ROS. For

example, Tokube et al. (118) found that hypoxanthine / xanthine oxidase treatments, as well as









hydrogen peroxide treatments inhibited the L-type Ca2+ channel current in cardiac myocytes.

Further, this inhibition was prevented by SOD. Guerra et al. (40) observed almost identical

findings, using dihydroxyfumaric acid (DHF). In addition, Coeteez et al. (22) described a

decrease in the peak Ca2+ current of L-type Ca2+ channels following hypoxanthine / xanthine

oxidase treatments.

Summary: Calpain and Calcium-Handling Proteins

There is recent evidence suggesting that calpain may cleave key SR Ca2+-handling proteins

during IR (64). Degradation of these proteins would lead to a disruption in Ca2+ transport within

the cell, further exacerbating the IR-induced increase in free cytosolic Ca2+. Importantly, there is

reason to believe that Ca2+-handling proteins within the SR may become targets for calpain

cleavage after they are oxidatively modified by ROS. This may provide a very important link

between the two predominant mechanistic theories of IR injury, the Ca2+-overload theory and the

oxyradical theory. Additionally, this would present an interesting scenario in which calpain can

could regulate its own activation in a feed-forward mechanism. Therefore, the first goal of this

these experiments will be determine the relationships between IR-induced ROS production, the

oxidative modification of Ca2+-handling proteins, calpain activation, and the calpain-mediated

cleavage of Ca2+-handling proteins.

Antioxidants and SR Dysfunction

Because of the possible link between the oxidation of Ca2+-handling proteins and their

subsequent degradation by calpain, alterations in the antioxidant status within the cell may play a

critical role in reducing calpain activation, Ca2+-overload, and the associated deleterious effects

within the myocardium. As discussed earlier, the addition of antioxidants has been shown to

significantly reduce Ca2+-overload in cardiac myocyte cultures and the intact heart following IR

(58, 61, 64, 102). Additionally, antioxidant treatments can also preserve Ca2+-handling protein









function in cells following oxidative stress. However, which antioxidants) are most effective in

providing protection to Ca2+-handling proteins in vivo are currently unknown. Nonetheless, an

antioxidant that is thought to play a critical role in protecting the myocardium against IR injury is

MnSOD (45, 93, 127). As discussed earlier, MnSOD may play a very important role in

protecting the heart against ROS because of its location within the mitochondria, a major source

of myocardial radical production. It is possible that an increase in myocardial MnSOD activity

may reduce the oxidative modification of critical SR and plasma membrane Ca2+-handling

proteins, maintaining their function and reducing the likelihood of their degradation by calpain.

This would in turn serve to attenuate any further IR-induced increase in free cytosolic calcium

and calpain activation. One well established model of increasing MnSOD protein content and

enzyme activity is endurance exercise training (55, 120, 122, 128).

Exercise-Induced Cardio-Protection Against IR Injury

Regular bouts of muscular exercise (e.g., 60 minutes of endurance exercise) is a well-

established means of inducing cardio-protection against IR-induced injury (71, 72, 93, 113, 114).

Work from numerous laboratories has consistently demonstrated exercise-induced cardio-

protection against IR insults of varying severities, ranging from minor injury to infarction.

Additionally, work from our laboratory has determined that three days of exercise training

provides the same degree of cardio-protection as long-term (weeks) training (14, 25, 44, 93).

Although there is little debate concerning the protective effects of exercise training, the

mechanisms through which it provides cardio-protection are not completely understood. Several

potential mechanisms to explain exercise-induced cardio-protection exist, including increases in

myocardial heat shock proteins (HSP's), increased antioxidant capacity, and reduced calpain

activation. The following sections will address each of these possibilities in greater detail.









Increased Myocardial Heat Shock Proteins

Heat shock proteins are a multifunctional group of proteins, which are stimulated by a

variety of stimuli such as heat, oxidative stress, Ca -overload, exercise training, and low pH.

Once active, these proteins serve several functions within the cell, including: chaperoning and /

or translocating proteins, folding and refolding proteins, scavenging free radicals, and even

facilitating protein synthesis. However, although the cardio-protective properties of various

HSPs have been demonstrated, the importance of HSPs to exercise-induced cardio-protection is

somewhat controversial. For example, work from our laboratory has suggested that an elevation

in HSPs is not essential for exercise induced cardio-protection (46). In these studies, hearts from

animals, which were exercise trained in a cold environment (40C), were compared to the hearts

from animals that were trained at room temperature. Hearts from the cold-trained animals

demonstrated a similar level of cardio-protection against IR injury compared to the warm-trained

animals, even without the exercise-induced increase in HSP protein content. These results have

been confirmed by other groups as well (114). Therefore, it appears that an elevation in

myocardial HSPs is not essential for exercise-induced cardio-protection. Another possible

mechanism of exercise-induced cardio-protection is through an up-regulation of endogenous

antioxidants.

Increased Myocardial Antioxidant Capacity

As discussed earlier, the cell contains several antioxidant defenses against IR-induced ROS

production. The primary antioxidant defenses are thought to include GSH, GPX, CAT, and

SOD. Although there is an abundance of research demonstrating the cytoprotective effects of

these antioxidants during IR, the question of which antioxidants may play a critical role in

exercise-induced cardio-protection is yet unanswered. Importantly, protein levels and activities

of only a few antioxidant enzymes have been shown to increase consistently following exercise









training (95, 96). For example, GSH content has been shown to be elevated following long-term

(8-10 weeks) exercise training ((52, 53, 56, 98) whereas short-term training has been found to

increase GSH protein content (75), decrease it (69), or result in no change (105). Therefore it

can be concluded that long-term exercise training elevates GSH but the conflicting evidence in

the literature does not permit a firm conclusion about the impact of short-term training on cardiac

levels of GSH. Moreover, most studies have concluded that exercise training does not elevate

GPX levels in the heart (25, 92, 94). The effect of exercise on CAT activity is also somewhat

unclear with some studies reporting increases and others reporting no change following training

(25, 44, 46, 109). In contrast, it is widely agreed that exercise elevates myocardial MnSOD

protein content and activity (25, 44, 45).

Increased MnSOD activity. Growing evidence suggests that endurance exercise may provide

protection, at least in part, by up-regulating the endogenous antioxidant MnSOD. As discussed

earlier, MnSOD may play an important cardio-protective role in the heart during IR due to its

localization in the mitochondria and ability to prevent oxidative stress induced by mitochondrial

superoxide production. Several studies have documented the protective effects of MnSOD. For

example, Chen et al. (20) demonstrated that MnSOD over-expression reduced infarct size

following IR injury. Further, Abunasra et al. (1) observed cytoprotection against IR injury using

adenoviral gene transfer of MnSOD. In addition, recent studies using a MnSOD mimetic, which

was directed almost exclusively into the mitochondria, observed that hearts from animals which

were given the mimetic showed a significant improvement in myocardial function and a

reduction in myocardial injury following IR (2).

Exercise-Induced Regulation of Calpain Activation

Recent work from our laboratory has revealed an exercise-induced decrease in calpain

activation following IR (30, 97). This work also demonstrated a decrease in calpain-mediated









cleavage and degradation of the Ca2+-handling protein SERCA2a (30). This suggests that

exercise may provide cardio-protection, at least in part, through the regulation of IR-induced

calpain activation. However, the mechanisms of exercise-induced calpain regulation are not

currently known. One possibility is that exercise-induced increases in MnSOD may reduce

oxidative modification to Ca2+-handling proteins, attenuating the IR-induced increase in free

Ca2+ and calpain activation. Therefore, the second goal of these experiments will be to

determine the relationships between MnSOD, oxidative modification of Ca2+-handling proteins,

calpain activation, and calpain-mediated degradation of Ca2+-handling proteins.

Summary

Myocardial IR injury is a complex problem involving both the generation of free radical

species (the oxyradical theory), as well as increases in free cytosolic Ca2 (the Ca2+-overload

theory), resulting in loss of myocardial function, damage and degradation of cellular proteins and

lipids, and cell death. Increased activation of the Ca2+-dependant protease, calpain during IR

may provide an important link between these two theories by preferentially cleaving Ca2+-

handling proteins which have been modified by free radicals, thus exacerbating the problem of

Ca2+-overload and calpain-mediated injury within the myocardium.

It is believed that much of the ROS production during IR originates from the mitochondrial

respiratory chain. The antioxidant MnSOD is localized in the mitochondria and can reduce the

generation of the free radical superoxide. Additionally, exercise training has been consistently

shown to provide cardio-protection against myocardial IR injury and is also associated with an

increase in MnSOD and a decrease in calpain activation. This increase in MnSOD may reduce

the oxidative modification of calcium handling proteins thus reducing calpain activation and

maintaining Ca2+ homeostasis.









CHAPTER 3
METHODS

The methods segment of this proposal will be divided into five sections with the first

providing details about the experimental animals, the second outlining the experimental designs,

the third and fourth detailing the general methods and dependant measurements used in these

experiments and the fifth describing statistical analyses.

Experimental Animals

Animal Model Justification

Adult (3-5 month old) male Sprague-Dawley (SD) rats were used for these experiments.

The animals were 3-5 months of age (young adult) at the time of sacrifice. The SD rat was

chosen for several reasons: first, the invasive nature of these experiments precludes the use of

human subjects. Second, the SD model is a well accepted model for the study of myocardial

ischemia reperfusion injury (14, 56, 70, 88, 92). Third, the SD rat does not display large inter-

animal variation in measures of cardiac contractility and/or collateral circulation. In addition, we

chose to study male rats to avoid the possibly confounding effects of varying estrogen levels

across the estrus cycle (116).

Animal Housing and Diet

All animals were housed at the University of Florida Animal Care Services Center.

Animals was maintained on a 12:12 hour light-dark cycle and provided food (AIN93 diet) and

water ad libitum throughout the experimental protocol.

Experimental Design

Experimental Design: Hypothesis One

Animals were randomly assigned to one of five experimental groups (Figure 3-1). The

Control group (hearts were quickly removed from anesthetized animals, i.e. no in-vitro









perfusion) served as a control for all dependant measures. A Control-perfused group was also

included to control for any dependant variable changes that result from perfusion on the isolated

working heart preparation. To investigate the effects of IR, three experimental groups (Control-

IR, Calpain-Inhibited-IR, and Vehicle-IR) were subjected to an in-vitro IR stress (i.e., no flow

ischemia, followed by reperfusion). At the conclusion of each experiment, segments of the left

ventricle were rapidly frozen in liquid nitrogen and stored at -80C until assay. Samples were

subsequently assayed to determine the levels of selected biochemical dependent measures.

Control Control Control Vehicle Calpain Inh
Perfused IR IR IR


Calpain Inhibition
In-vitro


Perfusion Ischemia-reperfusion
Only In-vitro



DEPENDANT MEASURES
*Contractile function / Ca2+-handling LVDP, +dp/dt, -dp/dt
*Oxidation of critical Ca2+-handling proteins WB for carbonyl & HNE formation
*Degradation of critical Ca2+-handling proteins WB

Figure 3-1. Experimental design for Hypothesis One.

Experimental Design: Hypothesis Two

Animals were randomly assigned to one of six experimental groups. Four groups were

exercise trained, as detailed below, while the other two groups remained sedentary (Figure 3-2).

To elucidate the role that MnSOD plays in exercise-induced cardio-protection, one exercise

trained experimental group received an antisense oligonucleotide against MnSOD following each

exercise training session. Importantly, our experience with this antisense oligonucleotide is that

this treatment consistently attenuates the exercise-induced increase in myocardial MnSOD










activity associated with exercise training without reducing MnSOD activity and/or protein levels

below those of control animals (45, 73). In addition, an oligonucleotide mismatch group of

trained animals was included to account for any extraneous effects of the oligonucleotide.

All experimental groups underwent either a sham surgery or an in vivo IR surgery, also

described below. The Control-Sham group served as a control for all dependant measures. At

the conclusion of each experiment, segments of the left ventricle were assayed to assess the

levels of numerous biochemical dependant measures.


Control Trained Control Trained Trained Trained
Sham Sham IR IR IR IR
MnSOD AS Mismatch


Exercise
Training


Sham IR
Surgery In-vivo





DEPENDANT MEASURES

Calpain activity WB for all-spectrin
*Oxidation of critical Ca"-handling proteins WB for carbonyl & HNE formation
*Degradation of critical Ca2+-handling proteins WB


Figure 3-2.Experimental design for Hypothesis Two.

General Methods

The Isolated Working Heart Preparation/IR Protocol (Hypothesis One)

To investigate myocardial function before and after an IR insult, we selected the in vitro

working heart model. This model is a highly reproducible preparation for examination of cardiac

performance, as cardiac preload and after-load pressures are maintained constant. Further, an









advantage of the working heart model versus an in vivo IR model is the elimination of the

confounding influence of other organ systems, systemic circulation, and peripheral

complications. This preparation has been successfully used by our laboratory for over five years

(30, 71-73, 92, 97).

In Vitro Ischemia-Reperfusion Protocol (Hypothesis One)

Simultaneous clamping of aortic and atrial lines was used to induce global, normothermic,

no flow ischemia. Following 30 minutes of perfusion during the pre-ischemic protocol, ischemia

was maintained for 25 minutes followed by 45 minutes of reperfusion. During ischemia, the

heart was enclosed in a sealed, water-jacketed chamber maintained at 37C. Following the

ischemic period, the heart was switched to the retrograde perfusion mode for 10 minutes

followed by 10 minutes of assist mode (retrograde perfused with the atrial cannula open) and 25

minutes of normal reperfusion. Upon the conclusion of non-perfusion, perfusion, or IR

treatments, the left ventricular free wall was immediately sectioned into four strips cut from base

to apex. Prior to storage, heart sections were rinsed in a cold antioxidant buffer (50mM

NaHPO4, 0. ImM butylated hydroxytoluene, and 0. ImM EDTA). These tissue sections were then

rapidly frozen in liquid nitrogen, and stored at -80'C until subsequent biochemical analysis.

Cardiac Performance Measurements (Hypothesis One)

Cardiac performance measurements were record every 5 minutes prior to ischemia and

during reperfusion. Measurements included: left ventricular developed pressure (LVDP), the

rates of pressure development (+dp/dt) and decline (-dp/dt), and heart rate. These variables were

measured via a calibrated pressure transducer (Harvard Instruments) connected to the aortic

cannula. Data was recorded and stored using a customized computer data-acquisition system.









Calpain Inhibition (Hypothesis One)

To determine the effects of IR-induced calpain activation on oxidation and degradation of

myocardial calcium handling proteins, calpain was inhibited (in vitro) using the selective

inhibitor MDL 28170, also known as "calpain inhibitor three" or (CI3) (EMD Biosciences, La

Jolla, CA). The efficacy of CI3 as a selective inhibition of calpain I and II has been well

established (18, 19, 30, 122, 125). The inhibitor was dissolved in Dimethyl Sulfoxide (DMSO)

and added to the perfusion buffer prior to heart perfusion at a concentration of 10[LM. In

preliminary experiments, this concentration of CI3 was shown to inhibit calpain I and II without

inhibiting the proteosome.

Exercise Training Protocol (Both Hypotheses)

Exercise trained animals began by performing 5 consecutive days of gradual habituation to

treadmill running. Treadmill habituation initiated with 10 minutes of training the first day and

was increased by 10 minutes each day, ending in 50 minutes of running on the fifth day.

Following habituation, exercise trained animals performed 3 consecutive days of treadmill

running (60 minutes/day) at an intensity of approximately 60-70% of VO2max.

In Vivo Ischemia-Reperfusion Protocol (Hypothesis Two)

The in vivo model of coronary artery ligation has been used successfully by our laboratory

for over 12 years. In our hands, ligation of the left main coronary artery (close to its origin)

using this in vivo preparation consistently results in ischemia in 60% of the ventricular free wall.

Rats were anesthetized (80 mg/kg sodium pentobarbital i.p.) and ventilated (Harvard Apparatus,

Holliston, MA) with room air via a tracheostomy tube. A saline-filled catheter attached to a

pressure transducer was placed in the carotid artery and interfaced with a computerized heart

performance analyzer for continuous monitoring of arterial blood pressures (Digi-Med,

Louisville, KY). Arterial blood (<100 tl) samples were obtained prior to ischemia to assess









blood gases and pH using a calibrated electronic blood gas analyzer (Instrumentation

Laboratories, Lexington, MA). When indicated, the tidal volume delivered by the ventilator was

adjusted to correct blood gas abnormalities. An additional catheter was placed in the jugular

vein for delivery of sodium pentobarbital (20 mg-kg-1) as needed. Following a left thoracotomy,

a ligature was placed around the left anterior descending coronary artery (LCA), close to its

origin. In sham surgery, the hearts were removed without occlusion of the LCA. For the IR

surgery, a soft piece of polyethylene tubing was threaded through the ligature, pressed on the

surface of the LCA, and secured with a small hemostat. Coronary occlusion was maintained for

50 min followed by 120 min of reperfusion. Electrocardiographic activity was continuously

monitored and recorded via an interfaced customized data acquisition program with data points

recorded every millisecond. Following reperfusion, hearts were removed, rinsed in a cold

antioxidant buffer (50 mM NaHPO4, 0.1 mM BHT, 0.1 mM diethylenetriaminepentaacetic acid,

pH 7.4), and quickly frozen in liquid nitrogen for later analysis.

Inhibition of MnSOD Protein Translation (Hypothesis Two)

Antisense oligonucleotides (AS-ODN) are single-stranded synthetic DNA that typically

contain a backbone with modification to a specific sequence to hybridize to a specific messenger

RNA. Hybridization of the ODN to mRNA inhibits the mRNA from initiating translation. To

block the translation of MnSOD protein, animals were injected (i.p.) with a 22-mer

phosphorothioate derivative of the AS-ODN (5'-CACGCCGCCCGACACAACATTG-3')

immediately post-exercise at a dose of 10 mg/kg. The injection time and dose of this specific

AS-ODN have been shown to provide optimal experimental conditions to inhibit the exercise-

induced increase of MnSOD activity in myocardial tissue (45, 73). This has been confirmed by

previous experiments from our laboratory. In addition, a mismatch control group (MM-ODN

(CAC TCC TCC CAG CAC AAC AGTC)) was included in these experiments to verify that the









inhibition of MnSOD translation was due to the specific AS-ODN sequence, as well as

accounting for any extraneous factors resulting from the delivery of the ODN.

Dependant Measures (Both Hypotheses)

Measurement of Calpain Activation

To assess calpain activity, calpain-specific cleavage product of the protein all-spectrin was

analyzed. Briefly, proteins were separated using standard SDS-PAGE techniques on a 4%-20%

polyacrylamide gel. Proteins were then transferred to nitrocellulose membranes and exposed to a

mouse monoclonal primary antibody to all-spectrin (SIGMA, St. Louise, MO). Following

washing, an anti-mouse IgG-HRP-conjugated secondary antibody was applied for

chemiluminescence detection (Amersham, Piscataway, NJ). Both all-spectrin intact and calpain-

cleaved fragments were analyzed using a Kodak imaging system. The cleaved band was

expressed as a percentage of the intact band and finally expressed as a percentage of the Perfused

Control group.

Western Blots for Calcium-Handling Proteins

Western blots were used to determine protein levels of the following Ca2+-handling

proteins: SERCA2a, phospholamban, L-type Ca2+ channels and the Na /Ca2+ exchanger. These

measurements were used to determine which Ca2+-handling proteins were degraded following

IR, as well which proteins were degraded specifically by calpain during IR (Hypothesis one).

Briefly, proteins were separated using standard SDS-PAGE techniques on 4%-20%

polyacrylamide gels. Proteins were then transferred to Polyvinylidene Difluoride (PVDF)

membranes and exposed to a monoclonal primary antibody. Following primary antibody

exposure, an anti-mouse, or anti-rabbit 800 (green) or 680 (red) infared secondary antibody (Li-

Cor, Lincoln, Nebraska) was applied for infared detection. Each blot was then analyzed using

an Odyssey infared imaging system (Li-Cor, Lincoln, Nebraska) and normalized to a commassie









blue protein stain in order to adjust for protein loading. Results were then expressed as a percent

of either Control-Perfused (Hypothesis one), or Control-Sham (Hypothesis two).

Immunoprecipitation of Calcium-Handling Proteins

To determine the effects of exercise, IR and calpain inhibition on the oxidation of Ca2+

handling proteins, each of the following proteins (SERCA2a, phospholamban, L-type Ca2+

channels and the Na /Ca2+ exchanger) were first isolated by immunoprecipitation. Briefly, heart

tissue was homogenized at a 1:10 dilution factor in a 100mM KPO4 buffer containing 1 |tm

lactacystine, 1 |tm MG-132 (SIGMA, St. Louise, MO), pH 7.4. The homogenate was then

centrifuged at 1000 g for 20 minutes to clear cellular debris. Approximately 1000 |tg of protein

was then transferred to a new tube and exposed to 10 [tl of primary antibody to the protein of

interest. Following an overnight incubation on a Fisher rocker, 40 [tl of protein A/G PLUS-

agrose (Santa Cruz Biotechnology, Santa Cruz, CA) were added and incubated overnight. Four

centrifugations (2500 rpm for 10 minutes) were then used to separate the agrose-bound

antibody/protein complex. Following each spin, the pelleted complex was suspended in 118 [tl

of KPO4 buffer. Finally, the Bradford protein assay was run to determine final protein

concentration and the samples were normalized to approximately 2 mg of protein / ml.

Measurement of Protein Carbonyl Formation on Calcium-Handling Proteins

Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive

indices of oxidative injury (15). Proteins, isolated via immunoprecipitation, were examined for

carbonyl formation using a commercially available Western Blot kit from Chemicon

International (Chemicon International, Temecula, CA). This allowed for the determination of

the level of oxidative modification to specific calcium-handling proteins.









Measurement of HNE Formation on Calcium-Handling Proteins

HNE (4-hydroxy-2-nonenal) formation is a major product of endogenous lipid

peroxidation. The w-6-family (linoleic and arachidonic acids) of polyunsaturated fatty acids

produce HNE as a result of free radical-induced lipid peroxidation. HNE is a highly reactive

aldehyde and can react with several functional groups on biological material, particularly

sulfydryl groups to form thioester adduct and then hemiacetals. HNE may also react with

histidine and lysine residues of proteins to form stable Michael addition-type of adducts. In

addition, HNE-modification of proteins may impair biological functions. Proteins, isolated via

immunoprecipitation, were examined for HNE formation using a commercially available

Western Blot kit from Calbiochem (SanDiego, Ca). This allowed for the determination of the

level of oxidative modification to specific Ca2+ handling proteins.

Data Analysis

To test our hypotheses, one-way ANOVA's were performed to assess IR, calpain

inhibition, and exercise training differences for the primary dependent measures. A Tukey post

hoc test was used to determine group differences when indicated. Significance was established a

priori at P < 0.05. The relationship between the oxidative modification carbonyll and HNE

formation) of Ca2+-handling proteins and the degradation of Ca2+-handling proteins was assessed

using a Pearson's simple correlation.










CHAPTER 4
RESULTS

Hypothesis One

Animal Characteristics

The physical characteristics for the animals in all experimental groups are presented in

Table 4-1. Although body mass differed between the experimental groups, heart weights and

heart-to-body weight ratios were similar.

Table 4-1. Animal body and heart weights.
Heart Weight Heart / Body Weight
Group Number Body Weight (g) (g) Ratio (mg/g)
Non-perfused Control 11 360 + 4.2 1.20 +.02 3.34 + .07
Non-perfused Trained 9 339 5.8 1.21 .03 3.57 .10
Perfused Control 11 354 4.4 1.16 .03 3.28 .09
Control-IR 11 347 + 8.4 1.22 + .03 3.53 + .11
Trained-IR 12 322 4.2 1.17 .02 3.65 .08
Calpain inhibited-IR 6 374 + 7.9 1.28 + .08 3.41 + .20
Vehicle-IR 6 401 8.0 1.25 .04 3.13 + .06
Values are means + SE. Significantly different from Trained-IR, Significantly different from Vehicle-
IR, P < 0.05. Note that Non-perfused Trained and vehicle-IR groups had significantly different body
weights and heart / body weight ratios compared to Non-perfused Controls, although heart weight did not
differ.



120


a-
o 100
-J
* 80

.- 60

, 40

4 20
0


Control-IR


Trained-IR


Inhibited-IR


Figure 4-1.% Recovery of left ventricular developed pressure (LVDP). Values are means + SE.
Significantly different from Control-IR, P < 0.05. Note that "inhibition" denotes
the inhibition of calpain using the inhibitor CI3.










Cardiac Performance Measures

Percent recovery of left ventricular developed pressure (LVDP)

LVDP is commonly used as an index of myocardial function. By comparing LVDP prior

to ischemia with post-ischemic LVDP we can quantify myocardial dysfunction. As expected, %

recovery of LVDP was significantly depressed in the Control-IR group (Figure 4-1). However,

both exercise training and calpain inhibition attenuated the loss of LVDP following IR. This

demonstrates the cardio-protective effects of exercise as well as the deleterious effects of the

Ca2+-activated protease calpain.



120


O.
+
80

0- 60

w 40

0 20

0
Co ntro 1-IR Tramine d-R Vehicle -R Inhibite d-IR

Figure 4-2.Rate of systolic pressure increase (+dp/dt). Values are means + SE. Significantly
different from Control-IR, P < 0.05. Note that "inhibition" denotes the inhibition of
calpain using the inhibitor CI3 and "vehicle" denotes vehicle treatment only without
CI3.

Percent of pre-ischemic +dp/dt and -dp/dt

The measurements of +dp/dt and -dp/dt are indicative of the rate of systolic pressure

development (i.e., rate of ventricular contraction) and the rate of systolic pressure decline (i.e.,

rate of ventricular relaxation), respectively. Because myocardial contraction is regulated by the










release and re-sequestering of Ca2+ within the sarcoplasmic reticulum, these measurements also

reflect myocardial Ca2+ release and uptake kinetics. Further, by comparing +dp/dt and -dp/dt

prior to and following ischemia, we can quantify the changes in myocardial

contraction/relaxation rates and/or Ca2+-handling kinetics.

As illustrated in Figures 4-2 and 4-3, both +dp/dt and -dp/dt decreased significantly

following IR in both the control-IR and vehicle-IR experimental groups, suggesting a decrease in

Ca2+-handling efficiency. However, both exercise training and calpain inhibition nearly

completely prevent this IR-induced dysfunction.


120 -
*
I 100- T

S 80 -

.- 60

(D 40
0.
II-
o 20

0
Control-IR Trained-IR Vehicle-IR Inhibited-IR

Figure 4-3.Rate of systolic pressure decline (-dp/dt). Values are means + SE. Significantly
different from Control-IR, P < 0.05. Note that "inhibition" denotes the inhibition of
calpain using the inhibitor CI3 and "vehicle" denotes vehicle treatment only without
CI3.

Oxidative Modification of Critical Calcium-Handling Proteins

The increase in free cytosolic Ca2+ and corresponding increase in calpain activity have

been shown to play a deleterious role in cardiac myocytes following IR. Because the oxidative

modification of Ca2+-handling proteins has been shown to lead to impaired Ca2+-handling, we

postulated that the oxidation of Ca2+-handling proteins within the cardiac myocytes may impair










function of the proteins and thus lead to increases in free cytosolic Ca2 the activation of calpain,

myocardial damage and contractile dysfunction.


Control Trained Control Control Trained Vehicle Inhibited
Perfused IR IR IR IR
LTCC
SERCA2
PLB
NCX

LTCC O SERCA2a O PLB m NCX
500
450 -
400-
O- *
o 0 *
.. 350 -
S300
0
C-) 250
O 200
150 -



0
Control Trained Control- Control-IR Trained-IR Vehicle-IR Inhibited-IR
Perfused

Figure 4-4. Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel
(LTCC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a),
Phospholamban (PLB) and Sodium/calcium exchanger (NCX). Representative blots
are displayed above. Values, below, are means + SE. Significantly different from
respective Control-Perfused group, P < 0.05. Note that "inhibition" denotes the
inhibition of calpain using the inhibitor CI3 and "vehicle" denotes vehicle treatment
only without CI3.

We assessed oxidative modification of calcium handling proteins by measuring both

carbonyl and HNE formation via Western Blotting of each individual Ca2+-handling protein.

Protein carbonyl levels of proteins are indicative of the magnitude of oxidative modification of

proteins whereas HNE-protein interaction is taken as an indication of protein reactions with the

reactive aldehyde, HNE. The measurement of both carbonyl and HNE formation are commonly

used to assess oxidative stress to proteins and lipids within the cell.










Control Trained Control
Perfused


SERCA2a
PLB
NCX


o SERCA2a [ PLB m NCX


Control Trained Control- Control-IR Trained-IR Vehicle-IR Inhibited-IR
Perfused


Figure 4-5.HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium
exchanger (NCX). Representative blots are displayed above. Values below are
means + SE. Significantly different from respective Control-Perfused group, P <
0.05. Note that "inhibition" denotes the inhibition of calpain using the inhibitor CI3
and "vehicle" denotes vehicle treatment only without CI3.

Carbonyl formation on calcium-handling proteins

IR resulted in an increase in carbonyl formation to all four Ca2+-handling proteins

measured (LTCC, SERCA2a, PLB, and NCX). Importantly, exercise training attenuated the IR-

associated increase in carbonyl formation in all four proteins (Figure 4-4). This suggests that all

four proteins are subject to oxidative modification following IR.

HNE formation on calcium-handling proteins

An increase in HNE formation on SERCA2a and PLB was also observed following IR.

However, the oxidative modification of these proteins was attenuated by exercise training


Control Trained Vehicle Inhibited
IR IR IR IR










(Figures 4-5). Note that no significant changes in HNE formation were detected on the NCX,

and there were not detectable levels of HNE formation on the LTCC.


Control Trained Control Control Trained Vehicle Inhibited
Perfused IR IR IR IR

LTCC W -
SERCA2a
PLB -




LTCC o SERCA2a o PLB u NCX

250 -


200 -


150 -
O

o 100






Control Trained Control- Control-IR Trained-IR Vehicle-IR Inhibited-IR
Perfused

Figure 4-6.Western blotting for intact Ca2+-handling proteins: L-type calcium channel (LTCC),
Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Phospholamban
(PLB) and Sodium/calcium exchanger (NCX). Representative blots are displayed
above. Values below are means + SE. Significantly different from respective
Control-Perfused group, P < 0.05. Note that "inhibition" denotes the inhibition of
calpain using the inhibitor CI3 and "vehicle" denotes vehicle treatment without CI3.

Calpain-Mediated Degradation of Calcium-Handling Proteins

We have previously demonstrated that IR results in increased calpain activation. Once

active, calpain degrades many proteins/protein complexes within the cell. We hypothesized that

calpain may cleave critical Ca2+-handling proteins, which have been oxidatively modified,









exacerbating IR-induced Ca2+-overload, calpain activation and myocardial dysfunction. To test

this hypothesis we performed Western Blot analyses to determine protein content of each of the

four Ca2+-handling proteins with and without calpain inhibition. Our results demonstrated a

decrease in intact LTCC, SERCA2a, PLB, and NCX following IR in both control-IR and

vehicle-IR groups. However, both exercise training and calpain inhibition prevented this IR-

induced degradation in all four proteins.

Table 4-2. Correlations Between the Oxidative Modification of Ca2+-handling
Proteins and Their Degradation Following IR
Carbonyl formation / Intact HNE formation / Intact
protein protein
(R2) (R2)
LTCC -.901
SERCA2a -.939 -.939
PLB -.827 -.955
NCX -.511 -.263
Pearson correlation R2 values depicting the relationship between oxidative modification carbonyll
formation and HNE formation) to critical Ca2+-handling proteins and the total amount of intact protein,
determined by Western Blot. All experimental groups were pooled for this analysis.

Our results indicate that all four of the measured Ca2+-handling proteins, LTCC,

SERCA2a, PLB and NCX, are degraded by calpain during IR (Figure 4-6). In addition, exercise

training attenuated the degradation of these proteins, most likely through a reduction in IR-

induced calpain activation. Consistent with this postulate, a strong negative correlation exists

between the oxidative modification carbonyll and HNE formation) and amount of intact protein

of each of the Ca2+-handling proteins (Table 4-2). This may suggest that oxidative modification

makes these proteins more susceptible to calpain-mediated degradation.

Hypothesis Two

Calpain Activation (Calpain-Cleaved aII-Spectrin)

The deleterious effects of IR-induced calpain activation have been well documented (55,

120, 122, 128). Additionally, we have previously demonstrated that exercise training attenuates









calpain activation and provides cardioprotection against IR injury (71, 72, 93, 96, 113, 114). The

mechanisms) of this protection, however, are currently unknown.


Control Trained Control Trained Trained-IR Trained-IR
Sham Sham IR IR Antisense Mismatch
250 kOD 0111M
145 kD i


400 -

E 350
300 -
i 250
0 200

0

50 -
0 --- --- -- -- --- -- -- -- -- -
Control Trained Control Trained Trained-IR Trained-IR
Sham Sham IR IR Antisense Mismatch

Figure 4-7.Western blotting for calpain-cleaved all-Spectrin (an in-vivo calpain substrate). A
representative blot, above, displaying intact (250 kD) and calpain-cleaved all-
Spectrin (145 kD). Values below are means + SE. Significantly different from
Control-Sham group, P < 0.05.

We hypothesized that exercise may provide protection through up-regulation of myocardial

MnSOD resulting in protection against IR-induced oxidative stress to Ca2+-handling proteins,

Ca2+-overload and calpain activation. Therefore, we performed Western Blotting for all-

Spectrin, a well-characterized in-vivo calpain substrate, in order to determine the effects of

MnSOD on IR-induced calpain activation.

As expected, IR resulted in an increase in calpain activation in control (sedentary animals),

which was attenuated by exercise training (Figure 4-7). Importantly, exercise trained animals

treated with the antisense oligonucleotide against MnSOD had similar levels of calpain










activation to control-IR animals. This finding is consistent with the hypothesis that MnSOD

plays a critical role in regulating IR-induced calpain activation.

Oxidative Modification of Critical Calcium-Handling Proteins

The increase in free cytosolic Ca2+ and corresponding increase in calpain activity have

been shown to play a deleterious role in the heart following IR.

Because the oxidative modification of Ca2+-handling proteins has been shown to lead to

impaired Ca2+-handling, we have hypothesized that the oxidation of Ca2+-handling proteins

within the myocardium may lead to increases in free cytosolic Ca2, the activation of calpain,

myocardial damage and contractile dysfunction.

Control Control Trained-IR Trained-IR Trained-IR
Sham IR Mismatch Antisense
LTCC
SERCA2a
PLB ... .
NCX

m LTCC o SERCA2a o PLB m NCX

450
*
400 -
E 350
S300 -
O 250 -
r 200 -
150o
O 100 T
S50


Control Control Trained-IR Trained-IR Trained-IR
Sham IR Mismatch Antisense

Figure 4-8.Carbonyl formation on critical Ca2+-handling proteins: L-type calcium channel
(LTCC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a),
Phospholamban (PLB) and Sodium/calcium exchanger (NCX). Representative blots
are displayed above. Values below are means + SE. Significantly different from
respective Control-Sham group, P < 0.05.










Carbonyl formation on calcium-handling proteins

IR resulted in an increase in carbonyl formation on all four Ca2+-handling proteins

measured in hearts from control (untrained animals): LTCC, SERCA2a, PLB, and NCX

(illustrated in Figure 4-8). Exercise training attenuated the IR-associated increase in carbonyl

formation in three of the four measured Ca2+-handling proteins: LTCC, SERCA2a, and PLB.

Importantly, exercise-induced protection against IR-induced carbonyl formation was abolished

by the antisense oligonucleotide against MnSOD in both the LTCC and SERCA2a, but not in

PLB. In addition, neither exercise training nor antisense treatment affected IR-induced carbonyl

formation on the NCX.

Control Control Trained-IR Trained-IR Trained-IR
Sham IR Mismatch Antisense
SERCA
PLB
NCX

o SERCA2a o PLB u NCX

300

250
E
200 *

2 150
O
o 100--
0



Control Control Trained-IR Trained-IR Trained-IR
Sham IR Mismatch Antisense

Figure 4-9.HNE formation on critical Ca2+-handling proteins: Sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA2a), Phospholamban (PLB) and Sodium/calcium
exchanger (NCX). Representative blots are displayed above. Values below are
means + SE. Significantly different from respective Control-Sham group, P < 0.05.










HNE formation on calcium-handling proteins

An increase in HNE formation on SERCA2a and PLB in the heart was also observed

following IR, which was attenuated by exercise training (Figures 4-9). In addition, exercise-

induced protection against IR-induced HNE formation on SERCA2a and PLB was abolished by

the antisense oligonucleotide against MnSOD. However, no significant changes in HNE

formation were detected on the NCX, and there was no detectable amount of HNE formation on

the LTCC.


Control Trained Trained Control Trained-IR Trained-IR
Sham Sham IR IR Mismatch Antisense
LTCC
SERCA2a -"o mlN
PLB


LTCC o SERCA2a o PLB m NCX

160
140 -
120
100 -
0
80 *
0
o 60
o 40
20

0-
Control Trained Trained Control Trained-IR Trained-IR
Sham Sham IR IR Mismatch Antisense


Figure 4-10. Western blotting for intact Ca2+-handling proteins: L-type calcium channel
(LTCC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a),
Phospholamban (PLB) and Sodium/calcium exchanger (NCX). Values are means +
SE. Significantly different from respective Control-IR group, P < 0.05.









Calpain-Mediated Degradation of Calcium-Handling Proteins

We and others (18, 19, 31, 97) have previously demonstrated that IR results in increased

calpain activation in the heart. Once active, calpain degrades calpain substrates (proteins /

protein complexes) within the cell. We hypothesized that calpain cleaves important Ca2+-

handling proteins, which have been oxidatively modified, exacerbating IR-induced Ca2+-

overload, calpain activation and myocardial injury. To test this hypothesis we performed

Western Blot analyses to determine protein content of each of the four Ca2+-handling proteins.

Our results demonstrated a decrease in myocardial levels of intact LTCC, SERCA2a, PLB,

and NCX following IR, which was attenuated by exercise training. Moreover, MnSOD antisense

oligonucleotide treatment abolished the exercise-induced cardioprotection against Ca2+-handling

protein degradation in all four proteins (Figure 4-10). These results are consistent with the

notion that MnSOD plays a critical role in both the regulation of calpain activation as well as the

preservation of Ca2+-handling proteins during IR. Additionally, these results suggest that

oxidative modification of these proteins leads to their degradation.

Table 4-3. Correlations between the oxidative modification of
calcium-handling proteins and their degradation following IR
Carbonyl formation / Intact HNE formation / Intact
protein protein
(R2) (R2)
LTCC -.809
SERCA2a -.802 -.789
PLB -.722 -.823
NCX -.810 -.792
Pearson correlation R2 values depicting the relationship between oxidative modification carbonyll
formation and HNE formation) to critical Ca2+-handling proteins and the total amount of intact protein,
determined by Western Blot. All experimental groups were pooled for this analysis.

Finally, there was a strong negative correlation between the oxidative modification and

amount of intact protein of each of the Ca2+-handling proteins (Table 4-3). This finding is









consistent with the concept that oxidative modification makes these proteins more susceptible to

calpain-mediated degradation.









CHAPTER 5
DISCUSSION

Overview of Principal Findings

These experiments examined both the mechanistic role that calpain plays in IR-induced

cardiac injury as well as the mechanism responsible for exercise-induced cardioprotection

against IR injury via two separate groups of experiments.

Experiments conducted to determine the mechanisms) responsible for calpain-induced

cardiac injury (Hypothesis One) tested the following separate hypotheses: (1) IR promotes

increased oxidative modification of important myocardial Ca2+-handling proteins; (2) oxidative

modification of Ca2+-handling proteins is associated with increased degradation of these

proteins; and (3) inhibition of calpain will attenuate the IR-induced degradation of myocardial

Ca2+-handling proteins. Our data confirm the deleterious effects of IR on the myocardium;

including impaired Ca2+-handling and contractile function, oxidation and degradation of critical

Ca2+-handling proteins, and increased calpain activation. In addition, we observed strong

negative correlations between the degree of Ca2+-handling protein oxidative modification and the

amount of intact protein, suggesting a link between oxidative stress and protein degradation

during IR. Importantly, our experiments established that inhibition of calpain protects the heart

against IR-induced contractile dysfunction as well as the degradation of Ca2+-handling proteins.

Our results provide the first evidence that calpain cleaves the following critical Ca2+-handling

proteins (LTCC, NCX and PLB) in the intact heart during IR. In addition, our data confirms

previous work describing the calpain-mediated cleavage of SERCA2a during IR.

Experiments completed to examine the mechanism responsible for exercise-induced

cardioprotection against IR injury (Hypothesis Two) tested the following postulates: (1) exercise

training attenuates IR-induced oxidation and degradation of critical myocardial Ca2+-handling









proteins; and (2) exercise-induced cardioprotection is dependent on an exercise-induced over-

expression of cardiac levels of MnSOD. Our results reveal that exercise attenuated IR-induced

calpain activation, oxidative modification of critical Ca2+-handling proteins and the degradation

of these proteins. Importantly, when the exercise-induced over-expression of MnSOD was

attenuated via gene silencing (i.e., antisense oligonucleotide against MnSOD), cardioprotection

against Ca2+-handling protein oxidation and degradation, as well as calpain activation was

abolished. These results support the concept that MnSOD plays a critical role in exercise-

induced cardio-protection against IR injury, at least in part, by preventing the IR-induced

oxidation and degradation of Ca2+-handling proteins and calpain activation. Moreover, these

findings represent the first in-vivo data demonstrating that oxidatively modified Ca2+-handling

proteins in the heart are more susceptible to degradation during IR.

In combination, the results of experiments suggest a chain of events during IR beginning

with an increase in oxidative damage of Ca2+-handling proteins, leading to impaired Ca2+-

handling and calpain activation, in turn resulting in the calpain-mediated degradation of critical

Ca2-handling proteins by calpain, exacerbating Ca2+-overload, and myocardial injury. In

contrast, exercise attenuates these events, due in a large part, to the up-regulation of MnSOD in

the heart. Increased myocardial MnSOD reduces the oxidation of Ca2+-handling proteins, and

the deleterious chain of events that follow by dismutating superoxide produced in the

mitochondria during IR.

Hypothesis One: The Effects of IR and Calpain Inhibition on Myocardial Calcium-
Handling Proteins

The following paragraphs provide a detailed discussion of the findings of experiments

designed to determine the role that calpain plays in IR-induced damage and removal of Ca2-

handling proteins in the heart.









Calpain Inhibition Protects Against IR-induced Damage and Removal of Calcium-
Handling Proteins.

Calpain can modify and damage the myocardium during IR via multiple pathways. For

example, calpains cleave several structural proteins leading to the release of myofilaments,

facilitating their degradation by the proteosome. In addition, calpains can also promote

apoptosis, through cleavage of Bid, mediating cytochrome c release from the mitochondria.

Moreover, calpains increase the expression of cell adhesion molecules, leading to an increase in

neutrophil-mediated oxidative damage. Each of these pathways has been shown to significantly

contribute to IR-induced injury (55, 120, 122, 128). Further supporting the deleterious role of

calpain during IR, inhibition of calpain has been previously reported, by our group and others, to

reduce many of the deleterious effects of IR including contractile dysfunction, infarct area and

apoptosis (18, 19, 122, 123). Our experiments contribute to previous studies, demonstrating that

calpain inhibition attenuates the decline in LVDP, +dp/dt and -dp/dt typically observed following

IR (Figures 4-1, 4-2, 4-3). Decreased peak pressures (LVDP), as well as decreased rates of

pressure development (+dp/dt) and relaxation (-dp/dt) typically occur following myocardial

ischemia and are indicative of impaired Ca2+ transport within the myocardium. Therefore, our

data suggests that calpain impairs myocardial Ca2+-handling following ischemia. To elucidate a

potential mechanism of calpain-induced impairments in Ca2+-handling we measured the protein

content of several critical myocardial Ca2+-handling proteins following IR, with or without

calpain inhibition.

Calpain Degrades Critical Calcium-Handling Proteins

We postulated that calpain degrades myocardial Ca2+-handling proteins during IR,

exacerbating Ca2+-overload and IR injury. Further, we hypothesized that IR-induced oxidation









of Ca2+-handling proteins would be associated with calpain-mediated degradation. We were lead

to these hypotheses by three key observations.

First, calpain cleaves Ca2+-handling proteins. There is growing evidence from in-vitro

studies suggesting that calpain can cleave Ca2+-handling proteins. For example, Belles et al. (8)

and De Jongh et al. (24) have reported that calpain degrades the LTCC in-vitro. Other in-vitro

experiments have demonstrated calpain-cleavage of the ryanodine receptor and the

sodium/potassium ATPase (126, 130). Our results demonstrated a decrease in intact Ca2+

handling proteins following IR, which was attenuated by calpain inhibition (Figure 4-6). These

findings indicate that calpain mediates the degradation of several critical myocardial Ca2+-

handling proteins, including the LTCC, SERCA2a, PLB and NCX, during IR. Although calpain-

mediated cleavage of various Ca2+-handling proteins has been demonstrated in-vitro, to our

knowledge, the present study is the first to investigate calpain-mediated degradation of these

Ca2+-handling proteins in intact hearts.

Second, Ca2+-handling proteins lose function when oxidized during IR. There is a strong

body of in-vitro evidence examining the effects of ROS on Ca2+-handling protein function. This

work is summarized in review papers detailing the ROS-mediated loss of function in all of the

Ca2+-handling proteins examined in these experiments (LTCC, NCX, SERCA, and PLB) (64,

129). In addition, several studies have demonstrated a loss in Ca2+-handling protein function

following IR (48, 62). In combination with the knowledge that widespread protein damage

occurs during IR, we postulated that Ca2+-handling proteins might also be oxidized during IR,

accounting for their loss of function. This postulate was supported by our findings that IR

resulted in increased oxidative modification of critical Ca2+-handling proteins (Figures 4-4, 4-5).









In addition, physiological measures of myocardial Ca2+-handling (+dp/dt, -dp/dt and LVDP)

were depressed following ischemia (Figures 4-1, 4-2, 4-3).

Third, oxidative modification of proteins can lead to degradation. Several studies have

demonstrated that oxidative modification predisposes cellular proteins for degradation. Recent

work by Zolotarjova et al. (130) demonstrated that when oxidized, the sodium/potassium

exchanger is increasingly prone to calpain-mediated degradation. Wu et al also found that

oxidation of the RYR leads to an increased incidence of degradation by calpain (126).

Supporting this work, our results demonstrated strong negative correlations between the amount

of oxidized calcium handling proteins (i.e., LTCC, NCX, SERCA, and PLB) and the level of

intact protein (Table 4-2), suggesting a link between the oxidative modification of Ca2+-handling

proteins and their degradation. Nonetheless, this finding should be viewed with caution because,

although strong correlations exist between the degree of Ca2+-handling protein oxidation and

degradation, a strong positive correlation alone does not confirm a causal relationship.

In combination, we feel that these data supports the idea that ROS produced during IR

oxidatively modify critical myocardial Ca2+-handling proteins, resulting in both a loss in Ca2+

handling ability, as well as calpain-mediated degradation of Ca2+-handling proteins, exacerbating

IR injury. If this line of reasoning proves correct, a reduction in IR-induced oxidative stress

would attenuate the oxidation and degradation of myocardial Ca2+-handling proteins, thereby

decreasing IR-induced Ca2+ overload and calpain activation, attenuating myocardial injury.

Exercise training has been shown to reduce oxidative stress within the myocardium during

IR, possibly through the over-expression of endogenous antioxidant enzymes such as MnSOD.

We have also previously demonstrated that exercise reduces IR-induced calpain activation. We

postulate that exercise regulates IR-induced calpain activation by reducing the oxidative









modification of myocardial Ca2+-handling proteins. This postulate was tested by a second series

of experiments and these results will discussed in the subsequent sections.

Hypothesis Two: IR, Exercise, MnSOD and Calcium-Handling Proteins

These experiments tested the hypothesis that exercise would provide cardio-protection

against the IR-induced oxidative modification and degradation of critical Ca2+-handling proteins

as well as calpain activation. In addition, we postulated that exercise-induced over-expression of

MnSOD is a critical component of this exercise-induced cardio-protection.

Exercise Training Provides Cardio-Protection

We and others have previously demonstrated that exercise protects the heart against IR

injury (71, 72, 93, 96, 113, 114). In addition, we have reported that exercise training prevents

IR-induced calpain activation (31). However, the mechanisms) responsible for this protection

have not been determined. Expanding on our previous work, the current experiments identified

one possible mechanism through which exercise may regulate calpain activation and provide

cardioprotection by the preservation of critical Ca2+-handling proteins within the myocardium.

Our results reveal that exercise provides cardioprotection against both the IR-induced oxidation

and degradation of Ca2+-handling proteins (Figures 4-8, 4-9, 4-10). In addition, exercise

attenuates IR-induced calpain activation (Figure 4-7), potentially through an improved regulation

of free cytosolic Ca2+ in cardiac myocytes. Although the mechanisms) responsible for these

protective effects are not completely understood, we hypothesize that one possibility is that an

exercise-induced over-expression of the antioxidant MnSOD may provide protection against

ROS-mediated Ca2+-handling protein degradation, calpain activation and Ca2+-handling protein

degradation. To further investigate this possibility, the exercise-induced over-expression of

MnSOD was prevented via a gene silencing using an antisense oligonucleotide against MnSOD.









Exercise-Induced Over-Expression of MnSOD Prevents the Oxidation of Calcium-
Handling Proteins.

As discussed previously, there is strong evidence that both ROS and free cytosolic Ca2+

increase dramatically during IR. Moreover, there is a biological rationale to support a causal

relationship between the IR-induced increase in ROS production and Ca2+-overload. In regard to

the source of radical production during both ischemia and reperfusion, the mitochondrial

respiratory chain has been identified as a major contributor to IR-induced free radical production

(9). Further supporting the importance of mitochondrial free radical production, studies using

antioxidants targeted specifically to the mitochondria have demonstrated a significant reduction

in oxidant-related damage within the myocardium, as well as improved myocardial function (4,

11,28, 86).

Results from our experiments demonstrated a reduction in the IR-induced oxidation of

Ca2+-handling proteins following exercise training (Figures 4-8, 4-9). Importantly, the exercise-

induced reduction in Ca2+-handling protein oxidation was abolished by the antisense

oligonucleotide against MnSOD. This suggests that mitochondrial superoxide production during

IR contributes to the oxidation of critical myocardial Ca2+-handling proteins.

The mechanism through which mitochondria-produced superoxide affects Ca2+-handling

proteins located in the SR and/or plasma membrane is currently not understood. Mitochondria-

produced superoxide has several potential fates including conversion to a less reactive oxygen

species, such as hydrogen peroxide (H202) by the antioxidant enzyme superoxide dismutase

(SOD), or conversion to a more reactive species such as the hydroxyl radical (OH'), in the

presence of iron, or peroxynitrite (ONOO*), through reaction with nitric oxide. Although highly

reactive, peroxynitrite and the hydroxyl radical have very short half-lives and therefore would

not be likely to oxidize Ca2+-handling proteins located in the SR and/or plasma membrane.









Hydrogen peroxide, however, has a longer half-life and could leave the mitochondria to directly

interact with Ca2+-handling proteins.

If hydrogen peroxide is responsible for oxidation of cytosolic calcium handling proteins

during an IR insult, an important question emerges. That is, what is the fate of the excess

hydrogen peroxide produced due to the exercise-induced over-expression of MnSOD? As

discussed earlier, MnSOD converts superoxide to hydrogen peroxide. Therefore, it is probable

that an exercise-induced over-expression of MnSOD would result in a marked increase in

hydrogen peroxide, which has also been shown to exacerbate myocardial injury. Since we have

consistently observed a decrease, not an increase, in IR-induced oxidative modification of

proteins with exercise training, we reason that exercise must also over-express or up-regulated

the activity of one or more hydrogen peroxide scavenging systems within the myocardium. One

possibility is an elevation in either protein content and/or activity of the hydrogen peroxide

scavenger, catalase (12, 31). Another possibility is that exercise training increases the hydrogen

peroxide buffering capacity of the glutathione system through an increase in the amount of

glutathione protein and/or an increase in the amount of glutathione reductase protein or activity.

Any of these alterations would allow the cell to more effectively manage increased amounts of

hydrogen peroxide. Nonetheless, previous experiments have failed to confirm that exercise

training results in an increase in myocardial levels of catalase, glutathione peroxidase, or

glutathione (44, 72, 87, 96, 117). Hence, it seems likely that another mechanism exists in cardiac

myocytes to remove hydrogen peroxide. Two recently discovered molecules involved in the

removal of hydrogen peroxide from the mitochondria include both periredoxin III and

thioredoxin (5, 23, 34, 54, 112, 121). However, at present, it is unclear if one or both of these









antioxidants are exercise-induced in the heart. Clearly, this is an important topic for future

research.

Exercise-Induced Over-Expression of MnSOD Attenuates Calpain Activation

In addition to protecting the heart against ROS-mediated damage to Ca2+-handling

proteins, exercise-induced over-expression of MnSOD also attenuated the IR-induced increase in

calpain activation (Figure 4-7). This supports the idea that IR-induced calpain activation, is at

least in part, due to ROS-mediated damage to Ca2+-handling proteins. It is possible that the

oxidative modification of Ca2+-handling proteins contributes to IR-induced Ca2+-overload,

resulting in greater calpain activation.

Exercise-Induced Over-Expression of MnSOD Prevents the Degradation of Calcium-
Handling Proteins

MnSOD also appears to play a role in the exercise-induced reduction of Ca2+-handling

protein degradation during IR (Figure 4-10). Indeed, prevention of exercise-induced increases in

myocardial MnSOD via antisense oligonucleotides eliminated the exercise-induced protection

against IR-induced degradation of these important proteins. This finding is consistent with the

concept that the oxidation of these Ca2+-handling proteins may make them more susceptible to

cleavage by calpain.

Degradation of Calcium-Handling Proteins is Associated with Oxidation

As previously discussed, research links the IR-induced oxidation of Ca2+-handling proteins

with a loss of function both in-vitro as well as in the intact heart (62, 64, 129). Additionally

there is evidence supporting the idea that oxidation of these proteins may facilitate their

degradation by calpain (126, 130). Our experiments provide two lines of evidence to support

these ideas. First, strong correlations were observed between the amount of protein oxidation

and degradation of Ca2+-handling proteins following IR, suggesting that oxidized Ca2+-handling









proteins are more likely to be degraded (Tables 4-2, 4-3). Second, exercise-induced protection

against both the oxidation and degradation of Ca2+-handling proteins was almost completely

prevented by MnSOD antisense oligonucleotide treatment. This finding is consistent with the

notion that a causal relationship exists between the oxidative modification of Ca2+-handling

proteins and their degradation.

Summary and Future Directions

This project utilized two separate experiments to investigate the relationships between

ROS, Ca2+-handling protein oxidation and degradation as well as calpain activation during IR.

Major findings include the following: (1) IR results in contractile dysfunction and impaired

Ca2-handling, calpain activation and the oxidation and degradation of critical myocardial Ca2+-

handling proteins; (2) calpain degrades several important Ca2+-handling proteins during IR

including LTCC, SERCA2a, PLB, and NCX; (3) exercise training attenuates IR-induced

oxidation of these calcium handling proteins and preserves their levels in the heart; (4) exercise-

induced cardioprotection is critically dependant on an up-regulation of MnSOD. In combination,

these findings are consistent with the mechanistic series of events, which is depicted in Figure 5-

1. We postulate that IR results in an increase in mitochondrial superoxide production, which

leads to the oxidation of critical Ca2+-handling proteins, resulting in increased free cytosolic Ca2+

and calpain activation and finally the calpain-mediated degradation of critical Ca2+-handling

proteins.

The results of these experiments provide a unique contribution to the existing research

describing the mechanisms of IR injury and the mechanisms of exercise-induced

cardioprotection against IR injury. Indeed, these new data provide a mechanistic link connecting

the relationship between IR-induced ROS and Ca2+-overload. This work is also the first to

demonstrate calpain-cleavage of several critical Ca2+-handling proteins. Finally, our work









provides additional insight into the mechanisms of exercise-induced cardioprotection as well as

the cardioprotective effects of MnSOD. In fact, these experiments may suggest a possible

beneficial clinical role for the acute use of calpain inhibitors and/or mitochondria-targeted

superoxide scavengers.

Future research is needed to determine if exercise training elevates mitochondrial or

cytosolic antioxidants capable of removing hydrogen peroxide. Moreover, more work is required

to clarify the oxidation pathway connecting mitochondrial superoxide production to the oxidative

damage of Ca2+-handling proteins. Finally, the sequence of events involving the oxidation and

calpain-mediated degradation of Ca2+-handling proteins is not yet completely understood and

warrants additional research.


T Calcium Handling Protein
Damage / Cleavage


I IR Inju
Figure 5-1.Proposed mechanisms underlying the IR-induced increase in calpain activity and
myocardial dysfunction.










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BIOGRAPHICAL SKETCH

Joel French was born in Sioux Falls, South Dakota. He attained two bachelor degrees from

the University of Sioux Falls (Exercise Science and Psychology) along with two minors in

Computer Science and English Literature. During this time, Joel also taught Tae-kwon-do,

started a personal training business and worked as a youth director at the Sioux Falls YMCA.

Following graduation, he then worked for two years as a rehabilitation specialist at Central

Plains Clinic and McKennan hospital in Sioux Falls. Joel graduated with his masters in Exercise

Physiology from St. Cloud State University (St. Cloud, MN) in 1998. He then worked for a year

at the US Olympic Training Center in Lake Placid, NY, returning to Minneapolis, MN for

another two years working in cardiac and orthopedic rehabilitation. Finally, deciding to focus

his career in basic science, Joel began his doctoral work at the University of Florida in 2001,

studying the mechanisms of heart disease and protection against myocardial infarction.