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Exercise Training Protects Myocardial Mitochondria against Ischemia Reperfusion-Induced Injury

Permanent Link: http://ufdc.ufl.edu/UFE0022117/00001

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Title: Exercise Training Protects Myocardial Mitochondria against Ischemia Reperfusion-Induced Injury
Physical Description: 1 online resource (89 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Coronary artery disease (CAD) is a major contributor to morbidity and mortality. The primary pathological manifestation of CAD is myocardial injury due to ischemia-reperfusion (IR). Given the worldwide prevalence of CAD and the associated IR-induced cardiac injury, understanding the mechanisms of myocardial IR injury as well as developing countermeasures to provide cardioprotection against IR-induced damage is important. In this regard, regular bouts of endurance exercise training (ExTr) protect the heart against IR-induced injury. However, it is unclear whether ExTr-induced cardioprotection against IR injury is mediated through mitochondrial adaptations. These experiments tested the hypothesis that exercise training protects cardiac mitochondria against IR-induced injury. To test this hypothesis, hearts were isolated from exercise-trained and sedentary control animals. Subsequently, hearts were exposed to either IR or continuous perfusion using an in vitro isolated working heart system, and cardiac contractile profiles were measured. At the end of IR or continuous perfusion, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from the hearts to assess mitochondrial respiratory function and ROS generation. Our results reveal that ExTr protects the heart against IR injury as shown by higher levels of contractile performance during recovery from an IR insult. Moreover, ExTr protects mitochondria, reflected in a higher respiratory control ratio in both SSM and IFM. Additionally, ExTr prevents IR-induced elevation of mitochondrial ROS production and the release of proapoptotic proteins (i.e., cytochrome c and AIF) during an IR insult. Moreover, ExTr increases mitochondrial antioxidant capacity and protects cardiac mitochondria against IR-induced oxidative damage. Collectively, these novel findings reveal that ExTr protects IR-induced mitochondrial injury and provide new insight into the mechanism responsible for exercise-induced cardioprotection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022117:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022117/00001

Material Information

Title: Exercise Training Protects Myocardial Mitochondria against Ischemia Reperfusion-Induced Injury
Physical Description: 1 online resource (89 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Coronary artery disease (CAD) is a major contributor to morbidity and mortality. The primary pathological manifestation of CAD is myocardial injury due to ischemia-reperfusion (IR). Given the worldwide prevalence of CAD and the associated IR-induced cardiac injury, understanding the mechanisms of myocardial IR injury as well as developing countermeasures to provide cardioprotection against IR-induced damage is important. In this regard, regular bouts of endurance exercise training (ExTr) protect the heart against IR-induced injury. However, it is unclear whether ExTr-induced cardioprotection against IR injury is mediated through mitochondrial adaptations. These experiments tested the hypothesis that exercise training protects cardiac mitochondria against IR-induced injury. To test this hypothesis, hearts were isolated from exercise-trained and sedentary control animals. Subsequently, hearts were exposed to either IR or continuous perfusion using an in vitro isolated working heart system, and cardiac contractile profiles were measured. At the end of IR or continuous perfusion, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from the hearts to assess mitochondrial respiratory function and ROS generation. Our results reveal that ExTr protects the heart against IR injury as shown by higher levels of contractile performance during recovery from an IR insult. Moreover, ExTr protects mitochondria, reflected in a higher respiratory control ratio in both SSM and IFM. Additionally, ExTr prevents IR-induced elevation of mitochondrial ROS production and the release of proapoptotic proteins (i.e., cytochrome c and AIF) during an IR insult. Moreover, ExTr increases mitochondrial antioxidant capacity and protects cardiac mitochondria against IR-induced oxidative damage. Collectively, these novel findings reveal that ExTr protects IR-induced mitochondrial injury and provide new insight into the mechanism responsible for exercise-induced cardioprotection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022117:00001


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EXERCISE TRAINING PROTECTS MYOCARDIAL MITOCHONDRIA AGAINST ISCHEMIA REPERFUSION-INDUCED INJURY By YOUNGIL LEE 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 2008 1

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2008 Youngil Lee 2

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To the numerous teachers and professors who co ntributed to my education; and to my wife, children, parents, and friends for their continuous support 3

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ACKNOWLEDGMENTS This project could not have been completed without the cooperation of many individuals. Above all, I would like to convey my most h eartfelt appreciation to my committee chair and mentor, Dr. Scott Powers, for his unending s upport and encouragemen t throughout my doctoral program at the University of Florida. His enthus iasm, availability, and dedication to work with me on the problems we encountered with the projects were unwavering. Watching the commitment and enthusiasm that Dr. Powers brin gs to work each day has had a great impact on my scientific career. I would also like to acknowledge supervisor y committee. I thank Dr. Stephen Dodd for his assistance during my doctoral career. I thank Dr David Criswell for his willingness to discuss science and the project. I thank Dr. James Jessup for providing me with re search ideas that link basic science questions to clinical problems. Additionally, I would like to thank two past postdoctoral fellows, Dr. Karyn Hamilton and Dr. John Quindry for their guidance and help during my first years in the laboratory. Many of my coworkers also cont ributed to the success of this project. These individuals include Kisuk Min, Erin Talbert, Andreas Kavazis, Joseph McClung, Melissa Whidden, and Darin Falk. Most importantl y, I would like to express my special gratitude to my wife, Hyunjoo, and my children, Austin an d Rachel, who have always been my greatest source of support, encouragement, and strength. A portion of this degree belongs to them. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........8LIST OF FIGURES.........................................................................................................................9ABSTRACT...................................................................................................................................11CHAPTER 1 INTRODUCTION................................................................................................................. .132 REVIEW OF RELATED LITERATURE..............................................................................16Introduction................................................................................................................... ..........16Myocardial Ischemia-Reperfusion Injury...............................................................................16Levels of Injury...............................................................................................................16Events Leading to Myocardial Ischemia-Reperfusion Injury.........................................17Ischemia Reperfusion-induced Cellular Oxidative Stress: An Overview.......................18Myocardial protein oxidation...................................................................................19Myocardial lipid peroxidation..................................................................................19Sources of ROS.......................................................................................................................20Mitochondria and IR Damage................................................................................................21Mitochondrial Antioxidants....................................................................................................22Superoxide Dismutase (SOD).........................................................................................22Catalase............................................................................................................................23Glutathione Peroxidase (GPx).........................................................................................23Glutathione (GSH)...........................................................................................................23Peroxiredoxin 3 (Prx3)....................................................................................................24Thioredoxin 2 (Trx2).......................................................................................................24Thioredoxin Reductase 2 (TrxR2)...................................................................................24Mitochondrial Antioxidant Network......................................................................................25Two Subpopulations of Mitochond ria Exist in Cardiomyocytes...........................................25Exercise-Induced Cardioprotection against IR Injury............................................................26Endoplasmic Reticulum (ER) Proteins............................................................................26Cyclooxygenase-2 (COX-2) Activity..............................................................................27Elevated Myocardial Heat Shock Proteins......................................................................27Sarcolemmal ATP-dependent Potassium Channel (sarcoKATP)......................................28Exercise Protects the Heart against IR-induced Calpain Activation...............................28Improved Antioxidant Capacity......................................................................................28Exercise and Mitochondrial Protection..................................................................................29Summary.................................................................................................................................30 5

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3 METHODS...................................................................................................................... .......34Experimental Overview..........................................................................................................34Animal Model Justification.............................................................................................34Animal Housing and Diet................................................................................................34Experimental Design and Primary Dependent Measures................................................34General Methods.....................................................................................................................35Exercise Training Protocol..............................................................................................35In Vitro Isolated Working Heart Preparation..................................................................35In Vitro Ischemia-Reperfusion Protocol..........................................................................36Cardiac Performance Measurements...............................................................................37Isolation of Subsarcolemma l Mitochondria (SSM) and Interfibrillar Mitochondria (IFM)............................................................................................................................37Mitochondrial Respiration (O xidative Phosphorylation)................................................38Mitochondrial H2O2 Production......................................................................................39Assessment of Mitochondrial Protein Oxidative Damage..............................................40Assessment of Mitochondr ial Lipidperoxidation............................................................41Assessment of the Release of M itochondrial Proapoptotic Proteins...............................41Assessment of Mitochondrial Antioxidant Proteins........................................................41Data Analysis..........................................................................................................................424 RESULTS...................................................................................................................... .........44Animal Characteristics............................................................................................................44Myocardial Performance during IR........................................................................................44Myocardial Functional Characteristics............................................................................44Percent Recovery of Cardiac Work (CW: Systolic Pressure x Cardiac Output).............44Percent Recovery of +dp/dt and -dp/dt............................................................................45Mitochondrial Measurements.................................................................................................45Subsarcolemmal and Interfibrillar Mito chondrial Protein Yi eld and Integrity...............45Mitochondrial Oxidative Phosphorylation (State 3 and State 4) and RCR of SSM........46Mitochondrial Oxidative Phosphorylation (State 3 and State 4) and RCR of IFM.........46P/O Ratio.........................................................................................................................47Mitochondrial H2O2 Production......................................................................................47IR-induced Oxidative Modifi cations in Mitochondria....................................................47The Release of Mitochondrial Proapoptotic Proteins......................................................48Mitochondrial Antioxidant Proteins................................................................................485 DISCUSSION................................................................................................................... ......69Overview of Principal Findings..............................................................................................69ExTr Provides Cardioprotecti on against an IR Insult......................................................69ExTr Protects Mitochondrial Respiratory Function........................................................70ExTr Retards IR-induced ROS Production.....................................................................71ExTr Attenuates IR-induced Oxidative Damage to Mitochondria..................................72ExTr Reduces the Release of Proapoptotic Proteins.......................................................73Potential Mechanisms Responsible for ExTr-induced Mitoch ondrial Protection...........74 6

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Conclusions and Future Directions.........................................................................................75LIST OF REFERENCES...............................................................................................................77BIOGRAPHICAL SKETCH.........................................................................................................89 7

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LIST OF TABLES Table page 4-1 Animal body and heart weights.........................................................................................504-2 Functional Characteristi cs of isolated, perfused hearts during IR.....................................514-3 Mitochondrial protein yield...............................................................................................524-4 P/O ratio.................................................................................................................. ...........534-5 Mitochondrial ROS production during state 4 respiration.................................................54 8

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LIST OF FIGURES Figure page 2-1 Illustration of the three le vels of myocardial injury...........................................................322-2 Illustration of the events lead ing to IR-induced cellular injury.........................................322-3 Illustration of interaction of endogenous antioxidant systems in the mitochondria of cardiac myocytes................................................................................................................333-1 Experimental design examining the role of exercise training in mitochondrial protection following IR......................................................................................................434-1 Percent recovery of cardiac work (systolic pressure x cardiac output).............................554-2 Percent recovery rate of +dp/dt (left ventricular sy stolic function)...................................554-3 Percent recovery rate of -dp/dt (left ventricular diastolic function)..................................564-4 Electron microscopic photographs of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondr ia (IFM) isolated from a rat heart................................................564-5 The rate of state 3 respira tion of SSM using pyruvate/malate...........................................574-6 The rate of state 4 respira tion of SSM using pyruvate/malate...........................................574-7 Respiratory control ratio of SSM, using pyruvate/malate.................................................574-8 The rate of state 3 respir ation of SSM using succinate......................................................584-9 The rate of state 4 respir ation of SSM using succinate......................................................584-10 Respiratory control ratio of SSM, using succinate............................................................584-11 The rate of state 3 respirati on of IFM using pyruvate/malate............................................594-12 The rate of state 4 respirati on of IFM using pyruvate/malate............................................594-13 Respiratory control ratio of IFM, using pyruvate/malate..................................................594-14 The rate of state 3 respir ation of IFM using succinate.......................................................604-15 The rate of state 4 respir ation of IFM using succinate.......................................................604-16 Respiratory control ratio of IFM, using succinate.............................................................604-17 The levels of H2O2 production from SSM, using CK clamp.............................................61 9

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4-18 The levels of H2O2 production from IFM, using CK clamp..............................................614-19 The levels of protein carbonyl formation in SSM.............................................................624-20 The levels of 4-HNE products of IFM following IR.........................................................634-21 The levels of 4-HNE pr oducts in IFM following IR..........................................................634-22 Levels of cytochrome c released from cardiac mitochondria............................................644-23 Levels of AIF released from cardiac mitochondria...........................................................644-24 Protein levels of manganese supe roxide dismutase (MnSOD) in SSM.............................654-25 Protein levels of copper zinc supe roxide dismutase (CuZnSOD) in SSM........................654-26 Protein levels of thio redoxin 2 (Trx2) in SSM..................................................................664-27 Protein levels of thioredoxin reductase 2 (TrxR2) in SSM................................................664-28 Protein levels of manganese supe roxide dismutase (MnSOD) in IFM..............................674-29 Protein levels of copper zinc supe roxide dismutase (CuZnSOD) of IFM.........................674-30 Protein levels of thioredoxin reductase 2 (TrxR2) in IFM................................................68 10

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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 EXERCISE TRAINING PROTECTS MYOCARDIAL MITOCHONDRIA AGAINST ISCHEMIA REPERFUSION-INDUCED INJURY By Youngil Lee May 2008 Chair: Scott K. Powers Major: Health and Human Performance Coronary artery disease (CAD) is a major contributor to morbidity and mortality. The primary pathological manifestati on of CAD is myocardial injury due to ischemia-reperfusion (IR). Given the worldwide prevalence of CAD and the associated IR-induced cardiac injury, understanding the mechanisms of m yocardial IR injury as well as developing countermeasures to provide cardioprotection against IR -induced damage is important. In this regard, regular bouts of endurance exercise training (ExTr) protect the heart against IR-indu ced injury. However, it is unclear whether ExTr-induced cardioprotection against IR injury is mediated through mitochondrial adaptations. These experiments te sted the hypothesis th at exercise training protects cardiac mitochondria agai nst IR-induced injury. To test this hypothesis, hearts were isolated from exercise-trained and sedentary control animals. S ubsequently, hearts were exposed to either IR or conti nuous perfusion using an in vitro isolated working heart system, and cardiac contractile profiles were measured. At the end of IR or continuous perfusion, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (I FM) were isolated from the hearts to assess mitochondrial respiratory function and ROS generation. Our results re veal that ExTr protects the heart against IR injury as s hown by higher levels of contract ile performance during recovery from an IR insult. Moreover, ExTr protects mitochondria, reflected in a higher respiratory 11

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control ratio in both SSM and IFM. Additionally, ExTr preven ts IR-induced elevation of mitochondrial ROS production and the release of pr oapoptotic proteins (i.e., cytochrome c and AIF) during an IR insult. More over, ExTr increases mitochondrial antioxidant capacity and protects cardiac mitochondria against IR-indu ced oxidative damage. Collectively, these novel findings reveal that ExTr protects IR-induced mi tochondrial injury and provide new insight into the mechanism responsible for exercise-induced cardioprotection. 12

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CHAPTER 1 INTRODUCTION Coronary artery disease remains a major cause of death in the Western world. The primary pathological manifestation of co ronary artery disease is myocardial damage due to ischemiareperfusion (IR) injury. The level of IR-induced myocardial injury can range from a small insult resulting in limited myocardial damage to a large injury culminating in myocyte death. Although IR-induced myocardial inju ry occurs due to the complex interaction of many factors, elevated production of reactive oxygen species (ROS) plays an im portant role in IR-induced myocardial injury (2, 13, 34, 102, 115). In this regard, mitochondria are targets and sources of incr eased ROS generation and thus induce oxidative damage and cell death during IR (15, 30, 31, 36, 88). Moreover, mitochondria have been shown as the mediators of life a nd death because damaged mitochondria release proapoptotic proteins that initiate cell d eath (32, 49, 60, 70, 90). Additionally, it has been demonstrated that an in vitro anoxia-reoxygenation challenge to mitochondria increases oxidative modification of mitoc hondrial proteins and lip ids (9). Collectively, it appears that IRinduced mitochondrial damage leads to cardiac c ontractile dysfunction, cellular damage and cell death. For this reason, developing countermeasures to protect mito chondria against IR injury is important to attenuate myocardial injury. Currently, the only practical strategy to protec t the heart against IR injury is endurance exercise training (ExTr). Indeed, regular exerci se training has been known to protect the heart against all levels of IR-in duced injury including myocar dial infarction (16, 42, 43, 47, 65, 66, 117, 118, 121, 123, 135, 143). Although the detailed mechanisms responsible for exerciseinduced cardioprotection remain unclear, numerous potentially cardioprotective candidates exist: increased myocardial levels of heat shock pr otein 72 (HSP72), increased endoplasmic reticulum 13

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(ER) stress proteins, elevated cyclooxygenase-2 (COX-2) act ivity, increased sarcolemmal potassium channels (sarcoKATP), increased mitochondrial potassium channels (mitoKATP), and improvements in cardiac antioxidant capacity. Of these proposed cardi oprotective candidates, studies suggest that HSP 72, ER proteins, and COX-2 activity are not essential to acquire exercise-induced cardiopro tection (65, 108, 122, 123, 135). In contrast, recent evidence suggests that Ex Tr-mediated cardioprotection is associated with improvement of myocardi al antioxidant capacity ( 42, 43, 47, 121). Specifically, these studies suggest that an ExTr-mediated increase in myocardial manganese superoxide dismutase (MnSOD), an endogenous mitochondrial antioxida nt enzyme, plays a crucial role in cardioprotection against IR inju ry (66, 143). For example, these studies demonstrate that when exercise-mediated increase in MnSOD is inhibi ted, exercise-induced cardi oprotection against IR insult is significantly reduced (66, 143). Consid ering the localization of MnSOD in mitochondria, it appears that ExTr may conf er mitochondrial protection against IR-induced oxi dative injury, which may lead to cardioprotection. Curre ntly, it is unknown whether ExTr provides mitochondrial protection against an IR insult. Furt hermore, it has not been investigated if ExTr protects either or both of the two morphologically and biochemica lly different subpopulations of mitochondria (i.e., subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM)). The purpose of the current experiments was to investigate whether ExTr confers mitochondrial (both SSM and IFM) protection against IR injury. Our hypothesis was that ExTr would protect myocardial mitochondria (SSM and IFM) ag ainst IR-induced respir atory dysfunction and oxidative injury and retard IR-induced mito chondrial ROS generati on and the release of proapoptotic proteins. We also postulated that if our hypothesis was correct, ExTr-mediated 14

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mitochondrial protection would be associated wi th exercise-induced elevation of mitochondrial antioxidants. 15

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CHAPTER 2 REVIEW OF RELATED LITERATURE Introduction Coronary artery disease (CAD) remains a major cause of death in the Western world and industrialized nations. The primary pathological consequence of CAD is myocardial damage due to ischemia-reperfusion (IR ) injury (124). The level of IR-in duced myocardial injury can range from a small insult resulting in limited myocardi al damage to a large injury culminating in myocardial cell death. Given the worldwide prev alence of CAD and the associated IR-induced cardiac injury, understanding the mechanisms of myocardial IR injury as well as developing countermeasures to provide cardioprotection against IR-induced damage is important. In this regard, numerous approaches to achie ve cardioprotection against IR injury have been investigated. To date the only practical and sustainable strate gy capable of providing cardioprotection is regular bout s of endurance exercise. Specifi cally, human epidemiological studies demonstrate that regular exercise reduces the risk of d eath during myocardial IR insult (73). Moreover, animal studies confirm that re gular bouts of aerobic exercise (i.e., treadmill running or swimming) protect the heart from IR-induced injury (9, 17, 19, 42, 43, 47, 66, 67, 83, 84, 96, 130, 131, 135, 136, 143). The objectives of this review are two-fold. The mechanisms responsible for IR-induced myo cardial injury are reviewed, and subsequently the current knowledge about the effects of exercise trai ning in providing cardiop rotection against IRinduced injury is reviewed. Myocardial Ischemia-Reperfusion Injury Levels of Injury Depending upon the duration of ischemia, three levels of IR-induced myocardial injury have been described (Figure 2-1). In general, re perfusion after 1-5 minutes of ischemia can result 16

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in ventricular tachycardi a or fibrillation without cell death or a deficit in ventricular contractile performance (44). Reperfusion after an ischemic period of 5-20 minutes results in the second level of myocardial injury known as myocar dial stunning(13, 44). My ocardial stunning is characterized by a deficit in myocardial contractility that occurs without myocardial cell death. Typically, IR-induced myocardial st unning results in ventricular cont ractile deficits lasting 24-72 hours after the IR event (13). The third and highest level of IR injury occurs when ischemia is extended beyond 20 minutes. In these circumstances cardiac myocytes become irreversibly damaged, resulting in cell death (i.e., myocardial infarction)(44). It is now clear that IR-induced cardiac myocyte cell death occurs due to both ap optosis and necrosis and that mitochondrial injury plays a major role in both forms of cell death (52, 70, 110). Events Leading to Myocardial Ischemia-Reperfusion Injury Despite the complexity in the mechanism(s) responsible for the IR-induced myocardial damage, essential factors leading to IR-induced cellular injury have been described (Figure 2-2). Evidence indicates that several intertwined factors, including a decrease in cellular ATP levels, production of reactive oxygen species (ROS), accumulation of hydrogen ions, generation of reactive nitrogen species (RNS), calcium overload, and calpain activ ation contribute to IR injury (51, 57, 69, 129, 149, 151). Collectively, these factors promote cellular injury and subsequent cardiac myocyte death. Importantly, the oxyradical theory of IR-indu ced myocardial injury was introduced in 1985 (102). This theory proposed that the elevation of ROS and free radical (i.e. superoxide anion, hydrogen peroxide, and hydroxyl ra dical) production during both ischemia and reperfusion contributes to myocardial inju ry. It has been also confirme d by using electron paramagnetic resonance that ROS production is highly elev ated during IR (6, 148, 150). Furthermore, ROS production in re-oxygenated cells is increased and the amount of ROS depends upon the duration 17

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of both anoxia and reoxygenation (10). A detailed discussion of ROS-induced oxidative stress follows. Ischemia Reperfusion-induced Cellula r Oxidative Stress: An Overview Radicals are chemically reactive molecules due to an unpaired electron. Superoxide anions are oxygen-derived radicals and can be produced during IR from the one electron reduction of molecular oxygen (102). Super oxide production can lead to the formation of many other ROS/RNS including the hydroxyl ra dical, hydrogen peroxide, a nd peroxynitrite (14, 44, 50, 63, 94, 102, 145, 147). Although radicals and other ROS can promote several types of cellular damage, two classes of biomolecules that are ta rgets for oxidant-induced damage are proteins and lipids. Radical-mediated oxidation of ami no acids can impair cellu lar protein function by altering their biochemical structure. Further, ox idized proteins become targets for proteolytic degradation; hence, IR-induced oxida tive injury to proteins within cardiac myocytes is associated with accelerated protein breakdown (26, 39, 40, 137). Lipid damage in cells can occur by radical species reacting with polyunsatur ated fatty acids in membrane s which results in propagation reactions and the formation of new radicals (63, 144). Lipid peroxi dation of the cellular membrane can result in altered fluidity a nd increased membrane permeability (63, 145). Experimental evidence for the involvement of ROS/RNS in myocardial IR injury includes detection of protein oxidation, lip id peroxidation, and protein nitr ation products in reperfused hearts (1, 42, 67, 121, 149). Furthermore, the importance of ROS-mediated damage to the heart after an IR insult has been confirmed by studi es indicating that an tioxidants can provide myocardial protection against IR-induced injury (66, 67, 143). Interestingly, it has been recently reported that ROS-mediated oxida tive stress also contributes to the calcium overload observed during IR (22) indicating that many of the inju rious effects of ROS on the myocardium reflect activation of the calcium dependent protease, calpain (26, 75, 128). 18

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Myocardial protein oxidation Many cardiac proteins can be oxidatively modified by free radi cals in the heart during IR. Included in the list of target proteins are enzyme s, structural proteins, contractile proteins, and membrane-bound proteins (64, 68, 126, 127). Of ten, the damage caused by free radical interaction is irreparable. In addition, many pr oteins which are oxidati vely modified become more susceptible to cellular prot eolytic pathway by way of calpain and proteosome (111). One example of oxidative modificati on to cellular proteins is the formation of carbonyl groups which can then be measured and provide an indirect indicati on of oxidative stress within the cell (20). Myocardial lipid peroxidation Polyunsaturated fatty acids are highly suscep tible to free radical modification at their unsaturated sites (4). ROS pull out electrons or hydrogen atoms from the methylene groups of fatty acids, and this reaction initiates a chain reaction where one modified fatty acid chain reacts with a neighboring chain. 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 necros is and/or apoptosis. Lipid peroxidation byproducts can be measured and used as a marker of oxidative stress within the cell (64). One such by-product is the formation of the reactive alde hyde, 4-hydroxy-2-nonenal (HNE). The omega-6 acyl groups of polyunsaturated fatt y acids (i.e., linoleic and arachi donic acids) produce HNE as a result of free radical attack. HNE is a highl y reactive compound and can react with several functional groups on biological material, particular ly sulfydryl groups, to form thioester adduct and then hemiacetals. HNE may also react with histidine and lysine residues of proteins to form stable -unsaturated carbonyl compounds. In addition, HNE-modification of proteins may impair biological functions. A discussion of the primary sources of ROS in cardiac myocytes during IR follows. 19

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Sources of ROS There are several potential sources of free ra dical production in the heart during IR. Major sources include: electron leak fr om the mitochondrial respiratory complexes, xanthine oxidase, enzymatic arachadonic acid oxygenation, the synt hesis of nitric oxide, catecholamine oxidation and oxidative burst from neut rophils (12, 24, 27, 44). Although each may play a role in IR damage, a large volume of evidence implicates mitochondrial production of radicals as the primary source of oxidants during both isch emia and reperfusion (30, 32, 34, 70, 86, 87) Therefore, a detailed discussion of mitochondrial ROS production follows. Mitochondria constitute about 30% of car diomyocyte volume and act as the cells powerhouses. They predominantly supply the en ergy (i.e., adenosine triphosphate (ATP) and phosphocreatine) required for cardiac muscle cont raction and relaxation. M itochondrial oxidative phosphorylation is the major ATP synthetic path way in eukaryotes. During this process, electrons liberated from reduced forms of ni cotine amide dinucleotides (NADH) and flavin amide dinucleotides (FADH) are delivered to oxygen via electron trans port chain (ETC). The ETC is composed of five complexes and creates an H+ gradient across the inner mitochondrial membrane. The electrochemical energy of this gr adient generated between intermembrane space and matrix is then utilized to drive ATP s ynthesis by complex V (ATP synthase). In these processes, it has been estimated that a pproximately 1-2% of the oxygen consumed by mitochondria is altered to superoxide due to th e escape of electrons from the chain (54). More specifically, it is believed that the majority of the superoxide is produced by combining electrons escaped from complex I and complex III with oxyge n (89). Importantly, the levels of superoxide from mitochondria are significantly elevated duri ng IR (3, 32). For this reason, mitochondria are considered as a major source of ROS production during IR. 20

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Mitochondria and IR Damage Mitochondria are both targets and sources of oxidative damage. As illustrated in figure 2-2, mitochondrial damage is induced by two major fact ors. For example, an IR insult can damage mitochondria via calcium overload and overproduc tion of ROS. Indeed, it is believed that mitochondria are associated with calcium homeos tasis and therefore are strategically located near the Ca2+ release channel (i.e., L-type Ca2+ channel) and endo/sarcop lasmic reticulum of cardiomyocytes. However, over-influx of Ca2+ into the mitochondria (e.g., IR) leads to the opening of mitochondrial permeability transition pore (mPTP) and dissipates membrane potential (41, 49, 82, 105, 107). As a result, potential oxidants such as superoxide, hydrogen peroxide, hydroxyl radicals, peroxyl radicals, and peroxynitr ite are generated via a series of chemical reactions (14, 44, 50, 63). Subsequen tly, these oxidants contribute to further impairing the ETC and a component of mPTP, cyclophilin D (11). The resulting damage initiates apoptosis by releasing pro-apoptotic proteins (11, 59). Indeed, it has been demonstrated that mitochondria after an IR insult increase ROS production, reduce oxidative phosphorylation, and release proapoptotic proteins (i.e cytochrome c, apoptosis inducing fact or (AIF)) into th e cytosol (32, 56, 77, 88, 90). Under normal conditions, cytochrome c is confin ed at the inner membrane of mitochondria by nonionic/electrostatic interact ions, and AIF is localized in the intermembrane space of mitochondria. However, mitochondrial membrane damage or opening of mPTP due to an IR insult causes the release of cytochrome c and AIF into the cy tosol from mitochondria initiating genomic DNA fragmentation a nd propagating cell death (52, 55) The cell death in which mitochondrial proapoptotic proteins are involved include both caspase-dependent and caspaseindependent pathways. Activation of the caspa se-dependent pathway occurs due to the translocation of cytochrome c to the cytosol activating caspase-9. Activated caspase-9 then 21

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activates caspase-3. Activated ca spase-3 then translocates to nucleus where it cleaves the DNA repairing enzyme, poly (ADP-ribose) polymerase (PARP) and activates endonucleases which cleave DNA. These events finally lead to cell death. On the other hand, the caspase-independent cell death pathway is initiated by AIF. For exam ple, AIF released from damaged mitochondria translocates to the nucleus and initiates apoptosis by cleaving chromatin and inducing large-scale DNA (~50 kbp) fragmentation in the absence of caspase activation (21, 38, 133). Collectively, given the mitochondrial vulnerabil ity to oxidative stress and Ca2+ overload and initiation of cell death signals during IR insult, mitochondrial dama ge appears to be a prime causative factor in myocardial injury and infarction (cell death) (32, 37, 52, 53, 55, 56, 88-91, 93). Although elevated ROS production from mitochondria is consid ered as a key factor in myocardial injury, mitochondria contain endogenous an tioxidants that react against ROS. Ther efore, the following section discusses mitochondrial antioxidants. Mitochondrial Antioxidants An antioxidant is characterized as a molecule capable of delaying or preventing oxidation of other molecules (62). A multitude of antioxi dants exists in mitochondria to provide a protective network against ROS injury (Figure 2-3). Primary enzymatic antioxidants include superoxide dismutase (SOD), glut athione peroxidase (GPx), cata lase, peroxiredoxin 3 (Prx3), thioredoxin 2 (Trx2), and thioredoxin reductase 2 (TrxR2). In addition, the non enzymatic antioxidants glutathione exists in mitochondria. The following discussion addresses the roles and function of individual antioxidants of mitochondria. Superoxide Dismutase (SOD) SOD was discovered in 1969 by McCord and Fridovi ch (103). SOD is characterized as an oxidoreductase and is a metalloenzyme containi ng metal ions in addition to amino acids. Two isoforms of SOD exist in mitochondria: manganese SOD (MnSOD) and copper-zinc SOD 22

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(CuZnSOD). MnSOD is located in the matrix, and CuZnSOD resides in intermembrane space (132). In mitochondria, SOD is to dismutates su peroxide into hydrogen pe roxide which is less reactive and non-radical. Therefore, both Mn SOD and CuZnSOD reduce mitochondrial free radical damage. The chemical reaction of SOD with superoxide is outlined below: O2 + O2 + 2H+ + SOD H2O2 + O2 Catalase Catalase is an antioxidant enzyme composed of four polypeptide chains containing four porphyrin heme groups that permit the enzyme to decompose hydrogen peroxide (139). The reaction of catalase in detoxifi cation of hydrogen peroxide is 2H2O2 + catalase 2H2O + O2 Glutathione Peroxidase (GPx) Glutathione peroxidase is a selenium-containing tetrameric glycoprotein that catalyzes the decomposition of hydrogen peroxide to two molecules of water. Although GPx has a much lower Km than catalase, GPx is more ubiquitous such that it also plays a critical role in reducing oxidative stress in mitochondria. The reaction re lies on the oxidation of reduced glutathione (GSH), producing oxidized glutathione (GSSG). A reaction in which GPx catalyzes the decomposition of hydrogen peroxide is H2O2 + 2GSH GSSG + 2H2O Glutathione (GSH) GSH is a tri-peptide containi ng a thiol group linked by cysteine and glutamate side chain. GSH exists in reduced and oxidized forms. GSH reduces any disulfide bonds formed by oxidative stress by donating electron from the thio l group. The thiol group of glutathione can be kept in a reduced form by a glutathione reductase that is constitutively active and inducible upon 23

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oxidative stress. In the reduced state, the thiol group of cystei ne donates a proton and an electron to other oxidized molecules. Importantly, since very little CAT resides within the mitochondria, GSH, in conjunction with Gpx, is the major mean s of decomposition of hydrogen peroxide in the mitochondria (104). Peroxiredoxin 3 (Prx3) Prx3 is a mitochondrial-specific antioxidant enzyme localized in the mitochondrial matrix that functions as an antioxidant enzyme removing mitochondrial H2O2 (23, 25). Indeed, the high abundance of Prx3 (~30 times more abundant than GPx) in mitochondria supports the importance of Prx3 in mitochondrial H2O2 detoxification (25). This reaction relies on the oxidation of reduced thioredoxin 2 (Trx2 red.) to oxidized thioredoxin 2 (Trx2 oxi.). A reaction where Prx3 catalyzes decompos ition of hydrogen peroxide is H2O2 + Trx 2 red. + Prx 3 H2O + Trx 2 oxi. Thioredoxin 2 (Trx2) Trx2 is an antioxidant protei n containing a dithiol-disulfid e active site. This enzyme facilitates the reducti on of other proteins linked by a disulfide bond by donating an electron (oxidation). Trx2 in conjunction with Prx3 plays an important role in removing hydrogen peroxide in mitochondria. Thioredoxin Reductase 2 (TrxR2) Maintaining reduced state of Trx2 is importa nt for Trx2 to continue to work as an antioxidant under the oxidative stress phase. Theref ore, reduced Trx2 should be regenerated from oxidized Trx2. TrxR2 is the only known enzyme th at reduces oxidized Trx2 in mitochondria via NADPH-dependent reaction (109). In this regard, TrxR2 contribut es to reducing the accumulation of hydrogen peroxide in mitochondria. 24

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Mitochondrial Antioxidant Network It is clear that mitochondrial antioxidants work as a unit to remove ROS precursors (i.e. O2 H2O2) (Figure 2-3) before they form more r eactive radicals (i.e. hydroxyl radical (HO.) and peroxyl radical (HOO.)). Namely, O2 -. molecules are dismutated by SOD to yield hydrogen peroxide and oxygen. The resulting hydrogen peroxide can be decomposed by actions of catalase, GPx, and Prx3 at the expense of GSH and Trx2, re spectively. With this antioxidative pathway, mitochondrial redox balance is maintained. Two Subpopulations of Mitochondr ia Exist in Cardiomyocytes Mitochondria of cardiomyocytes are composed of two spatially and functionally distinct populations. One is located under the sarcolemma referred to as subsarcolemmal mitochondria (SSM), and the other is interspa ced in contractile muscle fibe rs, defined as interfibrillar mitochondria (IFM). Morphological, biochemical, and functional differences between SSM and IFM have been reported (32, 89, 125). It has also been demonstrated that SSM and IFM respond differently to an in vitro IR insult (32) and aging (45). Ind eed, it is reported that IFM has an about 1.5 fold higher respirator y capacity (oxidative phosphoryla tion) and specific enzyme activities compared with SSM (114). It has been recently proposed with the use of advanced electron microscopic techniques that the structural difference in cristae may conf er distinct biochemical differences. For example, the more compact tubular structure of cristae in IFM may cont ribute to facilitating higher concentrations of H+ resulting in higher oxidative phosphoryl ation. It is also assumed that differences in the electron transport chain co mplexes may be involved in the biochemical difference between SSM and IFM (125). 25

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Exercise-Induced Cardioprotection against IR Injury It is well established that regular bouts of endurance exercise training (ExTr) is cardioprotective agai nst IR insult (17, 19, 42, 43, 47, 6567, 71, 83-85, 118-121). Indeed, ExTR protects the heart agai nst IR-induced oxidative injury, calci um overload and ce ll death (47, 121). Interestingly, short-term exercise training (i.e. 3-5 consecutive days) prov ides the same level of cardiac protection as that observed following lo ng-term training (i.e.10 weeks) (42, 43, 47, 83, 121). Recent findings indicate that ExTR-induced upregulation of antioxidants and heat shock proteins are associated with cardioprotect ion (19, 42, 43, 72, 106, 143). In addition, a recent study from our laboratory demonstrated that shor t term ExTR reduces calcium overload-induced cellular damage (47). Moreover, it has been repo rted that ExTR protects the heart against IRinduced apoptosis as evidenced by the observation that ExTR decreases the level of caspase-3 activity and TUNEL (terminal deoxynucleotidyl tran sferase nick end labe ling) positive stained nuclei. Nevertheless, the pathways responsible for ExTR-mediated cardioprotection against IR remain unclear. In this regard, the following sections will address possible mechanisms for exercise-mediated cardioprotection. Endoplasmic Reticulum (ER) Proteins Emerging evidence suggests that IR induces ER stress resulting in ER dysfunction. IRinduced ER dysfunction is involved in mitochondria-dependent and independent cell death due to disturbances in calcium homeostasis (142). Two proteins (GRP 78 and GRP 94) that are localized in ER and regulated by glucose are reported to protect the heart against IR-induced cell death (101). Therefore, it is possible that an ExTr-induced upregulation of GRP 78 and GRP 94 could induce cardioprotection. However, a recent study reveals that ExTr (short-term) does not elevate the ER proteins (108). Ther efore, it appears that ER proteins are not required to attain ExTr-induced cardioprotection. 26

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Cyclooxygenase-2 (COX-2) Activity COX-2 is the rate-limiting enzyme convert ing arachidonic acid to prostanoid. The beneficial actions of COX-2 on the heart appear to stem from prostaglandin (PGE2) and/or prostacyclin (PGI2) production. For example, the elevated PGE2 and PGI2 production protects cardiomyocytes against oxidative stress (1). None theless, a recent study reveals that ExTr does not elevate COX-2 levels in the rat heart (122). Therefore, it app ears that increases in cardiac levels of COX2 are not required to attain ExTr-induced cardioprotecti on against IR injury. Elevated Myocardial Heat Shock Proteins Heat shock proteins (HSP) are a multifunctiona l group of proteins which can be induced by a variety of stimuli such as heat, oxidative stress, calcium overload, ex ercise training, and low pH. Once active, these proteins serve several functions within the ce ll, including chaperoning and/or transporting proteins, fo lding and refolding proteins, s cavenging free radicals, and even facilitating protein synthesis (80, 97). Evidence exists that the eleva tion of HSP can confer cardioprotection against IR injury (99, 116). In addition, the cardioprotective properties of various HSP have been demonstrated. However, the importance of HSP to exercise-mediated cardioprotection has been controversial. Fo r example, exercise training in a warm 22 C environment elevated myocardial levels of HSP 90, HSP 72, and HSP 40 and concomitantly provided cardioprotection against IR injury. Howeve r, recent studies reveal that exercise training in a cold environment (5 C) where exercise-induced increases in HSP were prevented provides cardioprotection against an in vitro IR insult without an eleva tion of myocardial HSP (43, 123, 135). These results suggest that an elevation in myocardial HSP (HSP 10, HSP 27, HSP 40, HSP 60, HSP 72, HSP 73 or HSP 90) is not essential fo r exercise-mediated cardioprotection. In this 27

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regard, following section will discuss other possible mechanisms of exercise-mediated cardioprotection. Sarcolemmal ATP-dependent Potassium Channel (sarcoKATP) SarcoKATP is localized in the sarcolemma of cardiac myocytes. Emerging evidence indicates that opening of sarcoKATP during IR protects the heart ag ainst an IR insult by reducing cellular calcium overload (58, 79). N onetheless, the role that sarcoKATP plays in ExTr-induced cardioprotection remains to be investigated. To date, only one study reveals that ExTr increases sarcoKATP and when it was inhibited by a pharmacologi cal inhibitor, ExT r-induced reduction in cell death (i.e., infarct size) was compro mised (19). A recent study using cultured cardiomyocytes suggests that a sarcoKATPinduced protective mechanism against oxidative stress is accompanied by mitochondrial protection (i.e., i nhibition of mPTP opening) (100). Exercise Protects the Heart agains t IR-induced Calpain Activation It has been reported that IR leads to activa tion of the calcium-activated protease calpain, resulting in myocardial damage (78). On the other hand, ExTr has been shown to decrease calpain activation following IR (47, 121). These studies suggest that exercise may confer cardioprotection through the regulation of IR-induced calpa in activation. However, the mechanisms responsible for exercise-induced redu ction in calpain activation during IR remain unclear. One possibility is that exercise-induced increases in antioxidative capacity may reduce oxidative damage to Ca2+-handling proteins resulting in the maintenance of intracellular Ca2+ homeostasis and regulating calpain activation (French et al, a ccepted in FASEB journal, 2008). Improved Antioxidant Capacity Recent evidence indicates that over-expression of myocardial antioxidants (i.e. MnSOD) via transgenic animal generation or gene thera py attenuates IR-induced m yocardial infarction (34, 95, 141). In addition, a recent study demonstrated th at using a mitochondria-targeted antioxidant 28

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(Mito Q) results in cardioprotec tion against an IR insult (2). These results demonstrate that oxidative injury is a significan t contributor to myocardial IR injury. In this regard, growing evidence suggests that enduran ce exercise (both short-te rm and long-term) provides cardioprotection by improving myocardial antioxidant capacity. Specifically, studies indicates that exercise elevates myocardial levels of GSH and activities of Mn SOD and catalase (43, 117, 118). As discussed earlier, MnSOD may play an important cardioprotective role in the heart during IR. Its localization to the mitochondrial ma trix and ability to pr event oxidative stress induced by mitochondrial superoxide production indicates the importa nce of mitochondrial antioxidant capacity. Indeed, seve ral studies have demonstrated the cardioprotective effects of exercise-mediated improvement of MnSOD. For example, Yamashita et al. (34, 143) demonstrated that the prevention of exercise-m ediated increases in MnSOD (using an antisense oligodeoxyribonucleotide to MnSOD; ASODN-MnSOD) reduced exercise-induced cardioprotection. Further, Hamilton et al. (66) also showed th at the prevention of exercisemediated increase in MnSOD (using AS-ODN-M nSOD) attenuated the protection against IRinduced arrhythmia. It is unknown whether othe r antioxidants (i.e., Pr x3, Trx2 and TrxR2) are upregulated by ExTr and if they are associated with ExTr-induced cardioprotection against an IR injury. Exercise and Mitochondrial Protection It is well established that mitochondrial resp iratory dysfunction and damage due to an IR insult lead to cardiac injury (32, 88, 91). In contrast, recent evidence suggests that ExTr attenuates the da maging effects of in vitro anoxia-reoxygenation, calcium overload, and apoptotic stimuli on isolated ca rdiac mitochondria (8, 9, 76, 130) These studies demonstrated that ExTr reduces H2O2 production and the release of proa poptotic proteins and sustains 29

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respiratory function of cardiac mitochondria. Hence, it is feasible that ExTr-induced cardioprotection may be attained by mito chondrial protection against IR injury. Summary Myocardial IR injury resulting from coronary artery disease remains a primary cause of morbidity and mortality in indus trialized nations. Given the worldwide prevalence of IR-induced cardiac injury, understanding the mechanisms of myocardial IR injury as well as developing countermeasures to provide car dioprotection against IR-induced damage is important. Despite the complexity in the mechanism(s) responsible for the IR-induced myocardial damage, recent evidence suggests that overproductio n of ROS and calcium overload play a critical role in IRinduced myocardial injury. In th is regard, it is well documented that mitochondrial damage and dysfunction due to IR result in elevation of ROS production a nd contribute to IR-mediated myocardial oxidative injury a nd cell death. Additionally, it is not able that calcium overload in mitochondria plays an important role in me diating the production of ROS and initiating mitochondria-mediated apoptosis. Currently, the only practical strategy to protect the heart against IR-induced myocardial injury is ExTr. Indeed, it has been well documented that ExTr protects the heart against all levels of IR induced injury including myocardial infarction. Although the detailed mechanisms responsible for exercise-induced cardioprot ection remain unclear, numerous potentially cardioprotective candidates ex ist: HSP72, ER stress protei ns, COX-2 activity, elevated sarcoKATP and improvements in cardiac antioxidant cap acity. Of these proposed cardioprotective candidates, studies suggest that HSP 72, ER prot eins, and COX-2 activity are not essential to acquire exercise-induced cardioprotec tion. ExTr-induced increase in sarcoKATP and reduction in calpain activation are reported to contribute to cardioprotectio n. Importantly, recent evidence suggests that ExTr-mediated improvement of myocar dial antioxidant capacity plays a key role in 30

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cardioprotection. Specifically, these studies suggest that th e ExTr-mediated increase in myocardial MnSOD is a crucial factor inducing cardioprotection agains t IR injury including myocardial infarction. Collectively, these positiv e potential mechanisms appear to confer mitochondrial tolerance or adap tation to IR-induced oxidative injury, which may result in cardioprotection. Currently, it is unknown whether ExTr provi des mitochondrial protection against an IR insult, which may be a probable ca rdioprotective pathway. Furthermore, it has not been investigated whether ExTr protects either one or both of the subpopulations of mitochondria. 31

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0 5 20 Ischemia duration (minutes) Arrythmias Myocardial Stunning Infarction Levels of myocardial injury Figure 2-1. Illustration of the three levels of myocardial injury. L-type Ca2+ channel Caspase-9 Caspase-3 Cytc Cytc SERCA 2a calpain ROS [Ca2+] ROS B i d t B i dCa2+Lactate + H + N a2+ H + e x c h a n g e r Na2+H+Na2+ N a2+ C a2+ e x c h a n g e r Ca2+Ca2+ Na2+ Membrane damage m [Ca2+] Cytc ROS C y P D A N T V D A C A I F A I F ROS Ca2+Ca2+Ca2+ Ca2+ L-type Ca2+ channel Caspase-9 Caspase-3 Cytc Cytc SERCA 2a calpain ROS [Ca2+] [Ca2+] ROS B i d t B i dCa2+Lactate + H + N a2+ H + e x c h a n g e r Na2+H+Na2+ N a2+ C a2+ e x c h a n g e r Ca2+Ca2+ Na2+ Membrane damage m m m [Ca2+] Cytc ROS C y P D C y P D A N T V D A C A I F A I F ROS Ca2+Ca2+Ca2+ Ca2+ Figure 2-2. Illustration of the events leading to IR-induced cellular injury. 32

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NADPH NADP+ NADPH NADP+ H2O H2O H2O2 Catalase MnSOD CuZnSOD Gpx GR Prx3 Trx2 red Trx2 ox Trx R2 ONOOOxidative damage HO O2 GSH GSSG H2O NO Fe 2+ Figure 2-3. Illustration of intera ction of endogenous antioxidant sy stems in the mitochondria of cardiac myocytes. 33

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CHAPTER 3 METHODS These experiments tested the hypothesis that en durance exercise training will attenuate IRinduced mitochondrial injury in cardiac myoc ytes. This hypothesis was addressed by the experiments outlined in this section. This section is subdivided into two segments. The first segment will describe the animal model and expe rimental design. Note that dependent measures will be mentioned by name only. A general methods section will follow to provide methodological details. Experimental Overview Animal Model Justification Adult (3-5 months old) male Sprague-Dawley (S D) rats were used for these experiments. The SD rats were chosen for several reasons: fi rst, the invasive nature of these experiments precludes the use of human subjects. Second, the SD rat model is a well-accepted model for the study of myocardial ischemia reperfusion injury (17, 74, 81, 113, 117). Third, the SD rat does not display large inter-animal variation in m easures of cardiac contra ctility and collateral circulation. In addition, we chose to study male rats to avoid the possibly confounding effects of varying estrogen levels acro ss the estrus cycle (117). Animal Housing and Diet All animals were housed at the University of Florida Animal Care Service Center. Animals were housed on a 12:12 hour revers e light-dark cycle and provided food (AIN93 diet) and water ad libitum throughout the experi mental protocol. Experimental Design and Primary Dependent Measures The experimental design is illustrated (Figure 3-1). Briefly, 32 young male SpragueDawley rats were randomly assigned to either an exercise training group or a sedentary control 34

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group. The sedentary and exercise training groups were further s ub-divided into IR groups and IR sham groups of isolated working heart treatm ents (n=8). Animal sample size was determined based upon statistical powe r analysis using data from our laboratory. The IR sham groups received continuous perfus ion (85 minutes) without an IR insult. IR groups underwent 40 minutes of global isch emia and 45 minutes of reperfusion. During perfusion or an IR insult, working heart vari ables (i.e., cardiac output, cardiac work) were measured. After perfusion or an IR insult, two subpopulations of mitochondria were isolated. Then mitochondrial variables (i.e., oxygen consumption, ROS produc tion, mitochondrial proapoptotic proteins, oxidation of proteins and lipids, and antioxi dant enzymes) were measured. General Methods Exercise Training Protocol The animals assigned to the exercise training groups were habituated to treadmill running for five days. Treadmill habituation of the first day was started with 10 minutes of treadmill running at an intensity of ~ 70% of VO2 max, and from the second day exercise, exercise time was increased by 15 minutes each day, ending in 45 minutes of running on the forth and fifth day. Following habituation, animals were rested for 2 days and then performed 5 consecutive days of treadmill running at an intensity of approximately 70% of VO2max (60 min at 30 m/min) (43, 84). The sedentary control animals were pla ced in the non-moving treadmill for the duration of their matched exercise training animals runni ng to eliminate the possibility that handling or confinement may be a factor that induces cardioprotection. In Vitro Isolated Working Heart Preparation To investigate myocardial contra ctile 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 contractile performance, as cardiac pr eload and after-load pressures are maintained 35

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constant. Further, an advantage of in vitro workin g heart model versus an in vivo IR model is the elimination of the confounding influence of othe r organ systems, systemic circulation, and peripheral complications. This pr eparation has been successfully used by our laboratory for over five years (83, 117, 121). Briefly, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (100mg/kg) and when a su rgical plane of anesthesia was reached (an absence of foot and eye reflexes ), 100 IU heparin was injected into the hepatic vein through the opened abdominal area. The hearts were excised and quickly placed in cold 0.9% NaCl and then weighed. The aorta was secured on a cannula and perfused in a retrogr ade mode at 80 cmH2O with a modified Krebs-Henseleit buffer, c ontaining 1.25mM CaCl2, 130mM NaCl, 5.4mM KCl, 11 mM glucose, 0.5mM MgCl2, 0.5mM NaH2PO4, 25mM NaHCO3, aerated with 95% O2-5% CO2 gas at 37C. Then the left atrium was ca nnulated through the pulmonary vein. Following 10 minutes of retrograde perfusion and 5 minutes of assist mode perfusion (retrograde perfusion with the atrial cannula open), the perfusion was switched to working heart mode where the preischemic-function measurements were set at 13 cmH2O atrial filling pressure (preload) and 80 cmH2O of aortic column (after load). In Vitro Ischemia-Reperfusion Protocol After 15 minutes of stab ilization in working heart mode dur ing which pre-ischemic cardiac performance was measured, global, normo-th ermic, no flow ischemia was induced by simultaneously clamping atrial and aortic lines. Ischemia was maintained for 40 minutes in an environment where the heart was enclosed in a wa ter-jacketed, sealed glass chamber maintained at 37C. Following 40 minutes of ischemia, the heart was reperfused for 10 minutes via retrograde mode (with only the aortic cannula open). Then 25 minutes of reperfusion was continued via the working heart mode in which post-ischemic cardiac performance 36

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measurements were measured every 5 minutes for 20 minutes. At the end of IR or timed perfusion, the heart was cut and imme diately transferred to cold saline to stop the heart beat. Cardiac Performance Measurements Cardiac contractile performance measuremen ts were recorded every 5 minutes for 15 minutes prior to ischemia and every 5 minutes for 20 minutes during reperfusion. The same measurements were recorded for timed contro l groups. Measurements included: cardiac output, cardiac work, systolic/diastolic pressure, left vent ricular 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 Instrume nts) connected to the aortic cannula and collecting both coronary and aortic effluent. Data was recorded and stored using a customized computer data-acquisition system (Labview). Isolation of Subsarcolemmal Mitochondria ( SSM) and Interfibrillar Mitochondria (IFM) Cardiac mitochondria were isolated using th e procedure of Palmer (114) with slight modification. Trypsin was used as the protease (32) in order to isol ate IFM. At the end of either IR or timed perfusion, hearts were removed from the cannula and pl aced into saline at 4C. The hearts were transferred into isolation buffer (100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM MgSO47 H2O, 1 mM ATP, and 0.2% fatty acid free bovine serum albumin, pH 7.4) at 4C. Cardiac tissue was finely minced and homogenized with a polytron tissue processor (Virtis, Gardiner, NY) for 7 seconds at a setting of 50. The homogenate was centrifuged at 500g for 10 minutes. The supernatant was saved for isolati on of SSM. The remaining pellet was resuspended in isolation buffer and homogenized with the sa me polytron tissue processor for 5 seconds. Then the homogenate was incubated with 5 mg/g (wet weight) trypsin for 10 minutes at 4 C. The same amount of isolation buffer (10 ml/g wet weight) was added to deactivate trypsin activity. The homogenate was centrifuged at 500 g for 10 minutes. The supernatan t was saved for isolation of 37

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IFM. The supernatants saved for isolation of SSM and IFM were centrifuged at 3,000g for 10 minutes to sediment SSM and IFM. The supernatan t collected after isolation of SSM was further centrifuged at 20,000g for 30 minutes at 4C. The re sulting supernatant was used as the soluble cytosolic fraction. The pellets of SSM and IFM were washed tw ice and the final pelle ts were resuspended using a dounce homogenizer in resuspension buffer (220 mM manitol, 70 mM sucrose, 2 mM Tris base, and 20 mM HEPES, pH 7.4) at 4 C Mitochondrial proteins and soluble cytosolic proteins were measured by the Bradford protein assay method (18), using fatty acid free BSA as a standard. Mitochondrial Respiration (Oxidative Phosphorylation) Mitochondrial oxygen consumption was measured at 37oC by polarography using a Clark type electrode (Oxygra ph, Hansatech, Norfolk, UK). Experiments began with the addition of ~0.2 mg mitochondria in 1 ml of respiration buffer (in mM; 100 KCL, 5 KH2PO4, 1 EGTA, 50 MOPS, 10 MgCl2) containing 0.2% BSA. Since IR-indu ced mitochondrial damage occurs primarily in complex I and III within the ETC we studied mitochondrial respiration using selective respiratory substrates. Specifical ly, 2mM pyruvate and 2 mM malate (final concentration) were used as a complex I resp iratory substrate. 5 mM succinate with 5 M rotenone (to prevent electron backflow to comp lex I) was used as a complex II respiratory substrate. The maximal respiration (state 3), define d as the rate of respirat ion in the presence of ADP was initiated by adding 0.25 mM ADP to the respiration chamber containing mitochondria and respiratory substrates. State 4 (resting re spiration) was obtained when the complete conversion (phosphorylation) of ADP to ATP was reached. The respiratory control ratio (RCR) commonly referred to as an index of mitochondrial integrity (coupled resp iration) was calculated from the ratio of state 3 to state 4 respiration. 38

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In order to examine the efficiency of AT P synthesis coupled to mitochondria, the P/O ratio were calculated from the ratio of ADP untilized to O2 consumed during state 3 respiration. Mitochondrial H2O2 Production Mitochondrial H2O2 production was measured with the Amplex red-horseradish peroxidase (HRP) method (Molecular Probes, Eu gene, OR) in freshly isolated mitochondria. HRP catalyzes the H2O2-dependent oxidation of nonfluores cent Amplex red (146). CuZnSOD was added to convert al l superoxide into H2O2 because otherwise superoxide reacts very rapidly with HRP, resulting in underest imation of the actual rate of H2O2 production. Therefore, our results reflect the sum of both superoxide and H2O2 production are referred to as ROS rather than H2O2 production per se. More importantly, a subm aximal respiration rate conditions was used with a phosphocreatine (P Cr)/creatine (Cr) ratio of 0.5 to generate a free ADP concentration of about 67 M. This ADP concen tration was determined by progressive creatine kinase clamp experiment. This condition gives a steady state respiration rate of roughly 50% of state 3 respiration. Importantly, this method allows us to resolve one of the very valid criticisms about conventional in vitro mitochondrial ROS assays. For ex ample, many people only look at either state 4 (rest) or state 3 (all out) rates of ROS production. In an in vivo environment, mitochondria obviously operate at a rate interm ediate to these two extremes. Therefore, H2O2 production measured by this proposed technique allowed us to evaluate more physiological levels of mitochondrial H2O2 production. Briefly, experiments for the static cond ition began with the addition of 20 g mitochondria in 180 l of pre-warmed respir ation buffer at 37C (in mM; 100 KCL, 5 KH2PO4, 1 EGTA, 50 MOPS, 10 MgCl2 0.2% fatty acid free BSA, 50 M Amplex Red, and 5 mM succinate) containing CuZnSOD (40 unit/ml) and HRP (10 units/ml). For submaximal respiration condition, 5 mM ATP, 10 mM creatine phosphate 20 mM creatine were added to the same 39

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respiration buffer. H2O2 generation was determined by measur ing fluorescence (excitation at 544 nm emission at 590 nm) for 15 minutes at 37 C using a fluorometric device (Spectra Max, Molecular device). The sample values (nmol/m g/min) were calculated based on the values acquired from standard curve using H2O2 as a standard. Assessment of Mitochondrial Protein Oxidative Damage Proteins carbonyls, an index of oxidative modi fication of proteins in SSM and IFM, were detected using a protein oxida tion detection kit (Oxyblot, Chemicon, U.S.A). Briefly, 20 g of mitochondrial proteins were denatured by adding 12% SDS for a final concentration of 6% SDS. The protein samples were derivatized by adding 2,4-Dinitrophenyl hydrazine (DNPH). The samples were incubated for 15 minutes at room te mperature. Then the sa mples were neutralized by adding neutralization solution from the kit. Fina lly, 10 g of the prepared protein was loaded into 4-20% gradient acrylamide Criterion gel (Bio Rad). SDS-P AGE was performed at constant 200 Volts for 1 hour. The separated proteins were transferred to a pure nitrocellulose membrane by using a Transblot unit (Bio Rad) at 45 V for 2 hours. Equal loading was confirmed by staining the proteins with 0.1% Ponceau solu tion before the membrane was incubated in blocking buffer containing 5% non-fat milk with 0.05% Tween-20. The membrane was incubated in the primary antibody solution overnight at 4C with gentle shaking. The membrane was then rinsed with washing buffer and incuba ted with secondary antibody for 1 hour at room temperature. Finally, enhanced chemilumine scence detection reagents from Amersham (Amersham Pharmacia Biotech, Piscataway, NJ) were used to generate chemiluminescent signals and the bands were visual ized by exposing the membrane to light-sensitive film. The blot was analyzed using Kodak 1D image analysis software (Eastman Kodak, Rochester, NY). 40

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Assessment of Mitochondrial Lipidperoxidation 4-Hydroxynonenal (4-HNE or HNE), an index of lipid peroxidation chain reaction due to oxidative stress, was measured from SSM a nd IFM using a western blot technique. Equal amounts of mitochondrial protein were loaded and separated via SDS-polyacrylamide gel electrophoresis. The separated proteins were tr ansferred to a nitrocel lulose membrane. The membrane was blocked in PBS containing 0.05% Tween 20 and 5% nonfat milk and incubated overnight with primary antibodies targeted to 4-HNE (Ab 46545). The membranes was washed and incubated with horseradish peroxidase-conj ugated secondary antibodies for 1 hour. Finally, enhanced chemiluminescence detection reagents were used to gene rate chemiluminescent signals and the bands were visual ized by exposing the membrane to light-sensitive film. The blot was analyzed using Kodak 1D image analysis software. Assessment of the Release of Mito chondrial Proapoptotic Proteins Cytosolic cytochrome c and AIF were meas ured by western blot techniques. Briefly, cytosolic proteins were separated using SDS-PAGE under denaturing conditions and then transferred to nitrocellulose membranes. The membranes were blocked in PBS containing 0.05% Tween 20 and 5% nonfat milk and incubated overn ight with primary antibodies (cytochrome c: sc 8385; AIF: sc 9416). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Finally, enhanced chemiluminescence detection reagents from Amersham were used to generate chemilumine scent signals and the bands were visualized by exposing the membrane to light-sensitive film. Blots were analyzed using Kodak 1D image analysis software. Assessment of Mitochondrial Antioxidant Proteins MnSOD, CuZn SOD, catalase, Prx3, Trx2, and TrxR2 protein contents were measured by Western blot techniques. Briefly, mitochondria l proteins were separated using SDS-PAGE 41

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under denaturing conditions and then transf erred to nitrocellulose membranes. The membranes were blocked in PBS containing 0.05% Tween 20 and 5% nonfat milk and incubated overnight with primary antibodi es (MnSOD: sc 30080; CuZnSOD: sc 11407; catalase: ab 16731; Prx3: LF-P A0030; Trx2: sc 50336; TrxR2: LF-PA0024). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Finally, enhanced chemiluminescence det ection reagents were used to generate chemiluminescent signals and the bands were vi sualized by exposing the membrane to lightsensitive film. Blots were analyzed us ing Kodak 1D image analysis software. Data Analysis To test our hypothesis, we performe d a one-way ANOVA. When significance was indicated, Fishers LSD post hoc test was chosen to determine group differences. Significance was established at P < 0.05. 42

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Figure 3-1. Experimental design examining the ro le of exercise training in mitochondrial protection following IR. 43

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CHAPTER 4 RESULTS Animal Characteristics The physical characteristics for the animals in all experimental groups are presented in Table 4-1. No significant differences in animal body weight, heart weight, and heart/body weight ratio existed between experimental groups. Myocardial Performance during IR Myocardial Function al Characteristics The functional characteristics of the isolated, perfused hearts are pr esented in Table 4-2. Coronary flow (CF), cardiac output (CO), systolic pressure (SP), heart rate (HR), % left ventricular developed pressure (% LVDP), and ra te pressure product (RPP) were measured as indexes of myocardial function. Note that timed control groups (CP vs. EP) did not show any significant differences in myocar dial function, indicating that perfusion time (85 minutes of isolated perfused-working heart) did not affect cardiac function. IR compromised the cardiac contractile function, but ExTr prevented IR-induced contractile dysfunction. For example, CF, CO, SP, % recovery of LVDP, and RPP were significantly reduced in CIR group compared to EIR groups. Percent Recovery of Cardiac Work (CW: Systolic Pressure x Cardiac Output) CW is commonly used in an in vitro working heart model as an index of myocardial function. This marker of contractile performance is important, as it normalizes each group to its baseline (pre-ischemia) values. As illustrated in Figure 4-1, CW was not different between CP and EP groups. However, % recovery of CW fo llowing IR was significantly lower in CIR group compared to EIR group. 44

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Percent Recovery of +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 release and re-sequestration 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 following IR, we can quantify the changes in myo cardial contraction and relaxation rates and/or Ca2+-handling kinetics. As illustrated in Figure 4-2 and 4-3, no differences were found between CP and EP groups. However, +dp/dt and -dp/dt were significantly reduced in CIR group compared to EIR group. Mitochondrial Measurements Subsarcolemmal and Interfibrillar Mitochondrial Protein Yield and Integrity Mitochondrial protein yield is presented in Table 4-3. The protein yield of SSM in EP group was significantly increased compared to ot her groups. The protein yield of IFM of CIR group was significantly reduced compared to other groups. Additionally, the EIR group showed significant reduction in IFM protei n compared with EP group. Total mitochondrial proteins (the sum of SSM and IFM) were not significantly different between CIR and EIR group. The integrity of two subpopulations of isol ated mitochondria was confirmed by images obtained with transmission electron microscopy (Figur e 4-4). In addition, the integrity of isolated mitochondria was reconfirmed by measuring m itochondrial respiratory control ratio (RCR = state 3/state4 respiration). Indeed, RCR from both SSM (~4.5) and IFM (~10) demonstrate that isolated mitochondria were intact and well coupled. 45

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Mitochondrial Oxidative Ph osphorylation (State 3 and State 4) and RCR of SSM Changes in the rate of state 3 and state 4 ar e used as an index of mitochondrial damage. With pyruvate/malate as a substrate to donate re ducing equivalents to complex I, no differences existed between CP and EP in the rates of st ate 3 and 4 respiration. The rates of state 3 respiration following IR were decreased followi ng IR (Figure 4-5). The rates of state 4 respiration following IR were si gnificantly increased. However, co mpared to CIR group, the rate of state 4 respiration was significantly lower in EIR group (Figure 4-6). Thus, respiratory control ratio (RCR) was significantly higher the EIR group compared to CIR group (Figure 4-7). With succinate as a substrate to donate redu cing equivalents to complex II, the rates of state 3 and 4 respiration were not different be tween CP and EP groups. The rates of state 3 respiration following IR were si gnificantly reduced. However, EIR group maintained higher state 3 respiration rate compared to CIR group (Figure 4-8). The rates of state 4 respiration following IR were significantly elevated. However, EIR group sustained lower state 4 respiration rate compared to CIR group (Figure 4-9). As a resu lt, the RCR was significan tly higher in EIR group compared to CIR group (Figure 4-10). Mitochondrial Oxidative Ph osphorylation (State 3 and State 4) and RCR of IFM With pyruvate/malate, no differences existed between CP, EP, and EIR groups in the rates of state 3 and 4 respiration. The rate of state 3 respiration in following IR was significant reduced in CIR group compared to other groups (Figure 411). Moreover, the rate of state 4 respiration following IR was significantly elevated in CIR group compared to other groups (Figure 4-12). Therefore, the RCR of CIR group was significantly lower compar ed to other groups (Figure 413). With succinate, no differences existed in the rate of state 3 respiration between CP and EP groups. The rate of state 3 respiration followi ng IR was significantly decreased in CIR group 46

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compared to other groups (Figure 4-14). However, the rates of state 4 respiration were not different between groups (Figure 4-15). Th e RCR was significantly lower in CIR group compared to other groups (Figure 4-16). P/O Ratio Mitochondrial P/O ratios are presented in Ta ble 4-4. With pyruvate/malate as a complex I substrate, P/O ratio in SSM of CIR group wa s significantly lower compared to EIR group. However, no difference was found in IFM be tween CIR and EIR group. No significant differences were found in P/O ratio between gro ups when succinate (complex II) was used as a substrate. Mitochondrial H2O2 Production Horseradish peroxidase and Amplex red do not penetrate intact mitochondria (33). Therefore, only H2O2 released from mitochondria is de tected by this as say. Conventionally, H2O2 production is measured in a st atic condition (the presence of added substrate but not ADP). As shown in Table 4-5, the rate of H2O2 production was not different between groups when the static conditions were used. In contrast, in constantly active respiration c ondition where constant ADP concentration is maintained by crea tine kinase (CK) clamp, the rate of H2O2 production in SSM was significantly higher in CIR group compared to EIR group (Figure 4-17). Similarly, the rate of H2O2 production in IFM was also significantly higher in CIR group compared to EIR group (Figure 4-18). IR-induced Oxidative Modifi cations in Mitochondria Carbonyl formations in proteins are one of the features of oxidatively modified proteins. Our results show that the level of oxidatively modified proteins in SSM was significantly increased in CIR group compared with CP and EP groups (Figure 4-19). No differences existed in the level of protein carbonyls in IFM between groups. 47

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4-HNE is generated by the lipid peroxidati on chain reaction due to oxidative stress. Therefore, elevated levels of this marker indicat e an increase in lipid ox idation. Our results show that the level of 4-HNE products in SSM was significantly increas ed in CIR group compared to other groups (Figure 4-20). Moreover, the leve l of 4-HNE products in IFM was significantly elevated in CIR group compared to EP and EIR groups (Figur e 4-21). Additionally, CP group showed higher levels of 4-HNE formation compared to EP group. The Release of Mitochondrial Proapoptotic Proteins The release of pro-apoptotic proteins (i.e., cy tochrome c and AIF) from mitochondria into the cytosol is an index of mitochondrial damage and cell death. As illustrated in Figure 4-22, our results show that the level of cytochrome c released from mitochondria was significantly higher in CIR group compared to the other groups. Furthermore, the level of AIF released from mitochondria was also significantly higher in th e CIR group compared to other groups (Figure 423). Mitochondrial Antioxidant Proteins Mitochondrial antioxidant prot eins in both SSM and IFM were measured to determine whether exercise-mediated mitochondrial protect ion is associated with an increase in mitochondrial antioxidant capacity. Overproduction of superoxide anions from mitochondria during IR causes oxidative damage. In this re gard, MnSOD and CuZnSOD dismutate superoxide anions to a less reacti ve oxidant (i.e., H2O2). Our results show that both MnSOD (Figure 4-24) and CuZnSOD (Figure 4-25) in SSM were signifi cantly elevated in EP group compared with CP and CIR groups. In terms of IFM, the level of MnSOD proteins in IFM was significantly reduced in CIR group compared to EP group (Figure 428). The level of CuZnSOD proteins was significantly reduced in CI R group compared to othe r groups (Figure 4-29). 48

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49 Trx2 is a protein containing dithio l groups and thus acts as an an tioxidant. It is specifically involved in detoxifying H2O2. We measured the levels of Tr x2 protein contents utilizing immunoblotting techniques to examine if exercise training increases this protein and thus reduces oxidative stress. Our results indicate that the level of Trx2 proteins in SSM was significantly higher in EP and EIR groups compared with CP group. In addition, Trx2 in EIR group was significant higher than CI R group (Figure 4-26). Maintaining the reduced state of Trx2 is important for Trx2 to act as an antioxidant. In this regard, TrxR2 is the only known enzyme to re duce Trx2 and works as a unit with Trx2 to remove H2O2. The level of Trx2R proteins was signifi cantly reduced in CIR group compared to other groups (Figure 4-27). In regard to IFM, The level of TrxR2 was significantly lower in CIR group compared to EP and EIR groups (4-30). No significant differences were found in Pr x3 and catalase of SSM between groups (data not shown). In addition, no differences were found in Trx2, Prx3, and catalase in IFM between groups (data not shown).

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Table 4-1. Animal body and heart weights. Values are mean SE. No differences existed between the experimental groups in any variab les (P < 0.05). CP= control-perfused; EP= exercise-perfused; CIR= control-ischemia re perfusion; EIR= exerci se-ischemia-reperfusion. Group Number Body Weight (g) Heart Weight (g) Heart / Body Weight Ratio (mg/g) CP 8 422 8.2 1.82 0.06 4.31 0.19 EP 8 412 11.5 1.68 0.04 4.10 0.16 CIR 8 437 8.6 1.79 0.03 4.10 0.10 EIR 8 423 9.6 1.76 0.07 4.15 0.12 50

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Table 4-2. Functional Characteristics of isolated, perfused hearts during IR. Parameters CP EP CIR EIR CF (ml/min/g wet. wt.) 11.41 0.39 12.21 0.68 7.054 0.46 *** 10. 48 0.66 CO (ml/min/g wet. wt.) 38.76 1.36 42.38 1.20 11.71 3.51 *** 30.05 1.99 ** SP (mmHg) 93.13 3.34 87.50 1.59 69.88 1.48 *** 80.75 1.22 ** HR (beats/min) 317 12 339 7 292 23 324 10 % recovery of LVDP 101 3.6 95 3.5 21 9 *** 70 4 ** RPP (HR x SP) 29310 335 29680 546 20600 1800 *** 26160 494 ** Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. ** Signifi cantly different from CP and EP groups, P < 0.05. Significantly different from EP group, P < 0.05. CP = control-perfused; EP= exerci se-perfused; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion.51

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Table 4-3. Mitochondrial protein yield. Parameters CP EP CIR EIR SSM (mg/g.w.wt) 4.16 0.17 4.74 0.22 3.96 0.25 3.95 0.15 IFM (mg/g.w.wt) 9.48 0.25 9.96 0.28 7.92 0.51 *** 8.59 0.26 Total mitochondria (mg/g.w.wt) 13.64 0.27 14.70 0.37 11.89 0.67 ** 12.54 0.39 Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. ** Signifi cantly different from CP and EP groups, P < 0.05. Significantly different from EP group, P < 0.05. Significantly different from CP, CIR and EIR groups, P < 0.05. CP= control-perfused; EP= exercise-perfused; CIR= control ischemia-reperfusion; EIR= ex ercise ischemia-reperfusion.52

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Table 4-4. P/O ratio. SSM IFM CP EP CIR EIR CP EP CIR EIR Pyr/mal 4.2 0.1 4.3 0.1 3.8 0.2 *** 4.3 0.2 4.3 0.1 4.4 0.1 4.0 0.2 4.1 0.1 Succinate 2.0 0.1 2.0 0.1 1.9 0.2 1.8 0.1 2.3 0.1 2.3 0.1 2.20 0.1 2.1 0.1 Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. Signifi cantly different from EP group, P < 0.05. CP= control-perfused; EP= exercise-perfused; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion.53

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54Table 4-5. Mitochondrial ROS production during state 4 respiration. SSM IFM CP EP CIR EIR CP EP CIR EIR Static 373 91 331 81 472 55 461 79 372 91 306 70 457 52 391 59 Values are mean SE. CP= control-perfused; EP= exercise -perfused; CIR= control ischemia re perfusion; EIR= exercise ischemiareperfusion. No significant diffe rences were found in any of the variables measured (P < 0.05).

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** ***Figure 4-1. Percent recovery of cardiac work (systolic pressure x cardiac output). Values are mean SE. *** Significantly di fferent from CP, EP, and EIR groups, P < 0.05. ** Significantly different from CP and EP group, P < 0.05. CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. ***Figure 4-2. Percent recovery rate of +dp/dt (left ventricular syst olic function). Values are mean SE. *** Significantly different from CP EP, and EIR groups, P < 0.05. CP=controlperfused; EP=exercise-perfused; CIR=contro l ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 55

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# ***Figure 4-3. Percent recovery rate of -dp/dt (left ventricular dias tolic function). Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. # Significantly different from CP group, P < 0.05. CP=control-perfused; EP=exerciseperfused; CIR=control ischem ia-reperfusion; EIR= exercise ischemia-reperfusion. IFM SSM Figure 4-4. Electron microscopic photographs of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondr ia (IFM) isolated from a rat heart. 56

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** ** Figure 4-5. The rate of state 3 resp iration of SSM using pyruvate/malate. Values are mean SE. ** Significantly different from CP and EP groups, P < 0.05. P/M= pyruvate/malate; CP= control-perfused; EP= exercise-perfuse d; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion. ** Figure 4-6. The rate of state 4 resp iration of SSM using pyruvate/malate. Values are mean SE. *** Significantly different from CP, EP and EIR groups, P < 0.05. P/M=pyruvate/malate; CP= control-perfused; EP= exercise-perfused; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion. ** *** Figure 4-7. Respiratory contro l ratio of SSM, using pyruvate/malate. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. ** Significantly different from CP and EP groups, P < 0.05. P/M=pyruvate/malate; CP=controlperfused; EP=exercise-perfused; CIR=contro l ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 57

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*** ** Figure 4-8. The rate of state 3 respiration of SSM using succinate. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. ** Significantly different from CP and EP groups, P <0.05. succ=succinate; CP= control-perfused; EP= exercise-perfused; CIR= control ischem ia reperfusion; EIR= exercise ischemiareperfusion. *** Figure 4-9. The rate of state 4 respiration of SSM using succinate. Values are mean SE. *** Significantly different from CP, EP, a nd EIR groups, P < 0.05. succ=succinate; CP= control-perfused; EP= exercise-perfused; CI R= control ischemia reperfusion; EIR= exercise ischemia-reperfusion. *** **Figure 4-10. Respiratory control ratio of SSM, using succinate. Values are mean SE. *** Significantly different from CP, EP, and EIR group, P < 0.05. ** Significantly different from CP and EP group, P < 0.05. CP=control-perfused; EP=exerciseperfused; CIR=control ischem ia-reperfusion; EIR= exercise ischemia-reperfusion. 58

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*** Figure 4-11. The rate of state 3 resp iration of IFM using pyruvate/malate. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. P/M=pyruvate/malate; CP= control-perfused; EP= exercise-perfused; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion. ***Figure 4-12. The rate of state 4 respiration of IFM using pyruvate/malate. Values are mean SE. *** Significantly different from CP EP, and EIR groups, P < 0.05. P/M= pyruvate/malate; CP= control-perfused; EP= exercise-perfused; CIR= control ischemia reperfusion; EIR= exercise ischemia-reperfusion. *** Figure 4-13. Respiratory control ratio of IF M, using pyruvate/malate. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. P/M= pyruvate/malate; CP=control-perfused; EP=e xercise-perfused; CIR=control ischemiareperfusion; EIR= exercise ischemia-reperfusion. 59

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# ***Figure 4-14. The rate of stat e 3 respiration of IFM using succinate. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. # Significantly different from CP, P <0.05. succ=succinate ; CP= control-perfused; EP= exerciseperfused; CIR= control ischemia reperfus ion; EIR= exercise ischemia-reperfusion. Figure 4-15. The rate of stat e 4 respiration of IFM using succinate. Values are mean SE. No significant differences exist. succ=succina te; CP= control-perfused; EP= exerciseperfused; CIR= control ischemia reperfus ion; EIR= exercise ischemia-reperfusion. # *** Figure 4-16. Respiratory control ratio of IFM, using succinate. Values are mean SE. *** Significantly different from CP, EP, and EIR group, P < 0.05. # Significantly different from CP group, P < 0.05. CP=cont rol-perfused; EP=e xercise-perfused; CIR=control ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 60

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* *** Figure 4-17. The levels of H2O2 production from SSM, using CK clamp. Values are mean SE. *** Significantly different from CP, EP, and EIR group, P < 0.05. Significantly different from EP, P < 0.05. SSM=subs arcolemmal mitochondria; CK= creatine kinase; CP=control-perfused; EP=exerc ise-perfused; CIR=control ischemiareperfusion; EIR= exercise ischemia-reperfusion. *** Figure 4-18. The levels of H2O2 production from IFM, using CK clamp. Values are mean SE. *** Significantly different fr om CP, EP, and EIR groups, P < 0.05. IFM=interfibrillar mitochondria; CK= creatine kinase; CP=c ontrol-perfused; EP=exercise-perfused; CIR=control ischemia-reperfusion; EIR= exercise ischemia-reperfusion 61

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CP EP CIR EIR # Figure 4-19. The levels of protein car bonyl formation in SSM. Values are mean SE. # Significantly different from CP group, P < 0.05. Significantly different from EP group, P < 0.05. SSM=subsarcolemmal m itochondria; CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. 62

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***Figure 4-20. The levels of 4-HNE products of IFM following IR. Values are mean SE. *** Significantly different from CP, EP a nd EIR groups, P < 0.05. SSM=subsarcolemmal mitochondria; CP=control-perfused; EP=exe rcise-perfused; CIR=control ischemiareperfusion; EIR= exerci se ischemia-reperfusion. Figure 4-21. The levels of 4-HNE products in IFM following IR. Values are mean SE. Significantly different from EP group P < 0.05. Significantly different from EIR group < 0.05. IFM=interfibri llar mitochondria; CK= crea tine kinase; CP=controlperfused; EP=exercise-perfused; CIR=contro l ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 63

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***Figure 4-22. Levels of cytochrome c released from cardiac mitochondria. Values are mean SE. *** Significantly different from CP EP, and EIR group, P < 0.05. CP=controlperfused; EP=exercise-perfused; CIR=contro l ischemia-reperfusion; EIR= exercise ischemia-reperfusion. ***Figure 4-23. Levels of AIF released fr om cardiac mitochondria. Values are mean SE. *** Significantly different from CP, EP, a nd EIR groups, P < 0.05. CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. 64

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# Figure 4-24. Protein levels of manganese supero xide dismutase (MnSOD) in SSM. Values are mean SE. # Significantly differe nt from CP group, P < 0.05. Significantly different from CIR group, P < 0.05. CP=cont rol-perfused; EP=exercise-perfused; CIR=control ischemia-reperfusion; EIR= exercise ischemia-reperfusion. # Figure 4-25. Protein levels of copper zinc superoxide dismutas e (CuZnSOD) in SSM. Values are mean SE. # Significantly differe nt from CP group, P < 0.05. Significantly different from CIR group, P < 0.05. CP=cont rol-perfused; EP=exercise-perfused; CIR=control ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 65

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# # Figure 4-26. Protein levels of thiored oxin 2 (Trx2) in SSM. Values are mean SE. # Significantly different from CP group, P < 0.05. Significantly different from CIR, P < 0.05. IFM=interfibrillar mitochondria; CK = creatine kinase; CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. ***Figure 4-27. Protein levels of thioredoxin reductase 2 (TrxR2) in SSM. Values are mean SE. *** Significantly different from CP, EP, and EIR groups, P < 0.05. CP=controlperfused; EP=exercise-perfused; CIR=contro l ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 66

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*Figure 4-28. Protein levels of manganese supero xide dismutase (MnSOD) in IFM. Values are mean SE. Significantly different from EP group, < 0.05. CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. ***Figure 4-29. Protein levels of c opper zinc superoxide dismutase (CuZnSOD) of IFM. Values are means S.E.M. *** significantly different from CP, EP, and EIR groups, < 0.05. CP=control-perfused; EP=exercise-perfus ed; CIR=control ischemia-reperfusion; EIR= exercise ischemia-reperfusion. 67

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* Figure 4-30. Protein levels of thioredoxin reductase 2 (TrxR2) in IFM. Values are mean SE. Significantly different from EP, P < 0.05. Significantly different from EIR P < 0.05. IFM=interfibrillar mitochondria; CK= creatine kinase; CP=control-perfused; EP=exercise-perfused; CIR=control ischemia -reperfusion; EIR= exercise ischemiareperfusion. 68

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CHAPTER 5 DISCUSSION Overview of Principal Findings This experiment investigated the ability of ExTr to protect the tw o populations of cardiac mitochondrial (SSM and IFM) against damage i nduced by an IR insult. Four major findings emerged from our study. First, ExTr protected both SSM and IFM against IR-induced respiratory dysfunction resulting from long duration global ischemia (40 minutes) followed by reperfusion (45 minutes). Second, ExTr prevented an IR-i nduced increase in mitochondrial ROS production in both SSM and IFM. Third, ExTr attenuate d IR-induced oxidative damage to cardiac mitochondria. Finally, ExTr retarded the IR -induced release of pr oapoptotic proteins (cytochrome c and AIF) from cardiac mitochondria Collectively, our results indicate that ExTr induces a mitochondrial phenotype that resists IR-induced damage; hence, ExTr-induced mitochondrial adaptation may be the key cellula r alteration that explai ns exercise-induced cardioprotection. A detailed discussion of these and other related findings follows. ExTr Provides Cardioprotection against an IR Insult It is well established that regular ExTr pr ovides myocardial protection against IR-induced arrhythmias, myocardial stunning, and cell death (infarction) (65-67, 84, 85, 118, 121, 123, 135, 136, 143). Interestingly, as few as 3-5 exercise sessions (i.e., 3-5 consecutive days) confers the same level of cardioprotection comp ared to chronic exercise traini ng (i.e., weeks to months)(43). To confirm that our ExTr protocol was effec tive in providing cardiopro tection, we evaluated cardiac mechanical performance before and after an in vitro IR insult in hearts from both control and ExTr animals. Our results indicate that, comp ared to hearts from untrained animals, ExTr improved post-ischemia recovery of coronary flow, cardiac output, and cardiac work. In addition, our findings indicate that ExTr improves post-ischemia recovery of left ventricular systolic 69

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(+dp/dt) and diastolic function (dp/dt). Collectively, these results substantiate that our ExTr protocol was successful in providing cardioprotection against an IR-insult. Mitochondria are unique organell es that supply cellular energy and play a key role as the arbitrators of cellular life and death. Moreover, it is well established that IR-induced cardiac insult results in signifi cant mitochondrial damage and resp iratory dysfunction. Therefore, we reasoned that ExTr-induced mitochondrial adaptati ons could be a primary mechanism to explain why exercise training provides ca rdioprotection against IR injury. A detailed discussion of our findings related to exercise-i nduced mitochondrial protection during an IR insult follows. ExTr Protects Mitochondrial Respiratory Function Previous work indicates that ischemia alone or a combination of ischemia and reperfusion damages cardiac mitochondria (2, 32, 88). Speci fically, it has been reported that cardiac ischemia damages the mitochondrial electron tran sport chain resulting in respiratory dysfunction due to a decrease in state 3 respiration and an increase in state 4 respiration (28, 32, 91, 92). As a result, mitochondria become uncoupled and the re spiratory control ratio (RCR = state 3/state 4 respiration) is decreased. Prio r to the current study, only one published report has addressed the impact of ExTr on protection of mitochondria against anoxia-reoxygenati on injury. The earlier study indicated that ExTr provides protection to a combined pool of cardiac SSM and IFM against an in vitro anoxia-reoxygenation insult (9). While the report suggests that mitochondrial adaptation occurs with exercise training, the in vitro anoxia-reoxygenation protocol used in that study does not mimic the in vivo physiology of an IR insult that occurs within an intact heart. Indeed, many key elements that contribute to IR-induced mitochondrial damage are missing from the in vitro experimental model (e.g., cytosolic sources of ROS production, cytosolic calcium overload, etc.). Therefore, the current study significantly advances our knowledge in this 70

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area by investigating the ability of exercise training to provide protection to both subpopulations of mitochondria in an intact heart following an IR insult. Importantly, our results show that an IR insult applied to hear ts of untrained (CIR) animals results in diminished mitochondria l state 3 respiration and an increased state 4 respiration when both complex I (pyruvate/malate) and complex II s ubstrates (succinate) ar e used. In contrast, compared to CIR animals, ExTr provided significant protection against IR-mediated mitochondrial damage as indicated by a higher RCR in both SS and IFM. Collectively, these findings indicate that ExTr promotes a m itochondrial phenotype that resists IR-induced respiratory dysfunction. ExTr Retards IR-induced ROS Production It is well established that a myocardial IR insult promotes mitochondrial damage resulting in an increased production of ROS (31, 32, 140). In our experiments, the rate of mitochondrial ROS production was assessed using a novel experimental model that mimics in vivo mitochondrial function. This model, using a creatine clamp of resp iration, permits the measurement of ROS production in respiring mitochondria at a fixed submaximal rate (i.e., ~60%) of state 3 respiration. In this way, this in vitro creatine clamp model mimics the in vivo respiratory function of mitochondria in the hear t. Indeed, considering the fact that cardiac mitochondria are never exposed to state 2 respiration (without ADP) in normal physiology, the findings from the present study provide the first physiological assessment of cardiac mitochondrial ROS production following an IR insult. Our findings reveal that Ex Tr significantly reduces IR -induced mitochondrial ROS production. More specifically, ou r results provide novel evidence that ExTr decreases ROS production in both SSM and IFM following IR, which indicates that ExTr protects both 71

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subpopulations of mitochondria ag ainst IR-mediated damage. Similarly, Starnes et al. (130) reported that ExTr reduces mitochondrial H2O2 production during in vitro apoptotic challenges. In theory, ROS can be produced in mitochondria at nine different sites (5). Nonetheless, most studies suggest that complex I and complex III are the major sites of electron leaks leading to the univalent reduc tion of oxygen and the production of su peroxide (31, 33, 138). With regard to the mechanisms responsible for the IR-i nduced increase in mitochondrial ROS production, recent studies suggest that cytochrome c leak ages from the mitochondr ia along with oxidative damage to complex I and III lead to this el evated ROS production ( 29, 32, 88). Our findings reveal that ExTr prevents IR-induced cytoch rome c release from the mitochondria. This observation can explain, at leas t in part, why ExTr reduces the IR-induced increase in mitochondrial ROS production. ExTr Attenuates IR-induced Oxidative Damage to Mitochondria As mentioned earlier, ROS generated during an IR insult are implicated in mitochondrial dysfunction and damage. Using protein carbonyl form ation as a marker of protein oxidation, our findings show that IR increases protein oxidation in SSM of se dentary animals. These results may be explained by the fact that SS M and IFM respond differently to an in vitro oxidative challenge as SSM mitochondria appear to be more susceptible to damage (76). 4-HNE (a reactive aldehyde) and lipid hydr operoxides are major by-products of ROSmediated lipid peroxidation that occurs in cardiac mitochondria following IR (98, 140). Our results indicate that 4-HNE increases in mito chondria following an IR insult. Moreover, ExTr attenuates the increas e in 4-HNE levels in both SSM and IFM. Although our results do not provide a definitive explanation for these results, our data suggest that two possible interacting mechanisms could contribute to the protection of cardiac mitochondria against IR-induced oxidative damage. First, the ExTr-induced m itochondrial protection ag ainst oxidative injury 72

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could be mediated, at least in part, by ExT r-induced improvement in mitochondrial antioxidant capacity. For example, compared to cardiac mitochondria from the CIR group, both SSM and IMF from EIR animals containe d a higher protein abundance of several key antioxidants (i.e., MnSOD, CuZnSOD, TrxR2, etc.). Further, our find ings reveal that ExTr retards the IR-induced increase in mitochondrial ROS production. Hence, it is feasible that the mechanism to explain ExTr-induced mitochondrial protection agains t IR-induced oxidative damage is via a combination of increased m itochondrial antioxidants and a reduction in IR-induced mitochondrial ROS production. ExTr Reduces the Release of Proapoptotic Proteins IR-induced mitochondrial dysfunction and damage are closely linked to myocardial injury (2, 32, 46, 53-55, 61, 70, 77, 90, 91, 105). Studies indica te that mitochondria-mediated apoptosis is associated with IR-induced myocardial inju ry. Specifically, cytochrome c (2, 32) and/or AIF (77) release from mitochondria in to the cytosol are known to be a critical initiating step of the apoptotic processes during IR. R ecent studies report that ExTr attenuates myocardial apoptosis induced by an IR insult (121, 123). These studies show that ExTr decreases caspase-3 activity and the number of TUNEL positive nuclei. At pr esent, it is unknown why ExTr prevents IRinduced caspase-3 activation and ap optosis in the heart. In this regard, our findings show that ExTr prevents cytochrome c and AIF release fr om the mitochondria following IR. These findings are novel and indicate that ExTr prevents a critical step in th e initiation of both mitochondriamediated caspase-dependent (i.e., cytochrome c) and independent (i.e., AIF) apoptosis. Furthermore, considering the fact that AIF is activated by mitochondrial calpain due to mitochondrial calcium overload ( 7, 48, 112), our findings are cons istent with the notion that ExTr reduces mitochondrial calcium overload duri ng IR. Indeed, this postulate is supported by the observation that cardiac mPTP of exercise-tra ined animal is more resistant to exogenous 73

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calcium overload challenge (76). Taken together, our findings provide new evidence that regular bouts of ExTr prevent a critical step in initiating m itochondria-mediated apoptosis by inhibiting the release of key proapoptotic proteins during IR. Potential Mechanisms Responsible for ExTr-induced Mitochondrial Protection At present, the molecular mechanisms responsible for ExTr-induced mitochondrial protection against IR injury remain unknown. Given that IR-induced oxidative damages to mitochondria contribute to myocardial inju ry (30-33, 87, 91), and that ExTr improves antioxidant capacity (66, 76), we postulated that an ExTr-induced impr ovement in mitochondrial antioxidant capacity could protect mitochondria ( both SSM and IFM) against an IR insult. Our results are consistent with this notion as ExTr increased the protein abundance of MnSOD, CuZnSOD and Trx2 in SSM. Moreover, ExTr prevented IR-induced TrxR2 degradation in SSM. Although ExTr did not significantly increase antioxidants within IFM of the EP group, the protein abundance of MnSOD, CuZnSOD, and Tr xR2 was significantly higher in IFM from the EIR group compared to CIR. One interpretation of this finding is that an IR insult activates mitochondrial proteases that degrad e antioxidants. Although it is unclear if IR-induced protease activation plays a key role in degrading antioxida nt enzymes, numerous studies indicate that mitochondria contain endogenous pr oteases (i.e., mitochondrial calpain (7, 48, 112); HtrA2/Omi (134), etc.) that are activated by increased mitochondrial calci um levels and upon activation degrade oxidized proteins. If this is the case, activation of these proteases may explain our findings that, compared to EIR, lower levels of IFM antioxidants exist within the CIR group. Importantly, our results reveal that ExTr pr omotes an increase in both Trx2 and TrxR2 within SS mitochondria. This novel finding is significant because an increase in these proteins would increase the ability of mitochondria to remove H2O2. Specifically, thioredoxins are a class of small 12-kDa proteins present in all eukar yotic cells. Although thiore doxins are known for a 74

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variety of biological ac tivities, thioredoxins play an impor tant role in scavenging ROS and therefore can provide cellular pr otection against oxidative stre ss. In particular, Trx2 is a mitochondrial thioredoxin that provi des reducing equivalents to the H2O2 scavenging antioxidant enzyme, peroxiredoxin3 (23, 35). Although TrxR2 is not directly involved in scavenging oxidants, this key molecule plays a critical ro le in the peroxiredoxin/Tr x2 antioxidant couple by recycling reducing equivalents to Trx2. Hence, TrxR2 and Trx2 work as a unit to maintain peroxiredoxin3s H2O2 scavenging capacity. In summary, our collective findings indicate that ExTr-induced improvement of mitochondrial antioxi dative capacity may be involved in ExTrinduced mitochondrial protection (both SSM and IFM) against IR-induced oxidative injury. Conclusions and Future Directions These experiments provide the first direct evidence that ExTr supplies protection to cardiac mitochondria against IR-induced damage. Specifica lly, our findings reveal that ExTr protects both SSM and IFM against IR-indu ced injury and respiratory dysfunction. We interpret these results as proof that mitochondrial adaptation is a key cellular event that c ontributes to exerciseinduced cardioprotection. Although our experiments do not provide a defi nitive explanation for the mechanism(s) responsible for ExTr-induced protection of mitochondria, our data suggest that two key possibilities exist: 1) ExTr pr events the IR-induced increase in mitochondrial ROS production; and 2) ExTr promotes an increase in mito chondrial antioxidants. Collectively, these ExTr induced changes in mitochondrial phenotype resu lt in protection against IR-induced oxidative damage/respiratory dysfunction alon g with a retardation of the rel ease of proapoptotic proteins. Future experiments are required to define th e definitive mechanism(s) responsible for ExTr-induced mitochondrial protection. These ex periments could employ several strategies including siRNA to retard exercise-mediated e xpression of selected antioxidant proteins or 75

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transgenic animals to define the role that one or more specific proteins play in mitochondrial protection against an IR inju ry. Delineating the mechanism(s) responsible for mitochondrial protection during IR could lead to the de velopment of pharmacological or molecular interventions to protect the heart ag ainst IR injury. This is an exciting area for future research. 76

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LIST OF REFERENCES 1. Adderley SR and Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2mediated induction of cyclooxygenase-2. J Biol Chem 274: 5038-5046, 1999. 2. Adlam VJ, Harrison JC, Porteous CM, James AM, Smith RA, Murphy MP, and Sammut IA. Targeting an antioxidant to mito chondria decreases cardiac ischemiareperfusion injury. Faseb J 19: 1088-1095, 2005. 3. Aldakkak M, Stowe DF, Chen Q, Lesnefsky EJ, and Camara AK. Inhibited mitochondrial respiration by amobarbital dur ing cardiac ischaemia improves redox state and reduces matrix Ca2+ overload and ROS release. Cardiovasc Res 77: 406-415, 2008. 4. Alessio HM and Goldfarb AH. Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. J Appl Physiol 64: 1333-1336, 1988. 5. Andreyev AY, Kushnareva YE, and Starkov AA. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70: 200-214, 2005. 6. Angelos MG, Kutala VK, Torres CA, He G, Stoner JD, Mohammad M, and Kuppusamy P. Hypoxic reperfusion of the ischemic heart and oxygen radical generation. Am J Physiol Heart Circ Physiol 290: H341-347, 2006. 7. Arrington DD, Van Vleet TR, and Schnellmann RG. Calpain 10: a mitochondrial calpain and its role in calciu m-induced mitochondrial dysfunction. Am J Physiol Cell Physiol 291: C1159-1171, 2006. 8. Ascensao A, Ferreira R, Oliveira PJ, and Magalhaes J. Effects of endurance training and acute Doxorubicin treatment on rat heart mitochondrial alterations induced by in vitro anoxia-reoxygenation. Cardiovasc Toxicol 6: 159-172, 2006. 9. Ascensao A, Magalhaes J, Soares JM, Ferreira R, Neuparth MJ, Marques F, Oliveira PJ, and Duarte JA. Endurance training limits the f unctional alterations of heart rat mitochondria submitted to in vitro anoxia-reoxygenation. Int J Cardiol 109: 169-178, 2006. 10. Asimakis GK, Lick S, and Patterson C. Postischemic recovery of contractile function is impaired in SOD2(+/-) but not SOD1(+/-) mouse hearts. Circulation 105: 981-986, 2002. 11. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, and Molkentin JD. Loss of cyclophilin D reveals a critical role for mitochondrial permeabil ity transition in cell death. Nature 434: 658-662, 2005. 77

PAGE 78

12. Bolli R. Causative role of oxyradicals in m yocardial stunning: a proven hypothesis. A brief review of the evidence demonstrating a major role of re active oxygen species in several forms of postischemic dysfunction. Basic Res Cardiol 93: 156-162, 1998. 13. Bolli R. Mechanism of myo cardial "stunning." Circulation 82: 723-738, 1990. 14. Bolli R. Oxygen-derived free radicals and postischemic myocardial dysfunction ("stunned myocardium"). J Am Coll Cardiol 12: 239-249, 1988. 15. Borutaite V, Budriunaite A, Morkuniene R, and Brown GC. Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia. Biochim Biophys Acta 1537: 101-109, 2001. 16. Bowles DK, Farrar RP, and Starnes JW. Exercise training improves cardiac function after ischemia in the isolated, working rat heart. Am J Physiol 263: H804-809, 1992. 17. Bowles DK and Starnes JW. Exercise training improve s metabolic response after ischemia in isolated working rat heart. J Appl Physiol 76: 1608-1614, 1994. 18. Bradford MM. A rapid and sensitive method fo r the quantitation of microgram quantities of protein utilizing th e principle of protein-dye binding. Anal Biochem 72: 248254, 1976. 19. Brown DA, Chicco AJ, Jew KN, Johnson MS, Lynch JM, Watson PA, and Moore RL. Cardioprotection afforded by chronic exercise is mediated by the sarcolemmal, and not the mitochondrial, isoform of the KATP channel in the rat. J Physiol 569: 913-924, 2005. 20. Buss H, Chan TP, Sluis KB, Domigan NM, and Winterbourn CC. Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 23: 361-366, 1997. 21. Cande C, Cohen I, Daugas E, Ravagnan L, Larochette N, Zamzami N, and Kroemer G. Apoptosis-inducing factor (AIF): a novel caspase-independen t death effector released from mitochondria. Biochimie 84: 215-222, 2002. 22. Cande C, Vahsen N, Kouranti I, Schmitt E, Daugas E, Spahr C, Luban J, Kroemer RT, Giordanetto F, Garrido C, Penninger JM, and Kroemer G. AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene 23: 1514-1521, 2004. 23. Chae HZ, Kim HJ, Kang SW, and Rhee SG. Characterization of three isoforms of mammalian peroxiredoxin that reduce per oxides in the presence of thioredoxin. Diabetes Res Clin Pract 45: 101-112, 1999. 24. Chambers DE, Parks DA, Patterson G, Roy R, McCord JM, Yoshida S, Parmley LF, and Downey JM. Xanthine oxidase as a source of free radical damage in myocardial ischemia. J Mol Cell Cardiol 17: 145-152, 1985. 78

PAGE 79

25. Chang TS, Cho CS, Park S, Yu S, Kang SW, and Rhee SG. Peroxiredoxin III, a mitochondrion-specific peroxidase, regulat es apoptotic signaling by mitochondria. J Biol Chem 279: 41975-41984, 2004. 26. Chen M, He H, Zhan S, Krajewski S, Reed JC, and Gottlieb RA. Bid is cleaved by calpain to an active fragme nt in vitro and during myo cardial ischemia/reperfusion. J Biol Chem 276: 30724-30728, 2001. 27. Chen M, Won DJ, Krajewski S, and Gottlieb RA. Calpain and mitochondria in ischemia/reperfusion injury. J Biol Chem 277: 29181-29186, 2002. 28. Chen Q, Hoppel CL, and Lesnefsky EJ. Blockade of electron transport before cardiac ischemia with the reversible inhibitor amobarbital protects rat heart mitochondria. J Pharmacol Exp Ther 316: 200-207, 2006. 29. Chen Q and Lesnefsky EJ. Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. Free Radic Biol Med 40: 976-982, 2006. 30. Chen Q, Moghaddas S, Hoppel CL, and Lesnefsky EJ. Ischemic defects in the electron transport chain increase the producti on of reactive oxygen species from isolated rat heart mitochondria. Am J Physiol Cell Physiol 2007. 31. Chen Q, Moghaddas S, Hoppel CL, and Lesnefsky EJ. Ischemic defects in the electron transport chain increase the producti on of reactive oxygen species from isolated rat heart mitochondria. Am J Physiol Cell Physiol 294: C460-466, 2008. 32. Chen Q, Moghaddas S, Hoppel CL, and Lesnefsky EJ. Reversible blockade of electron transport during isch emia protects mitochondria and decreases myocardial injury following reperfusion. J Pharmacol Exp Ther 319: 1405-1412, 2006. 33. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, and Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 278: 36027-36031, 2003. 34. Chen Z, Siu B, Ho YS, Vincent R, Chua CC, Hamdy RC, and Chua BH. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol 30: 2281-2289, 1998. 35. Chiueh CC, Andoh T, and Chock PB. Induction of thioredoxin and mitochondrial survival proteins mediates preconditioni ng-induced cardioprotecti on and neuroprotection. Ann N Y Acad Sci 1042: 403-418, 2005. 36. Cho J, Won K, Wu D, Soong Y, Liu S, Szeto HH, and Hong MK. Potent mitochondria-targeted peptides redu ce myocardial infarction in rats. Coron Artery Dis 18: 215-220, 2007. 79

PAGE 80

37. Crow MT, Mani K, Nam YJ, and Kitsis RN. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res 95: 957-970, 2004. 38. Daugas E, Susin SA, Zamzami N, Ferri KF Irinopoulou T, Larochette N, Prevost MC, Leber B, Andrews D, Penninger J, and Kroemer G. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. Faseb J 14: 729-739, 2000. 39. Davies KJ and Goldberg AL. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. J Biol Chem 262: 82208226, 1987. 40. Davies KJ, Lin SW, and Pacifici RE. Protein damage and degradation by oxygen radicals. IV. Degradatio n of denatured protein. J Biol Chem 262: 9914-9920, 1987. 41. de Jesus Garcia-Rivas G, Guerrero-Hern andez A, Guerrero-Serna G, RodriguezZavala JS, and Zazueta C. Inhibition of the mitochondr ial calcium uniporter by the oxo-bridged dinuclear ruthenium amine complex (Ru360) prevents from irreversible injury in postischemic rat heart. Febs J 272: 3477-3488, 2005. 42. Demirel HA, Powers SK, Caillaud C, Coombe s JS, Naito H, Fletcher LA, Vrabas I, Jessup JV, and Ji LL. Exercise training re duces myocardial lipid peroxidation following short-term ischemia-reperfusion. Med Sci Sports Exerc 30: 1211-1216, 1998. 43. Demirel HA, Powers SK, Zergeroglu MA, Shanely RA, Hamilton K, Coombes J, and Naito H. Short-term exercise improves myocar dial tolerance to in vivo ischemiareperfusion in the rat. J Appl Physiol 91: 2205-2212, 2001. 44. Downey JM. Free radicals and their involvement dur ing long-term myocardial ischemia and reperfusion. Annu Rev Physiol 52: 487-504, 1990. 45. Fannin SW, Lesnefsky EJ, Slabe TJ, Hassan MO, and Hoppel CL. Aging selectively decreases oxidative capacity in ra t heart interfibri llar mitochondria. Arch Biochem Biophys 372: 399-407, 1999. 46. Fliss H and Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79: 949-956, 1996. 47. French JP, Quindry JC, Falk DJ, Staib JL, Lee Y, Wang KK, and Powers SK. Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise tr aining and calpain inhibition. Am J Physiol Heart Circ Physiol 290: H128-136, 2006. 48. Garcia M, Bondada V, and Geddes JW. Mitochondrial localiza tion of mu-calpain. Biochem Biophys Res Commun 338: 1241-1247, 2005. 49. Garcia-Rivas Gde J, Carvajal K, Correa F, and Zazueta C. Ru360, a specific mitochondrial calcium uptake inhibitor, improves cardiac post-ischaemic functional recovery in rats in vivo. Br J Pharmacol 149: 829-837, 2006. 80

PAGE 81

50. Garlick PB, Davies MJ, Hearse DJ, and Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res 61: 757-760, 1987. 51. Gottlieb E, Armour SM, Harris MH, and Thompson CB. Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ 10: 709-717, 2003. 52. Gottlieb RA. Mitochondria and apoptosis. Biol Signals Recept 10: 147-161, 2001. 53. Gottlieb RA. Mitochondria: execution central. FEBS Lett 482: 6-12, 2000. 54. Gottlieb RA. Mitochondrial signaling in apoptos is: mitochondrial daggers to the breaking heart. Basic Res Cardiol 98: 242-249, 2003. 55. Gottlieb RA. Role of mitochondria in apoptosis. Crit Rev Eukaryot Gene Expr 10: 231239, 2000. 56. Granville DJ and Gottlieb RA. Mitochondria: regulators of cell death and survival. ScientificWorldJournal 2: 1569-1578, 2002. 57. Green DR and Kroemer G. The pathophysiology of mitochondrial cell death. Science 305: 626-629, 2004. 58. Gross GJ and Peart JN. KATP channels and myocardi al preconditioning: an update. Am J Physiol Heart Circ Physiol 285: H921-930, 2003. 59. Halestrap A. Biochemistry: a pore way to die. Nature 434: 578-579, 2005. 60. Halestrap AP. Calcium, mitochondria and reperf usion injury: a pore way to die. Biochem Soc Trans 34: 232-237, 2006. 61. Halestrap AP, Clarke SJ, and Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion--a target for cardioprotection. Cardiovasc Res 61: 372-385, 2004. 62. Halliwell B. Mechanisms involved in the generation of free radicals. Pathol Biol (Paris) 44: 6-13, 1996. 63. Halliwell B, and J.M.C. Gutteridge. Free radicals in biology and medicine. New York: Oxford University Press, 1999. 64. Halliwell B and Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 57: 715S-724S; discussion 724S-725S, 1993. 65. Hamilton KL, Powers SK, Sugiura T, Kim S, Lennon S, Tumer N, and Mehta JL. Short-term exercise training can improve myo cardial tolerance to I/R without elevation in heat shock proteins. Am J Physiol Heart Circ Physiol 281: H1346-1352, 2001. 81

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66. Hamilton KL, Quindry JC, French JP, St aib J, Hughes J, Mehta JL, and Powers SK. MnSOD antisense treatment and exercise -induced protection against arrhythmias. Free Radic Biol Med 37: 1360-1368, 2004. 67. Hamilton KL, Staib JL, Phillips T, Hess A, Lennon SL, and Powers SK. Exercise, antioxidants, and HSP72: protection ag ainst myocardial ischemia/reperfusion. Free Radic Biol Med 34: 800-809, 2003. 68. Hein S, Scheffold T, and Schaper J. Ischemia induces early changes to cytoskeletal and contractile proteins in diseased human myocardium. J Thorac Cardiovasc Surg 110: 8998, 1995. 69. Hoffman JW, Jr, Gilbert TB, Poston RS, and Silldorff EP. Myocardial reperfusion injury: etiology, mechanisms, and therapies. J Extra Corpor Technol 36: 391-411, 2004. 70. Honda HM, Korge P, and Weiss JN. Mitochondria and isch emia/reperfusion injury. Ann N Y Acad Sci 1047: 248-258, 2005. 71. Hoshida S, Yamashita N, Otsu K, and Hori M. Repeated physiologic stresses provide persistent cardioprotection against ischemia-reperfusion injury in rats. J Am Coll Cardiol 40: 826-831, 2002. 72. Husain K and Hazelrigg SR. Oxidative injury due to chronic nitric oxide synthase inhibition in rat: effect of regular exercise on the heart. Biochim Biophys Acta 1587: 7582, 2002. 73. Ignarro LJ, Balestrieri ML, and Napoli C. Nutrition, physical activity, and cardiovascular disease: an update. Cardiovasc Res 73: 326-340, 2007. 74. Ji LL, Fu RG, Mitchell EW, Griffi ths M, Waldrop TG, and Swartz HM. Cardiac hypertrophy alters myocardial response to ischaemia and reperfusion in vivo. Acta Physiol Scand 151: 279-290, 1994. 75. Kakkar R, Wang X, Radhi JM, Ra jala RV, Wang R, and Sharma RK. Decreased expression of high-molecular-weight calmodu lin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart. Cell Calcium 29: 59-71, 2001. 76. Kavazis AN, McClung JM, Hood DA, and Powers SK. Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli. Am J Physiol Heart Circ Physiol 294: H928-935, 2008. 77. Kim GT, Chun YS, Park JW, and Kim MS. Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion. Biochem Biophys Res Commun 309: 619624, 2003. 78. Kitakaze M, Weisman HF, and Marban E. Contractile dysfunction and ATP depletion after transient calcium overlo ad in perfused ferret hearts. Circulation 77: 685-695, 1988. 82

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79. Kong X, Tweddell JS, Gross GJ, and Baker JE. Sarcolemmal and mitochondrial K(atp)channels mediate cardioprot ection in chronically hypoxic hearts. J Mol Cell Cardiol 33: 1041-1045, 2001. 80. Kwon JH, Kim JB, Lee KH, Kang SM, Chung N, Jang Y, and Chung JH. Protective effect of heat shock protein 27 using pr otein transduction doma in-mediated delivery on ischemia/reperfusion heart injury. Biochem Biophys Res Commun 363: 399-404, 2007. 81. 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. 82. Lemasters JJ. The mitochondrial permeability tran sition and the calcium, oxygen and pH paradoxes: one paradox after another. Cardiovasc Res 44: 470-473, 1999. 83. 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. 84. 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. 85. Lennon SL, Quindry JC, Hamilton KL, French JP, Hughes J, Mehta JL, and Powers SK. Elevated MnSOD is not required fo r exercise-induced cardioprotection against myocardial stunning. Am J Physiol Heart Circ Physiol 287: H975-980, 2004. 86. Lesnefsky EJ. Reduction of infarct size by cell-pe rmeable oxygen metabolite scavengers. Free Radic Biol Med 12: 429-446, 1992. 87. Lesnefsky EJ, Chen Q, Moghaddas S, Hassan MO, Tandler B, and Hoppel CL. Blockade of electron transport during ischemia protects cardiac mitochondria. J Biol Chem 279: 47961-47967, 2004. 88. Lesnefsky EJ, Gudz TI, Migita CT, Iked a-Saito M, Hassan MO, Turkaly PJ, and Hoppel CL. Ischemic injury to mitochondrial el ectron transport in the aging heart: damage to the iron-sulfur protein su bunit of electron transport complex III. Arch Biochem Biophys 385: 117-128, 2001. 89. Lesnefsky EJ, Gudz TI, Moghaddas S, Migi ta CT, Ikeda-Saito M, Turkaly PJ, and Hoppel CL. Aging decreases electron transport co mplex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site. J Mol Cell Cardiol 33: 37-47, 2001. 90. Lesnefsky EJ and Hoppel CL. Ischemia-reperfusion injury in the aged heart: role of mitochondria. Arch Biochem Biophys 420: 287-297, 2003. 83

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91. Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, and Hoppel CL. Mitochondrial dysfunction in cardiac disease: ischemia --reperfusion, aging, and heart failure. J Mol Cell Cardiol 33: 1065-1089, 2001. 92. Lesnefsky EJ, Tandler B, Ye J, Sl abe TJ, Turkaly J, and Hoppel CL. Myocardial ischemia decreases oxidative phosphory lation through cytochrome oxidase in subsarcolemmal mitochondria. Am J Physiol 273: H1544-1554, 1997. 93. Levraut J, Iwase H, Shao ZH, Vanden Hoek TL, and Schumacker PT. Cell death during ischemia: relationship to mito chondrial depolarization and ROS generation. Am J Physiol Heart Circ Physiol 284: H549-558, 2003. 94. Li C and Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 282: C227-241, 2002. 95. Li Q, Bolli R, Qiu Y, Tang XL, Murphree SS, and French BA. Gene therapy with extracellular superoxide dismutase attenuate s myocardial stunning in conscious rabbits. Circulation 98: 1438-1448, 1998. 96. Libonati JR, Gaughan JP, Hefner CA, Gow A, Paolone AM, and Houser SR. Reduced ischemia and reperfusion injury following exercise training. Med Sci Sports Exerc 29: 509-516, 1997. 97. Liu JC, He M, Wan L, and Cheng XS. Heat shock protein 70 ge ne transfection protects rat myocardium cell agains t anoxia-reoxygeneration injury. Chin Med J (Engl) 120: 578583, 2007. 98. Lucas DT and Szweda LI. Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction. Proc Natl Acad Sci U S A 95: 510-514, 1998. 99. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, and Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95: 1446-1456, 1995. 100. Marinovic J, Ljubkovic M, Stadnicka A, Bosnjak ZJ, and Bienengraeber M. Role of sarcolemmal ATP-sensitive potassium cha nnel in oxidative stress-induced apoptosis: mitochondrial connection. Am J Physiol Heart Circ Physiol 294: H1317-1325, 2008. 101. Martindale JJ, Fernandez R, Thuerauf D, Whittaker R, Gude N, Sussman MA, and Glembotski CC. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6. Circ Res 98: 1186-1193, 2006. 102. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159-163, 1985. 103. McCord JM and Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055, 1969. 84

PAGE 85

104. Meister A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmacol Ther 51: 155-194, 1991. 105. Motegi K, Tanonaka K, Takena ga Y, Takagi N, and Takeo S. Preservation of mitochondrial function may contribute to cardioprotective effects of Na+/Ca2+ exchanger inhibitors in ischaemic/reperfused rat hearts. Br J Pharmacol 151: 963-978, 2007. 106. Mungrue IN, Gros R, You X, Pirani A, Azad A, Csont T, Schulz R, Butany J, Stewart DJ, and Husain M. Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death. J Clin Invest 109: 735-743, 2002. 107. Murata M, Akao M, O'Rourke B, and Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2 +) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res 89: 891-898, 2001. 108. Murlasits Z, Lee Y, and Powers SK. Short-term exercise does not increase ER stress protein expression in cardiac muscle. Med Sci Sports Exerc 39: 1522-1528, 2007. 109. Mustacich D and Powis G. Thioredoxin reductase. Biochem J 346 Pt 1: 1-8, 2000. 110. Nakagawa T, Shimizu S, Watanabe T, Yam aguchi O, Otsu K, Yamagata H, Inohara H, Kubo T, and Tsujimoto Y. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434: 652-658, 2005. 111. Nakashima K, Nonaka I, and Masaki S. Myofibrillar proteoly sis in chick myotubes during oxidative stress. J Nutr Sci Vitaminol (Tokyo) 50: 45-49, 2004. 112. Neuhof C, Gotte O, Trumbeckaite S, Attenberger M, Kuzkaya N, Gellerich F, Moller A, Lubisch W, Speth M, Tillmanns H, and Neuhof H. A novel water-soluble and cell-permeable calpain inhibitor protects myocardial and mitochondrial function in p ostischemic reperfusion. Biol Chem 384: 1597-1603, 2003. 113. Oscai LB, Mole PA, and Holloszy JO. Effects of exercise on cardiac weight and mitochondria in male and female rats. Am J Physiol 220: 1944-1948, 1971. 114. Palmer JW, Tandler B, and Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252: 87318739, 1977. 115. Park JL and Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thorac Surg 68: 1905-1912, 1999. 116. Plumier JC, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, and Pagoulatos GN. Transgenic mice expressing the hu man heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 95: 1854-1860, 1995. 85

PAGE 86

117. Powers SK, Criswell D, Lawler J, Martin D, Lieu FK, Ji LL, and Herb RA. Rigorous exercise training increases superoxide di smutase activity in ventricular myocardium. Am J Physiol 265: H2094-2098, 1993. 118. Powers SK, Demirel HA, Vincent HK, Coom bes JS, Naito H, Hamilton KL, Shanely RA, and Jessup J. Exercise training improves myocardi al tolerance to in vivo ischemiareperfusion in the rat. Am J Physiol 275: R1468-1477, 1998. 119. Powers SK, DeRuisseau KC, Quindry J, and Hamilton KL. Dietary antioxidants and exercise. J Sports Sci 22: 81-94, 2004. 120. Powers SK, Lennon SL, Quindry J, and Mehta JL. Exercise and cardioprotection. Curr Opin Cardiol 17: 495-502, 2002. 121. Quindry J, French J, Hamilton K, Lee Y, Mehta JL, and Powers S. Exercise training provides cardioprotection against ischem ia-reperfusion induced apoptosis in young and old animals. Exp Gerontol 40: 416-425, 2005. 122. Quindry J, J. French, Hamilton, K.L., Lee, Y, Selsby, J., & Powers, S.K. Cyclooxygenase-2 is unaltered by ex ercise in the young and old heart. Medicine and Science in Sports and Exercise 2006. 123. Quindry JC, Hamilton KL, French JP, Lee Y, Murlasits Z, Tumer N, and Powers SK. Exercise-induced HSP-72 elevation and cardioprotection ag ainst infarct and apoptosis. J Appl Physiol 103: 1056-1062, 2007. 124. Reeve JL, Duffy AM, O'Brien T, and Samali A. Don't lose heart--therapeutic value of apoptosis prevention in the trea tment of cardiovascular disease. J Cell Mol Med 9: 609622, 2005. 125. Riva A, Tandler B, Lesnefsky EJ, Conti G, Loffredo F, Vazquez E, and Hoppel CL. Structure of cristae in cardi ac mitochondria of aged rat. Mech Ageing Dev 127: 917-921, 2006. 126. Romaschin AD, Rebeyka I, Wilson GJ, and Mickle DA. Conjugated dienes in ischemic and reperfused myocardium: an in vivo chemical signature of oxygen free radical mediated injury. J Mol Cell Cardiol 19: 289-302, 1987. 127. Romaschin AD, Wilson GJ, Thomas U, Feitler DA, Tumiati L, and Mickle DA. Subcellular distribution of peroxidized lipids in myocardial reperfusion injury. Am J Physiol 259: H116-123, 1990. 128. Sandmann S, Yu M, and Unger T. Transcriptional and tran slational regulation of calpain in the rat heart after myocardial infarction--effects of AT(1) and AT(2) receptor antagonists and ACE inhibitor. Br J Pharmacol 132: 767-777, 2001. 129. Solaini G and Harris DA. Biochemical dysfunction in he art mitochondria exposed to ischaemia and reperfusion. Biochem J 390: 377-394, 2005. 86

PAGE 87

130. Starnes JW, Barnes BD, and Olsen ME. Exercise training decreases reactive oxygen species generation but does not attenuat e Ca2+-induced dysf unction in rat heart mitochondria. J Appl Physiol, 2007. 131. Starnes JW, Taylor RP, and Ciccolo JT. Habitual low-intensity exercise does not protect against myocardial dysf unction after ischemia in rats. Eur J Cardiovasc Prev Rehabil 12: 169-174, 2005. 132. Sturtz LA, Diekert K, Jensen LT, Lill R, and Culotta VC. A fraction of yeast Cu,Znsuperoxide dismutase and its metallocha perone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J Biol Chem 276: 38084-38089, 2001. 133. Susin SA, Lorenzo HK, Zamzami N, Marz o I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, and Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441-446, 1999. 134. Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, and Takahashi R. A serine protease, HtrA2, is released from the mito chondria and interacts with XIAP, inducing cell death. Mol Cell 8: 613-621, 2001. 135. 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. 136. Taylor RP, Olsen ME, and Starnes JW. Improved postischemic function following acute exercise is not mediated by nitr ic oxide synthase in the rat heart. Am J Physiol Heart Circ Physiol 292: H601-607, 2007. 137. Tsuji T, Ohga Y, Yoshikawa Y, Sakata S, Abe T, Tabayashi N, Kobayashi S, Kohzuki H, Yoshida KI, Suga H, Kitamura S, Taniguchi S, and Takaki M. Rat cardiac contractile dysfunction induced by Ca2+ overload: possi ble link to the proteolysis of alpha-fodrin. Am J Physiol Heart Circ Physiol 281: H1286-1294, 2001. 138. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 552: 335-344, 2003. 139. Vainshtein BK, Melik-Adamyan WR, Bary nin VV, Vagin AA, and Grebenko AI. Three-dimensional structure of the enzyme catalase. Nature 293: 411-412, 1981. 140. Venditti P, Masullo P, and Di Meo S. Effects of myocardial ischemia and reperfusion on mitochondrial func tion and susceptibility to oxidative stress. Cell Mol Life Sci 58: 1528-1537, 2001. 141. Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong PC, and Zweier JL. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postischemic injury. Proc Natl Acad Sci U S A 95: 4556-4560, 1998. 87

PAGE 88

142. Wu J and Kaufman RJ. From acute ER stress to physiological roles of the Unfolded Protein Response. Cell Death Differ 13: 374-384, 2006. 143. Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, and Hori M. Exercise provides direct biphasic car dioprotection via manganese s uperoxide dismutase activation. J Exp Med 189: 1699-1706, 1999. 144. Yu BP. Cellular defenses against dama ge from reactive oxygen species. Physiol Rev 74: 139-162, 1994. 145. Yu BP, Suescun EA, and Yang SY. Effect of age-related lipid peroxidation on membrane fluidity and phospholipase A2: modulation by dietary restriction. Mech Ageing Dev 65: 17-33, 1992. 146. Zhou M, Diwu Z, Panchuk-Voloshina N, and Haugland RP. A stable nonfluorescent derivative of resorufin for the fluorometri c determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253: 162-168, 1997. 147. Zweier JL. Measurement of superoxide-derived fr ee radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem 263: 1353-1357, 1988. 148. Zweier JL, Duke SS, Kuppusamy P, Sylvester JT, and Gabrielson EW. Electron paramagnetic resonance evidence that cel lular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals. FEBS Lett 252: 12-16, 1989. 149. Zweier JL, Fertmann J, and Wei G. Nitric oxide and peroxynitrite in postischemic myocardium. Antioxid Redox Signal 3: 11-22, 2001. 150. Zweier JL, Kuppusamy P, Williams R, R ayburn BK, Smith D, Weisfeldt ML, and Flaherty JT. Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J Biol Chem 264: 18890-18895, 1989. 151. Zweier JL and Talukder MA. The role of oxidants and fr ee radicals in reperfusion injury. Cardiovasc Res 70: 181-190, 2006. 88

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BIOGRAPHICAL SKETCH Youngil Lee was born in Seoul, Korea. He atta ined a Bachelor of Science degree from Korean National Sport Univers ity-Seoul. After graduation, he pursued a masters degree in exercise biochemistry and graduated from Ko rean National University -Seoul in 1997. After finishing the masters degree, he became a lecturer at Kyung-Gi University and taught several classes for two years. Deciding to advance his scientific knowledge, Y oungil pursued his second masters degree in exercise physiology under the instruction of Dr. Roger P. Farrar and graduated from the University of Texas at Aust in in 2002. Deciding to focu s his career in basic science, Youngil began his docto ral work at the University of Florida in 2003 under the instruction of Dr. Scott K. Powers. Youngil focu sed his studies on the mechanisms responsible for exercise-induced cardioprotection against ischemia-reperfusion injury. He received his Ph.D. in 2008. 89