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Exploring the Mechanisms of Exercise-Induced Cardioprotection Against Myocardial Stunning

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Title:
Exploring the Mechanisms of Exercise-Induced Cardioprotection Against Myocardial Stunning
Creator:
Lennon, Shannon L. ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Antioxidants ( jstor )
Calcium ( jstor )
Free radicals ( jstor )
Heart ( jstor )
Ischemia ( jstor )
Myocardial stunning ( jstor )
Myocardium ( jstor )
Rats ( jstor )
Reperfusion ( jstor )
Superoxides ( jstor )

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University of Florida
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University of Florida
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Copyright Shannon L. Lennon. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
1/1/2004
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53185430 ( OCLC )

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EXPLORING THE MECHANISMS OF EXERCISE-INDUCED CARDIOPROTECTION AGAINST MYOCARDIAL STUNNING By SHANNON LIDUINA LENNON 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 2003

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Copyright 2003 by Shannon L. Lennon

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This dissertation is dedicated to David for loving me so completely and believing in me entirely. You are the best thing that ever happened to me and I thank heaven and the stars that you came into my life. I would not have made it through my last two years without your love and patience. You are my rock, baby. This is also dedicated to my folks Bill and Susanna. I want to thank my parents for being so supportive of my education. They were actually the first individuals to encourage me to pursue my PhD and it is wonderful to have parents who really advocate higher education. I couldn’t ask for better parents. Thank you.

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ACKNOWLEDGMENTS I would like to thank my mentor Dr. Scott Powers for taking a chance on me several years ago and bringing me into this lab. I had a tremendous learning experience as a graduate student and developing scientist because I was able to study and grow under his mentorship and guidance. His scientific knowledge, enthusiasm, and availability to work with me on the problems we encountered with the projects was unwavering. His willingness to come in on the weekend or a phone call to support me, clearly illustrates the type of mentor he is. For this, I am truly grateful. I would also like to acknowledge my committee. I thank Dr. Dodd for his assistance during my doctoral career. I thank Dr. Leeuwenburgh for his willingness to discuss science, the project, or running whenever I needed to. I thank Dr. Bailey for being a wonderful role model as a successful scientist and mother who seems able to balance all aspects of life extremely well. There are many people I want to acknowledge for their significant involvement in my dissertation. I would like to thank Dr. John Quindry and Joel French for their assistance on so many aspects of this project and the other two projects we completed simultaneously. Without them, I would not have finished. I would also like to thank Dr. Karyn Hamilton for her help with the training, the injections, and her support during the final months. I thank Andy Shanley for being a good friend throughout my time in the Powers laboratory. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION...........................................................................................................1 Specific Aims..................................................................................................................3 Significance.....................................................................................................................3 2 REVIEW OF LITERATURE..........................................................................................5 Introduction.....................................................................................................................5 Myocardial Stunning.......................................................................................................5 Mechanisms of Stunning.................................................................................................7 The Oxyradical Hypothesis......................................................................................8 The Calcium Hypothesis........................................................................................12 Calcium activated proteases.............................................................................13 Role of Ca 2+ in excitation-contraction (E-C) uncoupling................................14 Decreased Ca 2+ responsiveness by myofilaments............................................16 Combination of Oxyradical and Calcium Hypothesis...........................................17 Oxidative Stress............................................................................................................18 Radical Species......................................................................................................19 Superoxide (O 2 ).............................................................................................19 Hydroxyl radical (OH )....................................................................................19 Nitric oxide (NO )............................................................................................20 Non-radical Species...............................................................................................20 Hydrogen peroxide (H 2 O 2 )..............................................................................20 Peroxynitrite (ONOO ).....................................................................................21 Hypochlorous acid (HOCl)..............................................................................21 Damage Induced by ROS/RNS..............................................................................22 Lipids...............................................................................................................22 Proteins............................................................................................................23 Cellular Antioxidants....................................................................................................24 v

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Superoxide Dismutase (SOD)................................................................................24 Catalase (CAT).......................................................................................................25 Glutathione Peroxidase (GPx)................................................................................25 Glutathione (GSH).................................................................................................26 Other Antioxidants.................................................................................................27 Exercise and Cardioprotection......................................................................................27 Development of Collateral Vessels........................................................................28 Elevation in Myocardial Heat Shock Proteins.......................................................28 Myocardial Antioxidant Capacity..........................................................................29 Effect of Exercise on Antioxidant Status...............................................................29 Exercise and I-R injury..........................................................................................30 Manganese superoxide dismutase....................................................................32 Glutathione.......................................................................................................34 Summary.......................................................................................................................36 3 METHODS....................................................................................................................38 Animal Model...............................................................................................................38 Animal Model Justification....................................................................................38 Animal Housing and Diet......................................................................................38 Experimental Design.....................................................................................................38 Exercise Training Protocol.....................................................................................39 Inhibition of Myocardial MnSOD Protein Translation..........................................39 Overview of Sham and In vitro Working Heart I-R Protocol................................39 Details of Experimental Methods.................................................................................39 In vitro Working Heart Protocol............................................................................39 Sham Protocol........................................................................................................40 Cardiac Contractile Measurements........................................................................41 Exercise Training Protocol.....................................................................................41 Tissue Removal and Storage..................................................................................41 Inhibition of Myocardial MnSOD Protein Translation..........................................42 Biochemical Analysis of Antioxidant Enzyme Activity........................................42 Assessment of Reduced/Oxidized Glutathione......................................................43 Measurement of Lactate Dehydrogenase Activity.................................................43 Data Analysis.........................................................................................................43 4 RESULTS......................................................................................................................44 Animal Characteristics..................................................................................................44 Antioxidant Status of Animals in the Sham Group......................................................45 Cardiac Function of Isolated Perfused Hearts..............................................................45 Cellular Injury...............................................................................................................47 5 DISCUSSION................................................................................................................52 Overview of Principal Findings....................................................................................52 Exercise-induced Cardioprotection...............................................................................52 vi

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Mechanism(s) Responsible for Exercise-induced Cardioprotection.............................53 Other Potential Mechanisms of Exercise-induced Cardioprotection............................56 Catalase..................................................................................................................57 Inducible Nitric Oxide Synthase and Cyclooxygenase-2.......................................58 Critique of Experimental Model...................................................................................60 Conclusions...................................................................................................................61 LIST OF REFERENCES...................................................................................................63 BIOGRAPHICAL SKETCH.............................................................................................75 vii

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LIST OF TABLES Table page 4-1. Animal characteristics................................................................................................44 4-2. Effects of exercise training on left ventricular antioxidant status..............................45 4-3. Functional characteristics of pre-ischemic hearts.......................................................46 4-4. Functional characteristics of post-ischemic hearts.....................................................47 4-5. Percent recovery of functional characteristics of the heart.........................................47 viii

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LIST OF FIGURES Figure page 4-1. Percent recovery of cardiac work [cardiac output x systolic pressure (SP x CO)] at 30 minutes of reperfusion in I-R hearts......................................................................48 4-2. Changes in ventricular contractile function [cardiac output x systolic pressure (CO x SP)] relative to baseline from baseline (pre-ischemia) to reperfusion...................49 4-3. Effect of I-R injury on percent recovery of cardiac output (CO) at 30 minutes of reperfusion.............................................................................................................50 4-4. Effect of I-R injury on release of lactate dehydrogenase in the coronary effluent expressed as a percent of pre-ischemia values.......................................................51 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXPLORING THE MECHANISMS OF EXERCISE-INDUCED CARDIOPROTECTION AGAINST MYOCARDIAL STUNNING By Shannon L. Lennon May 2003 Chair: Scott K. Powers Department: Exercise and Sport Sciences Brief periods of ischemia followed by reperfusion results in stunning of the myocardium that is reversible within hours to days. This insult leads to contractile dysfunction and is associated with an increase in reactive oxygen species and calcium overload. Exercise has been shown to be cardioprotective against a stunning insult. The mechanisms behind this cardioprotection are unclear and are the focus of this experiment. These experiments tested the hypothesis that exercise-induced increases in manganese superoxide dismutase (MnSOD) activity and glutathione (GSH) levels in the heart are essential to achieve exercise-induced cardioprotection against myocardial stunning. To test this hypothesis, adult male rats were randomly assigned to 1 of 4 experimental groups: 1) sedentary, control with no treatment (S-C); 2) exercise trained, control no treatment (E-C); 3) exercise trained, treated with antisense oligonucleotide against MnSOD (E-AS); and 4) exercise trained, treated with mismatch oligonucleotide (E-MM). Groups were further subdivided into two surgery groups: sham or an in vitro working x

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heart ischemia-reperfusion (I-R) group to evaluate cardiac performance and contractile dysfunction. Exercise trained animals performed 60 minutes of treadmill running at ~70% VO 2 max for 3 consecutive days. Exercise—induced increases in myocardial MnSOD content and activity were inhibited using an AS oligonucleotide against MnSOD. A mismatch oligonucleotide was employed as a control to test the specificity of the AS oliogonucleotide against MnSOD. Exercise training increased MnSOD activity in the E-C and E-MM as compared to the S-C and E-AS groups (p < 0.05). In contrast, exercise did not alter (p > 0.05) myocardial GSH content in any experimental group. When hearts were subjected to an I-R insult of 25 minutes ischemia followed by 30 minutes of reperfusion, all trained groups showed significant recovery of pre-ischemic cardiac work (SP x CO) versus S-C. Additionally, the release of LDH, a marker of cellular damage, was significantly elevated in the S-C group as compared to the trained groups (p < 0.05). In conclusion, our results indicate that increases in myocardial GSH levels and MnSOD activity are not essential to achieve exercise-induced cardioprotection against stunning; therefore, exercise-induced cardioprotection must occur via other mechanisms. xi

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CHAPTER 1 INTRODUCTION Myocardial stunning is the mechanical dysfunction that occurs in the myocardium during reperfusion following a brief period of ischemia. An important distinction between myocardial infarction and myocardial stunning is that stunning does not result in irreversible damage to the myocardium. Brief periods of ischemia followed by reperfusion can occur frequently in many individuals before a major cardiac event such as a myocardial infarction or before a formal diagnosis of cardiovascular disease. Indeed, myocardial stunning is thought to be a part of the natural progression of coronary heart disease. Further, during treatment for various forms of coronary heart disease such as thrombolytic therapy, percutaneous transluminal coronary angioplasty, or coronary artery bypass graft surgery, brief ischemic periods can occur. These brief periods of ischemia followed by reperfusion result in stunning of the myocardium and reduced contractile function. Myocardial stunning has been postulated to occur via one of two mechanisms: the oxyradical hypothesis or the calcium overload hypothesis. The oxyradical hypothesis suggests that the generation of free radical species overwhelms the natural antioxidant defense systems in the myocardium and contribute significantly to the stunning and loss in contractile function. The calcium hypothesis argues that stunning is caused by a disturbance in the cellular calcium homeostasis that can result in impaired calcium sensitivity by myofilaments, excitation-contraction uncoupling, or decreased responsiveness of the contractile machinery to calcium. Both mechanisms are supported 1

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2 by numerous investigations but do not completely explain the mechanism independently. Therefore, it seems possible that a combination of these two hypotheses may best explain myocardial stunning. Exercise training, both short-term (days) and long-term (weeks), has been shown to improve myocardial I-R tolerance and this protection has been seen at all levels of I-R injury from stunning to infarction. Exercise training decreases cardiac arrhythmias, myocardial oxidative injury and cardiac contractile dysfunction during reperfusion following short-duration ischemia (20, 21, 33, 34, 51, 52, 62, 108, 127). The mechanism responsible for this exercise-induced cardioprotection remains unclear and is the focus of this investigation. Several mechanisms have been proposed including: 1) anatomic changes in the coronary arteries or increased collateral circulation; 2) induction of myocardial heat shock proteins; and 3) improved myocardial antioxidant capacity. Development of collateral circulation is not a viable mechanism, as exercise training does not alter collateral circulation in rats with short-term training (74). Further, exercise-induced cardioprotection has been shown in the absence of an elevation in myocardial heat shock proteins (51, 127). Hence, by elimination, exercise-induced increases in myocardial antioxidants could explain exercise-induced cardioprotection. Nonetheless, direct evidence for this supposition is limited. Induction of myocardial antioxidants by exercise is supported in the literature by numerous training studies (20, 33, 34, 51, 52, 62, 107, 108, 127). Specifically, exercise has been shown to increase levels of two important myocardial antioxidants: manganese superoxide dismutase (MnSOD) and glutathione (GSH) (107, 108, 138). Preliminary evidence from our laboratory suggests that exercise training increases both of these

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3 endogenous antioxidants and that they are essential for exercise-induced protection against myocardial stunning. Experiments in this study will rigorously test this hypothesis. Specific Aims The primary objective of these experiments was to determine if the exercise-induced cardioprotection against an in vitro I-R-induced myocardial stunning insult depends on both increased myocardial MnSOD activity and increased myocardial GSH content. To investigate the role of MnSOD activity in exercise-induced cardioprotection we administered an antisense oligonucleotide against MnSOD messenger RNA to inhibit the exercise-induced synthesis of this antioxidant protein. Cardiac performance in these hearts was evaluated after short-duration global ischemia in the in vitro working heart model. Aim one: To determine whether the exercise-induced cardioprotection during an in vitro myocardial I-R insult is dependent upon an increase in myocardial MnSOD levels. Hypothesis one: Prevention of exercise-induced MnSOD expression will eliminate exercise-mediated protection against myocardial stunning in those animals receiving the antisense oligodeoxynucleotide. Aim two: To determine whether the exercise-induced cardioprotection during an in vitro myocardial I-R insult is dependent upon an increase in myocardial GSH levels? Hypothesis two: Prevention of exercise-induced GSH synthesis will eliminate exercise-mediated protection against myocardial stunning in those animals receiving the pharamacological agent L-buthionine SR-sulfoximine. Significance Myocardial stunning is an important and common event in ischemia-reperfusion injuries. It is frequently seen during treatment for various forms of coronary heart disease such as thrombolytic therapy, percutaneous transluminal coronary angioplasty, or

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4 coronary artery bypass graft surgery, brief ischemic periods can occur. These procedures are common in individuals diagnosed with coronary heart disease (CHD). Coronary heart disease is the number one cause of death in the United States for both men and women. In 2000, the total cost of CHD in the U.S. was estimated at more than $100.8 billion and accounted for 459,841 deaths in the U.S. in 1998 (most recent AHA statistical information) translating to roughly 1 in every 5 deaths. Currently, it is estimated that 60,800,000 Americans have one or more types of cardiovascular disease (5). Importantly, it has been shown that regular exercise training is cardioprotective against a bout of I-R injury (20, 33, 34, 51, 52, 62, 107, 108, 127). What remains unclear is the mechanism behind this cardioprotection. These experiments were designed to advance our understanding of the mechanism responsible for exercise-induced cardioprotection against myocardial stunning by investigating two myocardial antioxidants that increase with exercise training.

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CHAPTER 2 REVIEW OF LITERATURE Introduction Short-duration myocardial ischemia followed by reperfusion is a relatively common event in many individuals before diagnosis of coronary heart disease. These brief periods of ischemia stun the myocardium for hours to days and reduce its contractile function as myocytes slowly recover from this insult. This phenomenon has been called myocardial stunning and interest in the mechanisms underlying the etiology of myocyte stunning has grown considerably. Exercise training has been shown to be cardioprotective and to reduce contractile dysfunction after an ischemic bout. The role of exercise in protecting role in the myocardium against contractile dysfunction associated with stunning drives this proposal. This review of literature is designed to explore the various topics associated with myocardial stunning, oxidative stress, and exercise-induced cardioprotection. Myocardial Stunning Braunwald and Kloner (23) first introduced the term myocardial stunning in 1982 but at the time, little was actually known about this phenomenon. However, in the late 1980s and continuing into the 1990s, interest in this area grew dramatically among both researchers and clinicians. Although much research has been done, it is still fragmented and linked to specific types of experimental settings and models. Myocardial stunning is the mechanical dysfunction that occurs with brief periods of ischemia and persists after reperfusion despite the absence of near irreversible damage and despite restoration of 5

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6 normal or near-normal coronary flow (13). Two key points of this definition are that post-ischemic dysfunction is fully reversible and that the dysfunction is not caused by a primary deficit of myocardial perfusion (16). Generally, it is thought that coronary occlusion of less than 20 minutes is not associated with development of myocardial necrosis. However, when occlusion is terminated, the function of the previously ischemic myocardium may be depressed for several days (23). Myocardial stunning is considered a heterogenous event because it is a syndrome that has been seen in a wide variety of clinical situations in which the myocardium is exposed to a transient ischemic period that does not result in frank infarction (13). Generally, the “classic model” of myocardial stunning has been a single, reversible ischemic episode lasting less than 20 minutes that does not result in any myocyte necrosis (16). Different experiments have shown variability in the duration of impairment to full recovery suggesting that stunning results in nonuniform impairment (14). Myocardial stunning can also occur after multiple, completely reversible ischemic episodes such as repeated (2-10 minute) bouts of occlusion that result in a longer prolonged contractile impairment with no permanent damage (6, 19, 27, 100, 116). This model differs from the single episode in that the mechanical dysfunction occurs more slowly and the total ischemic bout and its severity is not related to collateral perfusion during ischemia (19, 27). Some preconditioning can occur in response to the first few bouts of occlusion and result in no worsening of the overall severity of stunning; but after the third occlusion, this effect is negated (19). Myocardial stunning can also occur after in vitro global ischemia. The cellular viability in these preparations depends on many factors, such as species used (dog, ferret, rat, etc.); temperature; duration of ischemia; pH; and perfusate

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7 composition (13). In this model, it is not easy to verify the reversibility of contractile abnormalities, but some studies have shown normalization of phosphocreatine content and intracellular pH, suggesting that the heart was still viable (2, 3, 71, 91). Several factors contribute to the severity of the stunned myocardium. Stunning the heart makes it hyper-sensitive (13) and more susceptible to changes in temperature and pH that a healthy heart may better tolerate (19). Indeed, the severity and duration of impaired blood flow and temperature alterations are the most important factors in determining the dysfunction (13). The longer the ischemia and lack of blood flow, the greater the mechanical problems the heart will experience. Additionally, the inner layers of the ventricular wall are affected more severely than the outer layers (14). These factors play an important role in the stunning model. The next section will explore the mechanisms behind myocardial stunning. Mechanisms of Stunning In the 1980s, several hypotheses were posed to explain the etiology of myocardial stunning. Major hypotheses include the oxyradical hypothesis, the calcium hypothesis, excitation-contraction uncoupling due to sarcoplasmic reticulum dysfunction, insufficient energy production by mitochondria hypothesis, impaired energy use by myofibrils, and damage of the extracellular collagen matrix, to name a few. Most of these theories were subsequently abandoned. At present, only two viable theories remain regarding the pathogenesis of myocardial stunning: the oxyradical hypothesis and the calcium hypothesis. It is now thought that a combination of these two hypotheses is likely because in many regards they are not mutually exclusive. Each theory will be discussed in greater detail in the following sections.

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8 The Oxyradical Hypothesis The oxyradical hypothesis was first proposed in 1985 (97) when it was postulated that the generation of free radical species such as superoxide ion (O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH ) on reperfusion contributed significantly to the development of myocardial stunning. Many investigators then showed generation of free radicals in the “stunned” myocardium using a variety of models (11, 15, 63, 83, 131); showed improvement in post-ischemic contractile dysfunction using antioxidants (3, 12, 38, 64, 68, 97) or using free radical oxygen or metal scavengers to sequester ROS (4, 16, 98, 143). Free radicals generated during reperfusion can have several targets in the myocardium that contribute to the dysfunction. For example, two key cellular components, proteins and lipids, can be targets of free radicals. Reactive oxygen species (ROS) can oxidize amino acid side chains and can oxidize the protein backbone, leading to protein-protein cross-linking and protein fragmentation (9). The result of these actions can interfere with the normal activity of signaling proteins, potentially leading to organ failure. Damage to lipids can diminish membrane fluidity, increase membrane permeability, destabilize membrane receptors, and induce of an immune response to altered phospholipids (136). Damage to membranes can impair selective membrane permeability and can interfere with the function of various cellular organelles (16). Further, free radicals can interfere with cellular calcium transport and calcium-stimulated ATPase activity. Additionally, it has been suggested that oxidant stress-induced changes in ion translocating proteins can occur very quickly (<200 ms) before products of lipid peroxidation are detectable (118). Finally, DNA can also be targeted by modification of

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9 bases. This has been verified by the presence of 8-hyroxyguanine in hearts subjected to 30 min ischemia followed by 30 min reperfusion (30) The hypothesis that myocardial stunning is caused by production of ROS has been tested in several investigations. One approach has been to treat animals with superoxide dismutase (SOD) and catalase (CAT) 1 hour before a 15-minute occlusion (97). Superoxide dismutase and CAT had no noticeable effect on heart rate, aortic pressure, or left atrial pressure. Systolic wall thickening was similar in both groups at baseline and showed similar dyskinesia during ischemia. However, recovery of function was greater in treated dogs at 1 and 2 hours of reperfusion. This suggested that free radicals played an important role in myocardial dysfunction after a brief period of occlusion. Examining the effect of SOD and CAT infusion on infarct size further tested the use of antioxidants (64). Superoxide dismutase and CAT was infused before ischemia (group I), 15–min into reperfusion (Group II), or 40 min into reperfusion (Group III). Group I had the smallest infarct size at 19.4% (Group II was 21.8% and Group III was 47.6%). Group III was very similar to control at 43.6%. However, the percent of left ventricle at risk for infarction did not differ among groups. This study showed that free radical damage occurs within the first 40–min of reperfusion and those toxic oxygen metabolites are a secondary effect to the myocardial cellular damage incurred. Other investigators found similar results with SOD and CAT treatment using similar models (109). Administration of other types of oxidant scavenging molecules [such as dimethylthiourea and N-2-mercaptopropionylglycine (MPG)] that scavenge hydroxyl radical (OH ) produced significant and sustained improvement in the function of the stunned myocardium (11, 98). Administering MPG 1–min before reperfusion

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10 significantly attenuated myocardial contractile depression in the reperfused dog heart (11). Early administration of radical scavengers suggests that a critical window exists in the first minute of reperfusion (118). Hydroxyl radical can be produced from the Haber-Weiss reaction involving ferrous iron that is very reactive. Therefore, another approach is to give an iron chelator (such as desferrioxamine) early in reperfusion that has been shown to attenuate post-ischemic dysfunction (4, 16, 17). The use of antioxidants and oxidant scavenging molecules in the above-mentioned studies provided evidence of free radical generation in an ischemia-reperfused myocardium, but the evidence was indirect. To more directly link free radical generation with contractile dysfunction, investigators have used spin traps. Spin traps react with radicals to form a stable product that is detectable by electron spin resonance (ESR) — sometimes called electron paramagnetic resonance (EPR). Although ESR is the only technique that measures free radicals directly in vivo as it detects unpaired electrons, it is often not sensitive enough to detect the species directly (50). Typical traps used are 5,5-dimethylpyrroline-N-oxide (DMPO) and -phenyl-tert-butylnitrone (PBN). Zweier et al. (143) used EPR to directly measure free radical generation in rabbit hearts that underwent 10 min of global ischemia. The spectra of ischemic hearts exhibited a burst of oxygen-centered radicals peaking 10 seconds after flow was reintroduced. In another study, investigators were able to show a large initial burst of free radicals peaking at Minute 3 of reperfusion in a conscious open-chest dog model of stunning (83). This burst of free radicals then declined but remained detectable 1-3 hours later. A direct relationship was also seen between the magnitude of PBN adduct production and the severity of contractile dysfunction (83). Garlick et al. (46) also used

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11 the spin trap PBN to directly show free radical production during reperfusion in an isolated rat heart model. They found radical generation peaking at 4 minutes post-ischemia. However, if the heart was initially perfused with an anoxic buffer, no immediate burst of free radicals was seen. Upon return to an aerobic buffer, a quick burst of free radicals resulted. The investigators concluded sufficient oxygen appears essential to the production of these radicals. Bolli et al. (15), also using PBN, showed EPR signals characteristic of oxygenand carbon-centered radicals during 15-min of coronary occlusion and 30-min reperfusion in an open-chest dog model. Although PBN is a highly sensitive spin trap, a clear disadvantage is that it does not identify the oxyradical responsible for the damage. To pinpoint the type of ROS responsible for contractile dysfunction, Bolli et al. (12) combined PBN treatment with SOD and CAT infusion to target O 2 centered radicals in an open-chest dog model. He showed that SOD and CAT could effectively block free radical generation from the univalent reduction of O 2 , which improved functional recovery from myocardial stunning. Other techniques (such as the aromatic hydroxylation of phenylalanine) to detect OH were used in an open-chest dog model; hydroxylated derivatives of phenylalanine were detected in the first few minutes of reperfusion after 15 minutes of occlusion. Sun et al. (123) used a cocktail of antioxidants (including MPG, SOD, CAT, and desferrioxamine) to inhibit OH production in vivo. This resulted in attenuation of myocardial stunning and reduced the ventricular thickening fraction by almost 80%. While these models show free radical production during myocardial stunning, the source of these free radicals is not clear. Some potential sites include xanthine oxidase,

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12 peroxidation of arachidonic acid, autoxidation of catecholamines and other compounds, activation of various NAD(P)H oxidases, and the mitochondrial electron transport chain (13). Further, the evidence provided by Sun et al. accounted for 70-80% of the contractile dysfunction; but that does not address the mechanism behind the remaining 20-30% of dysfunction that occurs even in the presence of a cocktail of antioxidants. In his review, Shattock (118) concurs that free radicals are major initiators of stunning, but that cellular calcium overload is a serious factor in reperfusion injury and may account for a portion of the dysfunction. Although these studies appear to validate the oxyradical hypothesis, many gave only indirect evidence that oxygen acts as a harmful metabolite in myocardial dysfunction when using scavenging agents or preventing their generation (13). Also, the origin of scavenging agents is not clear nor is the mechanism underlying their effect on contractile dysfunction. Finally, studies using a variety of antioxidants and scavenging agents cannot completely retard the contractile dysfunction that occurs with stunning, suggesting that other mechanisms are at play in this complex paradigm. The Calcium Hypothesis Simply stated, the calcium hypothesis suggests that stunning is the result of a disturbance in cellular calcium homeostasis. Intracellular calcium levels are normally kept very low (in the 0.1 M range) while actual total cellular calcium is much higher. Most is sequestered in the mitochondria and the sarcoplasmic reticulum bound to cytoplasmic proteins (50). Studies have shown that Ca 2+ availability is not reduced in the stunned myocardium (43, 71). Also, estimates of maximal Ca 2+ -activated ventricular force in perfused hearts have demonstrated a decline that accounts for approximately half

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13 of the drop in pressure after stunning (73). Furthermore, several studies showed that intracellular Ca 2+ concentration increases during ischemia (69, 76, 90, 91, 122), remains elevated in early reperfusion (90) and returns to normal levels during late reperfusion (71). Therefore, Ca 2+ sensitivity may be impaired, uptake may be reduced, and/or Ca 2+ efflux via the Na + / Ca 2+ exchange may be marred. A cellular environment in which elevated calcium levels impair cardiac contractile function could occur by one of several mechanisms: 1) activation of calcium-activated proteases; 2) excitation-contraction uncoupling due to sarcoplasmic reticulum dysfunction; and 3) decreased responsiveness of the contractile machinery to calcium (13, 43, 45, 59, 89, 118). The following section addresses each of these possibilities and outline conclusions. Calcium activated proteases The two primary calcium-activated proteases found in the myocardium are -calpain (calpain I) and m-calpain (calpain II), are named for their sensitivity to intracellular calcium concentrations. Calpains are Ca 2+ -dependent cysteine proteases with four major properties including (in order of increased Ca 2+ concentration): 1) binding to subcellular organelles/plasma membrane; 2) binding to calpastatin; c) having proteolytic activity; and 4) self-autolysis (7). Unregulated calpain activity has been implicated in several cellular and pathological states such as Duchenne’s muscular dystrophy, Alzheimer’s disease, epilepsy, stroke, and apoptosis (ref). In myocardial I-R injury, an influx of Ca 2+ activates both and m-calpain (60, 67, 104, 132). Calpains play a potential role in Ca 2+ overload and in cytoskeletal and structural protein damage; they potentially activate apoptosis, as evidenced by DNA fragmentation and upregulation of Bax in myocardial I-R injury (67). The link between

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14 apoptosis and the calpains is not clear but one report links caspace-12 activation to m-calpain (99). The calpains are regulated in vivo by calpastatin, an endogenous inhibitor. When hearts are treated with calpain inhibitors (such as calpastatin or E64), contractile function is improved and infarct size is reduced (44, 60, 94, 132). Gao et al. (44) compared isolated stunned hearts (20 min I/20 min R) and skinned trabeculae exposed to calpain I. The skinned trabeculae exhibited troponin I (TnI) degradation similar to that seen in the stunned myocardium whereas adding calpastatin prevented TnI degradation. This study showed that TnI is selectively and partially degraded in the stunned myocardium, that calpain I could degrade TnI, and that a low Ca +2 reperfusion environment could prevent this. Van Eyk et al. (133) also found degradation of TnI using skinned rat trabeculae from stunned hearts. As the ischemic period increased from 15 to 60 minutes, there was a corresponding increase in TnI loss. A significant depression of force (~45%) was noted when ischemia was longer than 15 minutes with reperfusion but no depression was seen with 15 minutes of ischemia alone. This suggests that the cellular environment during reperfusion is key to the mechanisms underlying stunning. Another myofibrillar protein, -actinin had the same pattern as TnI, and myosin light chain-1 was degraded in the 60-minute ischemic group. Calpains also have been implicated in degrading other structural and cytoskeletal proteins (such as tropomyosin, C-protein, desmin, troponin (T, C, and I), filamin, nebulin, titin, -actinin, and myosin) (53, 104, 114, 125). Role of Ca 2+ in excitation-contraction (E-C) uncoupling The role of E-C uncoupling in calcium overload has been thought to occur from two potential mechanisms: either availability of Ca 2+ is restricted in the cell; or

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15 responsiveness of contractile machinery to Ca 2+ is decreased (13). Kusuoka et al. (73) found that stunned hearts show a decline in maximal Ca 2+ -activated force and a shift in the extracellular Ca 2+ concentration sensitivity. Gao et al. (43) measured intracellular Ca 2+ concentrations and force in thin ventricular trabeculae from stunned and control hearts and confirmed that Ca 2+ transients are not reduced in stunned myocardium. They showed a rightward shift of the intracellular Ca 2+ concentration-force relationship, suggesting that disruption of contractile proteins is involved. Research examining transporters involved in Ca 2+ movement in and out of the cell implicated the Na + /Ca 2+ transporter in mediating Ca 2+ influx during myocardial I-R, leading to calcium overload and injury (44, 94). The issue of calcium overload includes the Na + /Ca 2+ transporter and also other transporters. During the ischemic period, H + ions increase because of their greater reliance on anaerobic glycolysis and degradation of ATP. This cellular environment activates the Na + /H + transporter, causing Na + to move into the cell. Removal of this Na + from the cell is slowed as the Na + /K + ATPase transporter is inhibited during ischemia. This results in elevated levels of intracellular Na + (126) and ultimately higher intracellular Ca 2+ levels. This elevated Ca 2+ is thought to play a primary role in the myocardial injury accompanying stunning (31, 44, 69, 76, 90, 91, 94, 122, 128, 140). Alterations in sacroplasmic reticulum (SR) function can affect cellular Ca 2+ homeostasis. Temsah et al. (128) showed attenuated recovery of contractile function in rat hearts that had undergone a bout of I-R resulting in reduced SR Ca 2+ uptake, Ca 2+ release, and an alteration in the binding properties of the protein receptor ryanodine. Furthermore, mRNA levels and protein contents for the SR Ca 2+ ATPase pump and Ca 2+

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16 release channels were severely depressed in the I-R hearts, showing the importance of SR handling and release of Ca 2+ . In examining E-C uncoupling as the reason behind calcium overload, it is clear that Ca 2+ availability is the not the problem. Therefore, the dysfunction seen in E-C uncoupling must occur at the level of the contractile proteins; or perhaps the problem lies in a decreased responsiveness to Ca 2+ by the contractile machinery. Decreased Ca 2+ responsiveness by myofilaments The role of decreased Ca 2+ responsiveness of myofilaments in myocardial stunning is unclear. Evidence points to structural modifications of one or more of the myofibrillar proteins. A review by Kusuoka and Marban (72) suggests two mechanisms for the decrease in myofilament responsiveness to Ca 2+ : maximal force is decreased; and myofilaments are less sensitive even at submaximal intracellular Ca 2+ concentrations. Kusuoka et al. (73) previously showed a ~20% decrease in maximal Ca 2+ -activated force but the mechanism behind it was unclear. Kusuoka et al. (71) continued by using gated nuclear magnetic resonance (NMR) spectroscopy to determine Ca 2+ concentrations at various time-points. They found a decrease in myofilament sensitivity to Ca 2+ and an increase in Ca 2+ transient amplitude. Thus more ATP is spent on Ca 2+ sequestration, decreasing energy use by the myocardium, resulting in stunning. Using rat ventricular trabeculae strips, Gao et al. (45) also concluded that the decreased Ca 2+ responsiveness of the stunned myocardium was due to intrinsic alterations of the myofilaments. Van Eyk et al. (133) confirmed TnI and -actinin degradation in stunned hearts. Matsumura et al. (94) also specifically characterized several cytoskeletal proteins (such as -actinin, desmin, and spectrin) that were degraded during stunning.

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17 Although these isolated muscle preparations show strong support for dysfunction of contractile proteins, little evidence supports or negates this theory in vivo. Ito et al. (59) argued that an in vivo canine model of stunning, ventricular response to calcium was not impaired. However, these investigators did not quantify absolute force generation in their study; a limitation. In summary, the calcium hypothesis explains many aspects of myocardial stunning. Transient calcium overload has been implicated in the pathogenesis of stunning. Many thin-filament proteins are susceptible to proteolytic degradation although the functional consequences of that degradation are just beginning to be understood. Furthermore, the time course of stunning and its reversibility over several hours to days allows degraded contractile proteins to be replaced by newly synthesized ones to repair the myofilaments (13). However, calcium overload and proteolytic myofilament injury may not underlie all forms of stunning as evident in isolated heart models of global I-R. Finally, this hypothesis has yet to be tested in vivo and this is an issue that weakens the calcium hypothesis. Combination of Oxyradical and Calcium Hypothesis Because myocardial stunning is a multifactorial process that involves complex sequences of cellular perturbations and the interaction of multiple pathogenic mechanisms, these two hypotheses likely represent different parts of the same pathophysiological sequence (13, 16, 72, 118). Much evidence links the generation of ROS with alterations in calcium homeostasis in reperfusion injury. For example, oxyradicals can cause damage to the sacroplasmic reticulum and can alter calcium flux across the sarcolemma by slowing down transport action causing calcium overload (68). Free radicals may directly interact with the myofilament proteins and may compromise

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18 their function. Free radicals also could interfere with ATP delivery to the myofilaments by inhibiting creatine kinase and could initiate cellular overload that then activates Ca 2+ -dependent proteases to degrade structural and cytoskeletal proteins such as TnI (118). The calcium overload and increase in ROS could occur simultaneously; or one may precede the other. This viscous cycle ultimately injure the cardiac myocyte and results in myocardial stunning. Oxidative Stress Oxidative stress has been described as an imbalance between the pro-oxidants and antioxidants in the cell either by an increase in the pro-oxidants or a decrease in the antioxidant defenses thereby leading to oxidative damage (50). The pro-oxidants are ROS and RNS (reactive oxygen species and reactive nitrogen species) that inflict damage by targeting proteins, lipids, and DNA bases in the cell. The resultant cellular injury can transiently or permanently alter the homeostasis of the cell (101). The cell’s response to the injury may be reversible, entering a temporary or prolonged altered steady-state that does not lead to cell death. In some cases, the changes made by the cell become permanent (referred to as cellular adaptation). This can result in upregulation of antioxidant defense systems, heat shock proteins, or other proteins (50). When the cell cannot overcome the damage inflicted upon it by ROS/RNS, the cell will die. Because oxidative stress plays a central role in the contractile dysfunction seen during myocardial stunning, it is important to better understand the action of ROS/RNS. The following section discusses specific ROS/RNS and the metabolic pathways that generate them. Further, the next section discusses the endogenous myocardial antioxidant defense system and their role in protecting the myocardium.

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19 Radical Species Superoxide (O 2 ) Superoxide is formed by the addition of a single electron to an O 2 molecule. This results in an unpaired electron in the outer orbital that makes this molecule reactive. Superoxide is produced in vivo primarily by the electron transport chain in the mitochondria during aerobic metabolism. Superoxide can react with a variety of molecules (such as non-radical species, transition metals, or other radicals such as nitric oxide). Its reaction rates are much quicker with other radical species versus nonradicals. Superoxide plays an important role in myocardial I-R injury due to the aerobic nature of the myocardium. It has been reported to significantly increase immediately following reperfusion in in vitro models of I-R (15, 46). The use of scavenging molecules specific for oxygen-centered radicals have shown improved contractile recovery in stunned myocardium (3, 11, 12, 16, 17, 64, 97, 98). Hydroxyl radical (OH ) The hydroxyl radical is the most reactive oxygen radical. It reacts quickly with almost every type of molecule found in living cells (such as sugars, amino acids, phospholipids, and DNA bases) causing significant damage in the area surrounding it (50). Measurement of OH generation is difficult because of its high reactivity (rate constants of 10 9 to 10 10 M -1 s -1 ) and extremely short half-life (<~1nanosecond) (123). The OH radical has been purposed to be responsible for the majority of myocardial observed during I-R. It can be generated by the iron catalyzed Fenton’s reaction (or via the Haber Weiss reaction): 2O 2 + 2H + 2H 2 O 2 + O 2

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20 O 2 + Fe 3+ O 2 + Fe 2+ Fe 2+ + H 2 O 2 OH + OH + Fe 3+ Experiments using the iron chelator desferrioxamine and a radical scavenger MPG during reperfusion have shown an attenuation of stunning (3, 11, 17, 98). Nitric oxide (NO ) Nitric oxide is moderately soluble in water and more soluble in organic solvents, which allows it to easily diffuse in and out of cells (50). It has an unpaired electron making it a radical species. Nitric oxide is produced by family of nitric oxide synthases including: neuronal NOS (nNOS) found mostly in neurons, endothelial NOS (eNOS) produced by endothelial cells, and inducible NOS (iNOS) which is found ubiquitously. Nitric oxide acts as a vasodilator released by the endothelial cells. It causes smooth muscle cells to relax and lowers blood pressure, and inhibits platelet aggregation. Elevated levels of NO during oxidative stress can cause damage through its reaction with transition metals and O 2 . Its reaction with O 2 is very important because this pairing results in the production of peroxynitrite, which has been implicated in intimal damage to endothelial tissue (77). Further, in isolated heart preparations using EPR and chemiluminescence studies, NO adduct signals were seen during the first 2 min of reflow. Use of a NOS inhibitor, L-NAME significantly decreased NO concentration post-ischemia throughout 15 minutes of reperfusion as compared to control (142). Non-radical Species Hydrogen peroxide (H 2 O 2 ) Hydrogen peroxide is produced by a variety of molecules (such as xanthine, urate, and D-amino acid oxidases). It is also formed each time O 2 is dismutated. It is a weak

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21 oxidizing and reducing agent and not very reactive. However, it plays an important role in OH radical and HOCl production contributing to oxidative damage in the cell. Peroxynitrite (ONOO ) Peroxynitrite is formed by the reaction of O 2 with nitric oxide. This reaction is relatively fast and comparable to the rate at which O 2 reacts with SOD. O 2 + NO ONOO However, when NO and O 2 are both present, ONOO will be formed. The interaction of NO with O 2 is important for two reasons: NO and O 2 can antagonize one another’s biological actions and (50); a protonated form of ONOO is a powerful oxidizing cytotoxic species (ONOOH). It can deplete –SH groups and other antioxidants, oxidize lipids, cause damage to DNA bases and irreversible damage to proteins by methionine oxidation, carbonyl formation, and nitration of tryptophan and tyrosine side-chains (50). Methionine oxidation has been linked to impairment of calcium signaling involving inactivation of calmodulin (42). Peroxynitrite has been implicated in atherosclerotic lesions from adults in causing oxidative damage to LDL (77). Hypochlorous acid (HOCl) This non-radical species is produced by myeloperoxidase (MPO), found in neutrophils. Hypochlorous acid is very reactive and oxidizes molecules in a two-electron reaction directly or by decomposing to form chlorine. HOCl can oxidize thiols, ascorbate, and NAD(P)H. This can lead to chlorination of DNA bases and tyrosine residues in proteins; generating products such as 3-chlorotyrosine (50). H 2 O 2 + Cl HOCl + OH

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22 Damage Induced by ROS/RNS This review will focus on oxidative damage specific to the myocardium during an I-R insult. It has been thought that the I-R induced damage occurred in the reperfusion phase only but recently free radical production during ischemia has been shown (134). The first few minutes of reperfusion appear most critical, as some research has shown a rapid burst of free radicals in the first 4 minutes of reperfusion and then a rapid decline (46). There are several targets of this free radical burst and they be will discussed in the next sections. Lipids. During these bursts of free radical production, the cellular targets are often lipids or phospholipids found in cellular membranes. They are particularly vulnerable to attack because of their structure. In the outer membrane, hydrogen atoms attached to methylene groups are easily pulled off by interaction with a free radical. This attack weakens the energy of attachment of the subsequent hydrogen present on the next carbon (called allylic hydrogens) particularly if there is a double bond on either side of the methylene group (50). During this process of weakening hydrogen attachment, a chain reaction starts. This is lipid peroxidation. In addition to free radicals, other molecules (such as iron salts, simple chelates, heme, heme-containing proteins, and phenylalanine hydroxylase) can start this process (50). Further, reactive aldehydes (such as 4-hydroxy-2-trans-nonenal (HNE)) formed by peroxidation of n-6 polyunsaturated fatty acids (PUFAs) (such as arachidonic acid) cause significant damage. There are several markers of lipid peroxidation commonly used. Eight-isoprostane, a member of the F 2 -isoprostane family, can provide reliable evidence of lipid peroxidation from arachidonic acid in the myocardium (32, 61, 112).

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23 Malondialdehyde (MDA) is another commonly assayed measure of lipid peroxidation that is formed in small amounts from peroxidation of PUFAs (such as linolenic, arachidonic and docosahexaenoic acids). Lipid peroxidation decreases membrane fluidity, increases leakiness of the membrane bilayer to substances that do not normally pass through and inactivates membrane-bound enzymes. Further, cross-linking of membrane proteins decreases their ability to rotate and move within the membrane (50). Proteins. Another important target of ROS are proteins. They can be directly targeted or in a secondary fashion by products of lipid peroxidation such as MDA and HNE. Oxidation of amino acid residues is particularly common in the sulfur-containing amino acids, (methionine and cysteine) leading to disulfides and methionine residues (such as methionine sulfoxide) (9). Glycation reactions also damage proteins and particular amino acids such as tyrosine to form unusual products (such as the tyrosyl radical, 3-nitrotyrosine or o-o-dityrosine). Other aromatic amino acids are vulnerable as well. Tryptophan residues can be readily oxidized to formylkynurenine and kynurenine, phenylalanine to hydroxy derivatives, and histidine to 2-oxohistidine (9). The proteins involved in E-C coupling can be targeted. Myofilaments can be damaged by ROS and/or Ca 2+ overload. As state previously, calcium overload will activate the calpains to degrade myofibrillar proteins. Proteins can also be tagged or marked for degradation by another proteolytic system, the proteasome. The proteasome pathway uses ubiquitin to tag proteins and mark them for degradation (75). This ubiquitinated substrate will be hydrolysed by the 26S proteasome. Proteasomes degrade the majority of cellular proteins while another proteolytic system, the lysosomal proteolytic pathway, is responsible for extracellular and membrane proteins.

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24 Cellular Antioxidants The cell has many mechanisms that provide protection against free radicals. It has an elaborate defense network utilizing various antioxidants found in a variety of cellular locations with specific ROS/RNS they attack. Halliwell and Gutteridge (50) define an antioxidant as ‘any substance, that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents the oxidation of that substrate’. The major endogenous antioxidants will be discussed below. Superoxide Dismutase (SOD) The discovery of superoxide dismutase by McCord and Fridovich in 1969 (95) was an important discovery. This discovery led to the superoxide theory of oxygen toxicity and served as a basis for our understanding of antioxidant defense systems. Superoxide dismutase is an enzyme that catalyzes the reduction of superoxide to hydrogen peroxide, thus forming a non-radical species. O 2 + O 2 + 2H + H 2 O 2 + O 2 There are three isoforms of SOD: manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD) and extracellular superoxide dismutase (ECSOD). Copper-zinc superoxide dismutase was the first form to be identified and is found primarily in the cytosol. Manganese superoxide dismutase is located in the mitochondria and catalyzes the same reaction as CuZnSOD. Extracellular superoxide dismutase is also a copper-zinc containing secretory enzyme that is located in the extracellular fluid such as plasma and in the extracellular matrix of tissues. Superoxide dismutase is extremely important in the antioxidant defense system because of the excess production of O 2 that occurs in a variety of clinical diseases,

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25 exercise, and during respiration. The superoxide theory of oxygen toxicity proposes that O 2 is the major factor in O 2 toxicity and the various forms of SOD are critical in the defense against it. Catalase (CAT) Catalase is found primarily in the peroxisome and catalyzes the decomposition of hydrogen peroxide to water and oxygen. 2H 2 O 2 2H 2 O + O 2 In addition to CAT, peroxisomes also contain many cellular enzymes that generate H 2 O 2 such as glycolate oxidase, urate oxidase, and flavoprotein dehydrogenases involved in beta-oxidation of fatty acids (50). Within the human body, the liver contains the highest percentage of CAT with less found in brain, heart, and skeletal muscle. Glutathione Peroxidase (GPx) Glutathione peroxidase catalyzes a similar reaction as CAT. It handles H 2 O 2 by coupling its reduction to H 2 O with oxidation of reduced glutathione (GSH) to oxidized gluthatione (GSSG). H 2 O 2 + 2GSH GSSG + 2 H 2 O Glutathione peroxidase requires one atom of selenium in each of its four protein subunits and is present as selenocysteine. The ratio of reduced GSH to GSSG is assessed to determine the activity of GPx and the amount of H 2 O 2 present in the cell. Often this ratio is high in the cell and in rat myocardium it is greater than 10/1 (50). Due to the fact that both CAT and GPx remove H 2 O 2 from the cell, does cooperation exist between these two enzymes? The K m for CAT is very high which is seen by its high rate of H 2 O 2 destruction when levels of this non-radical species are elevated significantly. Glutathione peroxidase has a lower K m and due to its cellular

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26 location, handles the majority of H 2 O 2 that is generated from the mitochondria and endoplasmic reticulum (50). Additional factors that influence H 2 O 2 removal are the activity of GPx and glutathione reductase (GR), the rate of NADPH supply, and GSH content. Glutathione (GSH) Glutathione is the principal intracellular thiol-containing tripeptide (L--glutamyl-L-cysteinyl-glycine) found in cells at millimolar concentrations. Glutathione can be present in other forms within the cell such as GSSG or as other forms of disulfides, GSSR. The half-life of GSH is about 4 days in human erythrocytes and 3 h in rat liver (50). It has many important biological roles in the cell serving as a necessary substrate for both GPx and GSH S-transferase (GST), and scavenges OH and singlet oxygen. Further, it is involved in signal transduction, in gene expression, and apoptosis (121). Glutathione is synthesized in a two-step reaction involving the enzymes –glutamylcysteine synthetase (1 st reaction) and glutathione synthetase (2 nd reaction). L-glutamate + L-cysteine + ATP L--glutamyl-L-cysteine + ADP + P i L--glutamyl-L-cysteine + glycine + ATP GSH + ADP + P i The production of GSH can be inhibited by the use of a pharmacological agent, L-buthionine SR-sulphoximine (BSO), which inhibits the action of –glutamylcysteine synthetase. Glutathione is produced in the cytosol and supplies the mitochondria with its GSH. When cytosolic GSH levels fall, the mitochondria conserve its own supply by releasing very little to the rest of the cell. Since mitochondria contain very little CAT, they are completely dependent on the action of GSH to handle H 2 O 2 (96). Glutathione can also be degraded extracellularly by -glutamyltranspeptidase, which can transfer the

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27 glutamate residue to another amino acid (such as cysteine, methionine, or glutamine). The ratio of GSH/GSSG is used as a marker to estimate the redox environment of the cell. This ratio is dependent upon the absolute concentration of GSH and a change in its level has a dramatic effect on the reduction potential of this ratio (115). Other Antioxidants The cell contains a variety of non-enzymatic antioxidants that play potentially critical roles in maintaining redox balance. Many of these antioxidants are vitamins that can be consumed in our diet. They are Vitamin E, Vitamin C, carotenoids, lipoic acid, and plant phenols to name the primary ones. Because of the extensive nature of this subject, these antioxidants will not be explored in further detail since they are not germane to the proposal. Exercise and Cardioprotection Exercise training has been shown to be protective against myocardial I-R injury at all durations of ischemia (5-120 minutes) (20, 21, 33, 34, 51, 52, 62, 85, 108, 127). Additionally, 3 days of exercise training provides the same amount of protection as long-term (weeks) exercise training does as evidenced by a reduction in myocardial oxidative injury, fewer arrhythmias, and improved cardiac contractile performance following an I-R insult (21, 33, 34, 51, 108). It is clear that exercise training improves myocardial tolerance to an I-R injury but understanding the biochemical mechanism has been more elusive. It is believed that 3 possible mechanisms exist to explain the improved contractile function: 1) anatomical changes in the coronary arteries (ie. collateral circulation) 2) increases in myocardial heat shock proteins (HSPs) and 3) improved myocardial antioxidant capacity. The following section will explore each of these mechanisms in greater detail.

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28 Development of Collateral Vessels The development of collateral vessels to improve contractile function does not seem plausible given that exercise training has not been shown to alter the vascular beds in adult dogs, pigs, or rats (74). A recent study showed the benefits of 1 day of exercise training in reducing myocardial injury with no change in coronary vascular adaptation (138). Elevation in Myocardial Heat Shock Proteins The second possibility is an upregulation of myocardial HSPs. Heat shock proteins are a group of proteins induced by a variety of stimuli (such as heat, oxidative stress, exercise, low pH, and elevated calcium). Heat shock proteins are identified based on their molecular weight and classified as such. The most well know is HSP72 and it belongs to the HSP70 Kda class. This class is responsible for protein folding, translocation, and protein synthesis in the cell. Numerous papers have shown that exercise training upregulates myocardial HSP72 content as well as other HSPs (33, 34, 39, 87, 88). The increase in myocardial HSP72 has been shown to be cardioprotective due to improved contractile dysfunction in trained animals subjected to a myocardial I-R stunning model as compared to controls. However, a paper by Taylor et al. (127) clearly showed an exercise-induced cardioprotection against an in vitro myocardial I-R insult in the absence of elevated myocardial HSP72 levels. Animals were trained at 4C and rectal temperatures confirmed no increase in core temperature as is typically seen in room temperature (25C) trained animals. These results were confirmed and extended in another study (51) that examined HSP10, 40, 60, 72, 73 and 90 content in cold (4C) and warm trained (25C) animals subjected to an in vivo myocardial I-R (20 min I/30 min R)

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29 insult. No HSPs were elevated in the cold trained animals yet they exhibited the same cardioprotection. Therefore, HSPs are not essential to exercise-induced cardioprotection. The antioxidant MnSOD did increase in both cold and warm trained animals suggesting that it may have played a critical role in the exercise-induced cardioprotection seen. Myocardial Antioxidant Capacity Having provided evidence that development of collateral vessels and protection by HSPs are not viable options; the last remaining possibility is an improved myocardial antioxidant capacity. Exercise training has been shown to increase myocardial antioxidant capacity (34, 51, 52, 62, 86, 107, 108, 111). However, there appears to be some variability in upregulation of antioxidant enzymes depending upon the length of exercise training (acute vs. chronic training), the intensity of training, and the model of I-R chosen to study exercise-induced cardioprotection. Effect of Exercise on Antioxidant Status Powers et al. (107) examined the role of intensity (low, moderate, and high) and duration (30, 60, and 90 min day) during a chronic 10-week training protocol on left ventricular antioxidant capacity. The activity of CAT and GPx did not change with any duration or intensity of exercise while total superoxide dismutase (T-SOD) activity increased in the high intensity groups of all durations. Low and moderate intensity of 90-minute duration also raised T-SOD activity. This study showed that exercise of high intensity or longer duration could upregulate T-SOD activity in the rat myocardium while CAT and GPx remained unaltered. Another long-term training study (8-week) (86) of moderate to high intensity found elevations in antioxidant-related compounds (such as -tocopherol, total ubiquinone, and ubiquinol). In this study, SOD, CAT, and GPx activity were not

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30 evaluated. No change was seen in GSH or the ratio of GSH/GSSG. In the acute exercise group (1 bout to exhaustion) GSH did increase. Leeuwenburgh et al. (78) trained rats for 10 weeks and looked at antioxidant adaptations in various muscles and organs. Heart tissue showed an elevation in GSSG and GST activity with a reduction in the GSH:GSSG ratio as compared to control. No change was observed in CuZnSOD, MnSOD, T-SOD or GPx activity. From the above experiments, it is clear that exercise increases antioxidant capacity however the variation from study to study makes it challenging to make definitive conclusions. The divergence may be explained by training protocols. In the next section, the link will be made between antioxidants and exercise-induced cardioprotection Exercise and I-R injury The above studies clearly showed an increase in antioxidant capacity with exercise training, yet the link between these antioxidants and protection against a myocardial insult was not tested. Therefore, the next set of experiments showed that exercise was cardioprotective. Libonati et al. (85) treadmill trained rats for 6 weeks using either a low-intensity exercise protocol or sprint protocol. At the end of 6 weeks, the hearts of these animals were subjected to an in vitro I-R injury (20 min I/30 min R). Sprint-trained animals had a significantly higher recovery of left ventricular developed pressure (LVDP) and rate of pressure development/decline (dP/dt) during reperfusion as compared to the control and low-intensity group. Using a 10-week high intensity training protocol, Powers et al. (108) subjected rats to an in vivo I-R insult (20min I/10min R). Compared to control, trained hearts

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31 maintained significantly higher peak systolic pressure and a greater rate pressure product during both ischemia and reperfusion. Further, T-SOD, MnSOD, and CuZnSOD activities were significantly elevated following training. The left ventricle of trained animals experienced less oxidative stress during I-R as evidenced by lower levels of lipid peroxidation. These two investigations clearly showed that exercise training was beneficial against an I-R insult. The work by Libonati et al. showed exercise was cardioprotective and the Powers et al. study linked the exercise-induced cardioprotection to an increase in SOD. This was further supported by a decrease in lipid peroxidation in the trained group. These two investigators clearly showed that exercise training was beneficial against an I-R insult. Harris and Starnes (52) found slightly different results in myocardial antioxidants measured in trained rats at 4C or 25C for 3, 6, and 9 weeks. Increases in CuZnSOD were shown at 3 weeks for warm trained animals while MnSOD decreased in cold trained animals. Catalase was significantly increased at 3 weeks in the warm trained only. By 9 weeks of training, the antioxidant capacity of both trained groups had returned to baseline (86). Further, only the 9-week warm trained group had a higher percent recovery during reperfusion compared to their warm control counterparts. The results of this study are in contrast to a study by Hamilton et al. (51). They found elevations in MnSOD in both warm and cold trained groups that paralleled the protection against I-R-induced lipid peroxidation in an in vivo I-R model. Further, neither CuZnSOD nor CAT activity was different among groups while GPx activity rose in the cold trained only. There are potentially two main reasons why these papers differ in their results. Hamilton et al. (51) measured enzyme activities in sham animals to reflect exercise changes while Harris and

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32 Starnes measured enzyme activities from hearts that had undergone an I-R protocol. Second, Harris and Starnes exercised their animals for 3 weeks at 20 mmin -1 at a 6% grade while Hamilton trained animals for 3-5 consecutive days at 30 mmin -1 , 0% grade. Manganese superoxide dismutase may be upregulated early on in exercise and provide critical protection but return to basal levels as the exercise training continues. Manganese superoxide dismutase While the research above does not clearly designate one particular antioxidant as being most critical to exercise-induced cardioprotection, a key study appeared in 1999 that suggested MnSOD is essential to this protection in the myocardium. Yamashita et al. (138) showed that exercise-induced regulation of myocardial MnSOD provided essential protection against infarction. The exercise stimulus was a single bout of 25-30 min of treadmill running at 27-30 mmin -1 . While this exercise protocol is rather short, their data is difficult to ignore. They looked at the time-course of MnSOD activity and protein content following the exercise bout at various timepoints. Manganese superoxide dismutase activity peaked at half-hour and again 48 hours after exercise. The protein content of MnSOD gradually increased and peaked at 48 hours. To critically evaluate the role of MnSOD in exercise-induced cardioprotection, they used a novel approach. They employed an antisense oligonucleotide (AS-ODN) against MnSOD mRNA that blocked translation of the MnSOD protein. This abolished the expected decrease in infarct size due to exercise-induced protection at 48 hours of reperfusion suggesting that MnSOD is essential to the myocardium during infarction. The idea that MnSOD might be extremely important in the myocardium originated from other experiments using different stressors and models. Cultured

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33 heat-shocked myocytes showed a 1.8-fold increase in MnSOD mRNA after 40 minutes as compared to control (137). The use of hyperthermia as a means to increase cardiac tolerance to an I-R insult resulted in increased MnSOD activity and content at all temperatures (40, 41, and 42C) tested (139). These preliminary studies led to the choice of exercise as the next potential stressor and mediator of MnSOD activity as described above in the 1999 paper. Manganese superoxide dismutase overexpression in transgenic models has resulted in greater protection against both in vivo and in vitro myocardial I-R injuries (25). The transgenic mice had a 325% increase in activity with no change in other antioxidants (CuZnSOD, CAT, GPx or GR). The in vitro Langendorff model showed functional recovery of 52% in transgenics versus 31% in controls. A reduction in infarct size using the in vivo model was seen at 14.62.2% for transgenics versus 22.42.4% in the controls. In summarizing the MnSOD experiments, training studies show an exercise-induced increase in MnSOD activity and this has been linked to cardioprotection. Further, the Yamashita et al. (138) study showed an abolishment in exercise-induced cardioprotection against infarction by blocking the exercise-induced increase in MnSOD. These results have led us to the postulate that MnSOD is essential in protecting the heart during an I-R injury. Pilot work done in our lab using the antisense technology in one trained animal clearly showed a reduction in protection during an in vivo I-R insult. However, we do not believe that MnSOD acts alone. We hypothesize that GSH is also very important based on the work of Ji et al. and others (26, 62). In pilot work, two animals were given a pharmacological inhibitor of GSH synthesis, BSO. Both trained

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34 animals receiving BSO showed a reduction in protection against I-R injury providing evidence for the importance of myocardial GSH. The role of GSH in cardioprotection will be addressed further. Glutathione Glutathione is the main non-protein thiol source in the cell and plays a critical role in the antioxidant defense system in the heart serving as the major cellular thiol-disulfide redox buffer (115). Glutathione content in an untrained rat heart is roughly 1-2 mM (50) with the greatest concentration found in the liver (~ 5-6 mM). The heart turns over GSH rather quickly compared to skeletal muscle but is still slower than liver or kidney. Mice treated with BSO i.p. (2mmol/kg) showed GSH levels 8% of control after 500 hours (93). BSO blocks -glutamylcysteine synthetase, the first enzyme in the synthesis of GSH. It has been shown to be very specific, nontoxic, and reversible once administration of BSO has been halted (96). Following cessation of BSO treatment, GSH levels are seen to rebound more quickly in the heart than skeletal muscle. The myocardium maintains its intracellular concentrations by a constant turnover of the -glutamyl cycle (35, 48). To investigate the myocardial response to a reduction in GSH levels, Leeuwenburgh et al. (79) used BSO to induce a 90% reduction in mouse heart GSH content. The reduction in myocardial GSH content was significantly correlated with a reduction in endurance exercise time. Further, BSO appeared to directly inhibit myocardial -glutamylcysteine synthetase (GCS) activity, the first enzyme in the synthesis of GSH and therefore, the turnover rate of the -glutamyl cycle (79). Gamma-glutamyltranspeptidase (GGT), the enzyme responsible for breaking down GSH activity

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35 remained constant and the supply of cysteine and glutamate was adequate for potential GSH synthesis. The effect of exercise training on myocardial GSH content has not been straightforward. Several studies have shown an increase in myocardial GSH content with long-term (8-10 weeks) of training (54, 55, 62, 78, 111) while one study showed no elevation (57). This is in contrast to studies using short-term exercise training protocols. The research in this area is more divided. Short-term exhaustive exercise has been shown to increase GSH content (86), decrease it (79), or result in no change (117). In conclusion, it does not appear that short-term training consistently increases GSH content in the myocardium. While exercise may not consistently elevate myocardial GSH content, it does play an important role in recovery from I-R injury. GSH depleted rats undergoing global I-R showed impaired systolic recovery during reperfusion and increased coronary resistance. Further, introduction of GSH into the reperfusate improved these functional parameters (10). Ji et al. (63) found myocardial GSH content to fall and the GSH:GSSG ratio to decrease following an in vivo I-R insult (30 min I/15 min R). Further, activities of GPX, GR, and CAT fell. Increased MDA, a marker of lipid peroxidation, was seen in GSH depleted hearts. A recent study by Leichtweis and Ji (80) studied GSH depletion using an in vivo I-R model. Rats were injected with BSO or saline 24 hours prior to I-R injury. Glutathione depleted hearts showed greater cardiac dysfunction compared to controls as evidenced by a lower cardiac contractility, LVDP, and RPP. Therefore, a fall in myocardial GSH content appears to be the result of oxidative damage incurred during an I-R injury. This notion is further supported by evidence that perfusate containing GSH

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36 detoxified potential ONOO damage during an in vitro I-R injury and improved post-ischemic mechanical function (26). A study by Ramires and Ji (111) has made the story of GSH more complex and interesting. They found no difference in left ventricular systolic pressure or contractility between trained and control hearts both supplemented with GSH during an I-R insult. This indicated that GSH alone plays a very important role in the myocardial antioxidant defense system in protecting the heart against oxidative damage caused by I-R independent of exercise. Rate pressure product was significantly higher in the trained and control GSH groups as compared to the unsupplemented trained and control groups. Total superoxide dismutase, GPx, and GR activities were elevated in both trained groups. This study found that supplementation with GSH provided the same elevation in myocardial GSH levels as did exercise-induced synthesis of GSH and provided protection against myocardial I-R injury. Therefore, these conclusions regarding GSH and the work cited from the literature has led us to propose that both MnSOD and GSH are essential to the exercise-induced cardioprotection seen during myocardial stunning. Summary Myocardial stunning is a complex clinical problem involving both generation of free radical species and Ca 2+ overload leading to cardiac myocyte damage and contractile dysfunction. Countermeasures against this dysfunction have been investigated and it has been shown that exercise training enhances myocardial antioxidant capacity leading to a decrease in dysfunction as a result of myocardial I-R injury. The mechanisms behind this exercise-induced cardioprotection remain unclear and provide an interesting and exciting avenue to investigate. Through previous investigations, it has become clear that MnSOD

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37 and GSH play pivotal roles in this protection. Indeed, a training induced increase in both MnSOD and GSH is logical given that these two antioxidants can work as a unit to remove oxidant precursor (i.e. O 2 , H 2 O 2 ) before they form more reactive radical species (i.e. OH , ONOO ). Superoxide radicals are dismutated by SOD to form H 2 O 2 and O 2 . GPx at the expense of GSH can remove the resulting H 2 O 2 . Therefore, it is plausible that an increase in the MnSOD activity and GSH content is required to provide myocardial antioxidant protection against I-R mediate oxidative injury resulting in myocardial stunning. This proposal is designed to specifically test the role of these two exercise-induced myocardial antioxidants in their role against myocardial stunning.

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CHAPTER 3 METHODS Animal Model The animal model chosen in these experiments was the male Sprague-Dawley (SD) rat. The animals were approximately 4 months of age at the time of sacrifice and this age represents a young adult in the rat. Animal Model Justification The SD rat was chosen as the experimental model because: 1) the nature of these experiments is very invasive and prevents the use of humans as models; 2) the SD rat does not display large inter-animal variation in the coronary collateral circulation (47); and 3) the SD rat is a well accepted model for the study of exercise-induced myocardial adaptations (21, 62, 81, 102, 107). Further, we chose to study the male rat to avoid the varying estrogen levels in female rats across the estrus cycle (130). Animal Housing and Diet Animals were housed at the Animal Care Services at the University of Florida upon arrival from the vendor. Animals were housed two to a cage, maintained on a 12:12 hour light-dark cycle and provided food (AIN93 diet) and water ad libitum throughout the experimental protocol. Experimental Design The experimental design is shown below. 1) Control – no exercise or treatment 2) Trained control – no treatment 38

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39 3) Trained antisense Administration of antisense oligodeoxynucleotide (AS-ODN) against MnSOD mRNA protein translation 4) Trained mismatch Treatment with mismatch oligodeoxynucleotide (MM-ODN) Each of these four groups was divided into one of two surgery groups: sham surgery or short term in vitro global ischemia (25 min ischemia/30 min reperfusion). Exercise Training Protocol Exercise trained animals performed 3 consecutive days of treadmill exercise (60 minday -1 @ 60-70% of VO 2 max). Inhibition of Myocardial MnSOD Protein Translation Antisense oligodeoxynucleotides are single stranded sequences of DNA that are designed to inhibit messenger RNA expression for a specific protein. To block the expression of MnSOD, animals were injected (i.p.) with AS-ODN immediately following exercise. We included a mismatch oligodeoxynucleotide (MM-ODN) as a control to verify that the antisense treatment was effective and not having nonspecific effects. Overview of Sham and In vitro Working Heart I-R Protocol At the end of the experimental period, ten animals from each group underwent a sham surgery (ie. no ischemia) and the hearts from these animals were used to provide baseline data for biochemical measurements. Another ten animals from each group underwent an in vitro working heart I-R protocol. These animals provided the cardiac contractile measurements. Details of Experimental Methods In vitro Working Heart Protocol Myocardial function was evaluated using an isolated, working heart preparation (20, 21, 127). Hearts were perfused at 37C with a modified Krebs-Henseleit buffer

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40 containing in (mM) 1.25 Ca 2+ Cl 2 , 130 NaCl, 5.4 KCl, 11 glucose, 0.5 MgCl 2 , 0.5 NaH 2 PO 4 , 25 NaHCO 3 and aerated with a 95% O 2 5%CO 2 . Buffer pH was ~7.4-7.45. Animals were anesthetized with sodium pentobarbital at a dose of 100 mgkg and a 100 IU injection of heparin was made into the hepatic vein. The heart was rapidly excised and placed in cold saline (4C) on tared electronic balance for accurate heart weight. The aorta was secured on a stainless steel catheter of the perfusion apparatus and perfused in a retrograde, or Langendorff mode at 80 cm H 2 O. Excess tissue was trimmed, weighed, and subtracted from the gross weight to get a final wet weight. All subsequent values were normalized for heart weight. The left atrium was cannulated through the pulmonary veins and tied off securely. After 10 min of the Langendorff mode, the heart was switched to the working heart mode and preischemic function was evaluated at 14 cm H 2 O (atrial filling pressure) with an 80cm-high aortic column. Global, normothermic ischemia was induced by simultaneously clamping both the aortic and atrial lines for 25 min. During ischemia, the heart was enclosed in a sealed, water-jacketed chamber maintained at 37C. Following the ischemic period, hearts were initially perfused in the Langendorff mode for 15 minutes to allow recovery from the insult and then switched to working heart mode for additional 15 minutes at 80 cm H 2 O. The heart was removed from the apparatus, dissected, washed in antioxidant buffer, and stored at C. Sham Protocol The protocol for the sham surgery was similar to the above protocol in terms of preparation of the heart however, it was cannulated in the Langendorff mode only and

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41 perfused for 5 minutes. The heart was removed from the apparatus, dissected, washed in antioxidant buffer, and stored at C. Cardiac Contractile Measurements Cardiac contractile measurements were made every 5 minutes during baseline (preischemia) and reperfusion. A timed collection of coronary flow (CF) and aortic overflow (AF) were made from the effluent dripping off the heart and the aortic overflow, respectively. Cardiac output was defined as the sum of these two flows (CF + AF). Peak systolic pressure, diastolic pressure, the rate of pressure development and decline ( dP/dt), and heart rate were measured via a pressure transducer (Harvard Instruments) connected to a T connector off the aortic cannula. Data was recorded using a customized data acquisition system (customized by Curtis Weldon using Lab View Software). Exercise Training Protocol The animals assigned to the exercise training groups were habituated to the treadmill for 5 days increasing the duration slowly (5, 10, 15, 30, and 45 minutes). Following this acclimation to the treadmill, exercise groups performed 3-consecutive days of endurance treadmill exercise (60 minday -1 @ 70% of VO 2 max). We have found that this exercise protocol increases myocardial antioxidant capacity and provides cardioprotection during an in vivo I-R insult ((34, 51)). All exercise training took place at 25C. Mild electrical shocks were used sparingly to motivate the animals to run. Tissue Removal and Storage At the completion of either the sham or I-R protocol, hearts were removed, the left ventricle was immediately sectioned into five vertical strips cut from base to apex,

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42 frozen in liquid nitrogen, and stored at C for subsequent biochemical analysis. Prior to freezing, the heart was rinsed with a cold antioxidant buffer containing 50mM NaHPO 4 , butylated hydroxytoluene (0.1mM), diethylenetriaminepentaacetic acid (0.1mM), and 3-aminotriazole (10mM). Inhibition of Myocardial MnSOD Protein Translation Antisense oligodeoxynucleotides (AS-ODN) are single-stranded synthetic DNA that usually contain a backbone with modification to a specific sequence to hybridize to a specific messenger RNA. Hybridization of the ODN to mRNA inhibits the mRNA from initiating translation (105). To block the translation of the MnSOD protein, animals were injected (i.p.) with a 22-mer phosphorothioate derivative of the AS-ODN (5’-CACGCCGCCCGACACAACATTG-3’) immediately post-exercise at a dose of 10 mg kg . The injection time and dose of this specific AS-ODN has been shown to provide optimal experimental conditions to inhibit the exercise-induced increase of MnSOD activity in myocardial tissue (138). Preliminary experiments in our laboratory have confirmed this. Further, we used a mismatch control in our experiment to verify that the AS-ODN was effectively blocking MnSOD expression by use of both a MM-ODN (CAC TCC TCC CAG CAC AAC AGTC). This control was administered in the same fashion as the AS-ODN. Biochemical Analysis of Antioxidant Enzyme Activity A section of left ventricle was mixed and homogenized in 100 mM cold phosphate buffer with 0.05% bovine serum albumin (1:20 wt/vol; pH 7.4). Homogenization was achieved by 20 passes of the homogenate in a Potter-Elvehjem homogenizer. Homogenates were centrifuged at 400g for 10 min at 4C. The

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43 supernatant was decanted and assayed to determine total protein content along with the activities of MnSOD, CuZnSOD, GPx, and CAT activities. Protein content was determined using methods described by Bradford (22). MnSOD activity was measured by the method of Oyanagui (103). GPx activity was measured by the method of Flohe and Gunzler (40) and CAT by the method of Aebi (1). All biochemical analyses were performed in quadruplicate at 25C and samples from all experimental groups were assayed on the same day to avoid interassay variation. Assessment of Reduced/Oxidized Glutathione Left ventricular reduced and oxidized glutathione concentration was assayed using a kit (Cayman Chemical Company; Ann Arbor, Michigan). Measurement of Lactate Dehydrogenase Activity. Lactate dehydrogenase (LDH) activity of the I-R hearts was determined by the method of Bergmeyer et al. (8). Briefly, a volume of coronary effluent was incubated with pyruvate and NADH. The rate of NADH oxidation to NAD was measured spectrophotometrically in duplicate at 340 nm. This rate of oxidation is directly proportional to the amount of LDH present. LDH activity was corrected for heart weight and CF from preischemia and reperfusion. Data Analysis To test our hypotheses we performed a one-way ANOVA to determine if group differences existed. Significant group differences were evaluated via post-hoc analysis (Tukeys test). Significance was established at p < 0.05.

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CHAPTER 4 RESULTS Animal Characteristics The physical characteristics of the animals in both the sham and ischemia-reperfusion (I-R) groups are shown in Table 4-1 . Heart weights only are given for the I-R animals because their heart weight was used to normalize cardiac output. Although animals were randomly assigned to experimental groups, one group of animals differed in weight from several of the other groups. Specifically, the sedentary control (S-C) sham group contained the heaviest animals and their weight was significantly greater several other experimental groups. Note that young adult male Sprague Dawley rats tend to gain weight rapidly at this age and these group variances in weight do not reflect differences in age between the animals. Table 4-1. Animal characteristics Group Number Weight (g) Heart weight (g) Body/heart weight ratio (g/mg) Sham groups S-C 10 3584.7 E-C 10 3382.9* E-MM 10 3415.5 E-AS 10 3414.7 I-R groups S-C 10 3223.4* 1.010.02 3.200.05 E-C 9 3245.3* 1.030.02 3.150.04 E-MM 9 3214.5* 1.010.02 3.180.03 E-AS 9 3264.2* 1.040.02 3.130.03 Values are means SEM. C-control; E-C-exercise trained control, no treatment; E-MM-exercise trained treated with mismatch oligonucleotide; E-AS-exercise trained, treated with antisense oligonucleotide; I-R-ischemia-reperfusion. *Significantly different from S-C, p < 0.05. Significantly different from E-AS-sham, p < 0.05. 44

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45 Antioxidant Status of Animals in the Sham Group Table 4-2 contains both antioxidant enzyme activities and glutathione (GSH) concentrations from the left ventricle of the myocardium. Antioxidant activities were measured in the sham animals only as I-R can up-or down-regulate the activity of these enzymes. Compared to S-C, manganese superoxide dismutase (MnSOD) activity was elevated in the hearts of E-C and E-MM groups while MnSOD did not differ between E-AS and S-C animals. These results indicate that the antisense oligonucleotide was effective in decreasing the exercise-induced elevation of MnSOD activity while the mismatch oligonucleotide (control) had no effect in the E-MM group. Note that catalase was significantly elevated in all trained groups compared to S-C. Finally, exercise did not alter myocardial GPx activity or GSH levels in any experimental group. Table 4-2. Effects of exercise training on left ventricular antioxidant status Enzyme, activity per mg protein Sedentary control Exercise control Exercise mismatch oligonucleotide Exercise antisense oligonucleotide Total SOD, units 107.81.9 117.94.43 116.23.85 114.26.75 MnSOD, units 10.421.54 18.32.98* 18.61.8* 6.11.31 CuZn SOD, units 97.42.11 99.65.6 97.64.71 108.16.66 GPx, molmin -1 0.0920.002 0.0880.003 0.0880.002 0.0890.003 CAT, units 0.820.03 1.070.06* 1.050.02* 1.070.02* Total GSH, mM 1.940.07 1.850.05 1.820.07 1.790.06 GSH, mM 1.780.09 1.710.05 1.650.08 1.670.08 GSSG, mM 0.170.02 0.170.03 0.180.02 0.170.02 Values are means SEM. *Significantly different from control, p < 0.05. Significantly different from E-AS, p < 0.05 Cardiac Function of Isolated Perfused Hearts Table 4-3 contains cardiac performance parameters measured during pre-ischemia (i.e. baseline). Importantly, no significant differences existed between groups during pre

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46 ischemia. Table 4-3. Functional characteristics of pre-ischemic hearts Pre-ischemia heart parameters Sedentary control Exercise control Exercise mismatch oligonucleotide Exercise antisense oligonucleotide HR (bpm) 342.28 3458.5 341.14.4 328.46 CF (mlmin -1 g wet weight-1) 150.61 15.020.43 13.60.57 14.740.78 CO (mlmin -1 g wet weight-1) 47.10.84 47.020.71 44.21.3 43.61.3 SP 95.11.9 96.22.1 93.80.98 971.7 PP 57.72.9 58.43.6 54.51.6 59.82.6 Cardiac work (SPxCO) 4476118 451889 4150147 4222132 RPP (HRxSP) 32464582 33058220 31984404 31781404 Values are means SEM. HR= heart rate; CF=coronary flow; CO=cardiac output (CF + AF); AF= aortic flow; SP=systolic pressure; PP=pulse pressure, RPP=rate pressure product. The functional characteristics of the post-ischemic hearts can be found in Table 44 . Note that I-R injury decreased the amount of contractile work performed in each experimental group. When comparing across groups, hearts from E-C animals performed more cardiac work and had a greater systolic pressure and rate pressure product than S-C animals. Further, although cardiac performance parameters in both the E-MM and E-AS groups tended to be greater than the S-C group, these differences did not reach significance. However, when cardiac work (SP x CO) was expressed as percent recovery of baseline, all exercise-trained groups were able to produce more cardiac work as compared to S-C. This marker of contractile performance is important as it normalizes each group to its baseline (pre-ischemia) values ( Table 4-5 ). This data is also represented in figure format to more clearly illustrate differences between the groups [ Figure 4-1 (SP x CO)]. In addition, Figure 4-2 shows left ventricular contractile performance of the

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47 groups over time from pre-ischemia to reperfusion. All exercise-trained groups were able to perform more cardiac work than S-C at all time-points measured during reperfusion. Finally, Figure 4-3 illustrates the percent recovery of cardiac output (CO = AF + CF) during reperfusion. Note that the E-AS group had significantly improved recovery of cardiac output compared to S-C. Table 4-4. Functional characteristics of post-ischemic hearts Post-ischemia heart parameters Sedentary control Exercise control Exercise mismatch oligonucleotide Exercise antisense oligonucleotide HR (bpm) 32716.3 32112.2 32410.2 3347.3 CF (mlmin -1 g wet weight-1) 15.71.4 151.5 12.61.1 12.90.78 CO (mlmin -1 g wet weight-1) 36.51.4 40.71.3 38.62.3 40.61.1 SP 832.5 92.32.8* 88.52.3 87.21.6 PP 37.94 51.74.4* 46.23.5 44.22.5 Cardiac work (SPxCO) 3010140 374485* 3409212 3541131 RPP (HRxSP) 26817791 29351482* 28492595 29034496 Values are means SEM. *Significantly different from S-C, p < 0.05 Table 4-5. Percent recovery of functional characteristics of the heart At 30 min of reperfusion Sedentary control Exercise control Exercise mismatch oligonucleotide Exercise antisense oligonucleotide % recovery of CO 77.84.2 86.72.4 86.93.4 93.21.2* % recovery of cardiac work (SPxCO) 67.73.5 83.12.4* 81.62. 7* 842.3* % recovery of RPP 82.92.8 88.81.3 89.11.5 91.51.9* Values are means SEM. *Significantly different from S-C, p < 0.05 Cellular Injury To determine if disruption of the cellular membrane occurred in the cardiac myocytes during the I-R insult, LDH activity was measured in the coronary effluent from hearts during pre-ischemia and from minute 5 to 7 during reperfusion ( Figure 4-4 ). The activity of LDH was normalized for heart weight and coronary flow. Note that LDH

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48 release was significantly lower in the exercise-trained groups as compared to the S-C group (p < 0.05). S-CE-CE-MME-AS% recovery of cardiac work(SP x CO) 020406080100 *** Figure 4-1. Percent recovery of cardiac work [cardiac output x systolic pressure (SP x CO)] at 30 minutes of reperfusion in I-R hearts. Values are means SEM. *Significantly different from S-C, p < 0.05.

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49 TIME (min) BaselineIschemiaR5R10R15SP x CO(% of pre-ischemic function) 020406080100120 ***E-AS S-C E-C E-MM ###!!! Figure 4-2. Changes in ventricular contractile function [cardiac output x systolic pressure (CO x SP)] relative to baseline from baseline (pre-ischemia) to reperfusion. Values are means SEM. Pre-ischemia values were similar in all groups (p > 0.05). *E-C significantly different from S-C, p < 0.05; #E-MM significantly different from S-C, p < 0.05; and !E-AS significantly different from S-C, p < 0.05.

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50 S-CE-CE-MME-AS% recovery of CO 020406080100 * Figure 4-3. Effect of I-R injury on percent recovery of cardiac output (CO) at 30 minutes of reperfusion. Values are means SEM. *Significantly different from S-C, p < 0.05.

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51 LDH Activity(% Difference) 0100200300400500600700 S-CE-CE-MME-AS*** Figure 4-4. Effect of I-R injury on release of lactate dehydrogenase in the coronary effluent expressed as a percent of pre-ischemia values. Values are means SEM. *Significantly different from S-C, p < 0.05.

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CHAPTER 5 DISCUSSION Overview of Principal Findings These experiments tested two hypotheses: 1) exercise-mediated cardioprotection against myocardial stunning is dependent upon exercise-induced increases in myocardial manganese superoxide dismutase (MnSOD) activity; and 2) exercise-induced cardioprotection is also dependent upon an increase in myocardial levels of glutathione (GSH). While the results of these experiments demonstrated that short-term exercise training is protective against myocardial stunning in an in vitro model of I-R injury, our results do not support the hypotheses that exercise-induced increases in myocardial MnSOD activity and GSH are essential to achieve this protection. Indeed, despite the prevention of the exercise-induced increase in MnSOD activity via antisense oligonucleotide, short-term exercise training resulted in cardioprotection against I-R-induced myocardial stunning. Further, our experiments demonstrated that short-term exercise training is not associated with an increase in myocardial GSH content. Hence, this protein thiol is not essential to exercise-induced cardioprotection against myocardial stunning. A brief discussion of these and other related issues follows. Exercise-induced Cardioprotection The current data support the notion that endurance exercise training provides cardioprotection against myocardial stunning (20, 21, 33, 85, 108). Indeed, exercise-induced cardioprotection has been observed in moderate I-R insults resulting in stunning (5-20 minutes), and more severe insults resulting in infarction (30-60 minutes) (20, 33, 52

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53 51, 62, 108, 127). Interestingly, as little as 3 consecutive days of exercise training has been shown to provide the same magnitude of cardioprotection (e.g. reduced arrhythmias and oxidative injury) against I-R-induced injury as long-term (8-10 weeks) exercise training (21, 33, 34, 51, 108). Mechanism(s) Responsible for Exercise-induced Cardioprotection To date, the mechanisms responsible for exercise-induced cardioprotection continue to remain elusive. Several cardioprotective mechanisms have been proposed and include changes in collateral circulation, elevation in myocardial heat shock proteins and/or an increase in myocardial antioxidants. Aerobic exercise training has been shown to increase coronary vascular transport capacity by increasing cross-sectional area, increasing arteriolar density, and altering vascular control (24, 82). While this development of collateral circulation may occur after months of training, it does not appear to increase with short-term training (74). Furthermore, a recent study demonstrated that the benefit of 1 day of exercise training in reducing myocardial injury was not due to a change in coronary vascular adaptation (138). Hence, changes in myocardial collateral circulation do not appear to be a primary mechanism for exercise-induced cardioprotection. Heat shock proteins (HSPs) are a class of stress proteins that respond to disturbances in cellular homeostasis. HSPs are induced by stressful conditions such as elevations in body temperature and exercise (58, 108). Indeed, numerous papers have shown that exercise training upregulates myocardial HSP72 content as well as other HSPs (33, 34, 39, 87, 88). Overexpression of myocardial HSP72 has been convincingly shown to be cardioprotective in studies using cell transfection and transgenic mouse models (58, 92, 106). However, recent work (51, 127) clearly demonstrates that

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54 exercise-induced cardioprotection against I-R injury can occur in the absence of elevated myocardial HSP72 levels. Hence, myocardial HSPs are not essential to exercise-induced cardioprotection against stunning. Collectively, the aforementioned studies disrepute the possibility that exercise-induced increases in collateral circulation or myocardial HSPs are responsible for exercise-induced cardioprotection. These results led us to postulate that exercise-induced cardioprotection is linked to exercise-mediated increases in myocardial antioxidant capacity. In particular, the current experiments tested the hypothesis that a training-induced increase in both myocardial MnSOD activity and GSH concentrations are required to provide the cardioprotection associated with exercise. Specifically, we postulated that an increase in each of these antioxidants is an essential component of the exercise-induced protection against I-R-induced myocardial stunning. The key points that lead to this hypothesis are as follows. First, radicals and other reactive oxygen species are important contributors to I-R-induced injury in the heart (3, 4, 12, 13, 15, 16, 46, 64, 83, 98, 109, 123, 143). Secondly, increasing myocardial antioxidant capacity (e.g. gene transfer or exogenous antioxidants) can reduced I-R-induced cardiac damage (25, 28, 29, 38, 65, 111). Also, the cardioprotection associated with long-term (weeks) exercise training is accompanied by an increase in myocardial antioxidant capacity (i.e. MnSOD activity and [GSH]) (34, 51, 54-57, 62, 107, 108, 111). A training-induced parallel increase in both MnSOD and GSH is logical given that MnSOD and GSH work as a unit to remove oxidant precursors [i.e. superoxide (O 2 ), hydrogen peroxide (H 2 O 2 )] before they form more reactive cytotoxic oxidants [i.e. hydroxyl radical (OH )]. That is, superoxide radicals are dismutated by SOD to yield

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55 H 2 O 2 and O 2 . The resulting H 2 O 2 can be removed by the action of glutathione peroxidase (GPx) at the expense of GSH. Although exercise training does not increase myocardial GPx activity, an increased total ability to remove H 2 O 2 can be achieved by increasing cellular levels of GSH (26, 111). Hence, it is plausible that an increase in both MnSOD activity and [GSH] is required to provide myocardial antioxidant protection against I-R mediated oxidative injury resulting in myocardial stunning. Nonetheless, while theory supports the notion that elevated myocardial levels of MnSOD and GSH should promote cardioprotection, the results of this investigation clearly demonstrate that exercise-induced increases in myocardial [GSH] and MnSOD activity are not essential to provide cardioprotection against myocardial stunning. Indeed, the current results indicate that 3 days of exercise training do not increase GSH levels in the rat heart. A thorough review of the literature reveals that the impact of exercise on myocardial levels of GSH may be dependent on the duration of the exercise-training program. For example, short-term exhaustive exercise has been reported to either increase (86), decrease (79) or result in no change (117) of myocardial levels of GSH in mice. In contrast, several studies indicate that 8 weeks of treadmill exercise significantly elevates myocardial GSH levels in the rat (55, 56, 111). Regardless, in the current experiments, an increase in GSH content was not essential to achieve exercise-induced cardioprotection against stunning. Similar to GSH, MnSOD has been implicated as a key molecule in exercise-induced cardioprotection. Nonetheless, in the current experiments, antisense treatment prevented exercise-induced increases in cardiac MnSOD activity but did not ablate exercise-induced cardioprotection against myocardial stunning. In contrast to

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56 these results, Yamashita et al. demonstrated that antisense inhibition of exercise-induced MnSOD activity removed the normally observed exercise-induced cardioprotection against infarction (138). The explanation for these discrepant results is unclear but may be linked to differences in the experimental model. Indeed, the current study investigated myocardial stunning using an in vitro working heart model while Yamashita et al. (138) studied myocardial infarction using an in vivo model. Clearly, myocardial stunning and infarction are very different cardiac insults and the mechanisms for cell injury in these insults may differ. Infarction results in necrotic and apoptotic cell death while stunning causes temporary contractile dysfunction with little or no cell death. In this regard, it is possible that MnSOD plays a more important role in long-duration ischemia and reperfusion resulting in infarction due to greater oxidative damage inflicted upon myocardium. For example, during an in vivo I-R insult, the heart is subject to damage by inflammatory molecules such leukocytes and neutrophils that can exacerbate ROS mediated injury (66). In contrast, isolated and buffer perfused hearts such as the working heart model remove the influence of inflammatory molecules from I-R insults. Regardless of the explanation for these divergent findings between the current study and the work of Yamashita et al. (138), the present data clearly demonstrate that exercise-induced increases in myocardial MnSOD activity are not essential to produce exercise-induced cardioprotection against myocardial stunning. Other Potential Mechanisms of Exercise-induced Cardioprotection While our exercise-trained hearts were protected against an I-R insult, this protection was not a result of increased myocardial MnSOD activity or GSH content. This suggests that exercise provides protection against myocardial stunning via alternative mechanisms. Potential cardioprotective molecules induced by exercise

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57 include catalase, nitric oxide, and cyclooxygenase-2. A brief discussion of each of these cardioprotective candidates follows. Catalase The primary role of the enzymatic antioxidant catalase is to decompose H 2 O 2 . This H 2 O 2 can be generated from the dismutation of O 2 by SOD. It is noteworthy that GPx can also decompose H 2 O 2 . The km of GPx is several fold lower than catalase. However, when cellular levels of H 2 O 2 are high, catalase can become the more important antioxidant of the two (41). Besides its primary location in the peroxisome, catalase has been found in the matrix of the mitochondria (110) suggesting a potentially key role in detoxifying superoxide generated in the mitochondria during exercise. Therefore, catalase, which increased with exercise training in the current study, may be important in providing protection during myocardial I-R. In the current experiments, 3 days of exercise significantly elevated catalase activity in the left ventricle. A review of the literature suggests that the effect of exercise training on myocardial catalase activity appears to be specific to the training duration. Long-term training does not appear to result in an elevation in activity (57, 107, 108). This point was clearly demonstrated in a recent 9-week training study(52). In these experiments, myocardial catalase activity showed an initial increase through 3 weeks of training and a subsequent decrease as training continued to 9 weeks. Others have reported similar results (129). It is possible that the initial oxidative stress associated with exercise training may be the mechanism for increased myocardial catalase levels in the beginning but as the training progresses, other antioxidant defense systems are upregulated, therefore decreasing the requirement for catalase.

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58 Investigations into the antioxidant role of catalase during myocardial stunning have been studied for approximately 20 years. Many investigators have administered catalase in combination with SOD prior to ischemia, during ischemia, or post-ischemia to examine the role that free radicals play during myocardial stunning (3, 12, 64, 109, 123, 131). Recent work demonstrates that catalase activity increases in ischemic rabbit hearts following 15 minutes of regional ischemia (70). Gene expression research in human hearts with end stage heart failure showed a significant increase in catalase mRNA expression and protein content with no change in MnSOD, CuZnSOD or GPx (36). The authors suggested that the increase in catalase was a compensatory mechanism against the oxidative assault that occurred in these patients. In conclusion, catalase has been shown to be protective during an I-R insult and our results support a role for catalase in exercise-induced cardioprotection. Nonetheless, additional experiments are required to determine if exercise-induced increases in myocardial catalase activity is essential to provide exercise-induced cardioprotection against myocardial stunning. Inducible Nitric Oxide Synthase and Cyclooxygenase-2 In recent years, ischemic preconditioning (PC) and the role it plays in protection against I-R injury has gained much interest. PC refers to the process whereby a sublethal ischemic insult enhances the myocardium’s ability to tolerate subsequent ischemic stress (18). PC has been subcategorized into an early and late phase of preconditioning. The early phase lasts 2-3 hours while the late phase (delayed) occurs approximately 12-24 hours following the ischemic bout and lasts 3-4 days. It has recently been suggested that exercise training results in late phase PC. The connection between exercise and PC was observed in a study that exercised dogs for 5 periods of 5 minutes each followed by occlusion of the descending coronary artery immediately following exercise or 24 hours

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59 later (37). The authors found that exercise induced both the early and late phase of preconditioning. Further, postulated mediators of PC such as antioxidants, HSPs, cyclooxygenase-2 (COX-2), and the inducible form of nitric oxide synthase (iNOS) are upregulated in exercise trained hearts (37, 113, 120). The mechanism behind PC is quite complex with multiple “triggers” and “mediators”. Of the potential mediators, the most prominent is iNOS. At this time, the effect of exercise on iNOS activity is a relatively new area of investigation. Currently, there are only two published studies addressing exercise and iNOS (55). The first study reported significant increases in myocardial iNOS activity following 8 weeks of treadmill training (55) and the second study reported increased NOS activity and iNOS protein content in aortas from these trained animals (54). Further, an abstract published in the fall of 2001 (49) linked exercise training and increases in iNOS activity with protection against infarction using transgenic mice. Significant increases in iNOS protein expression and activity were correlated with a significant reduction in infarct size. This protection was completely lost in homozygous null iNOS mice. However, it is now thought that iNOS may act through another mediator COX-2 (120). Cyclooxygenase-2 has been implicated as another important mediator in this process and is thought to be downstream from iNOS (120). Cyclooxygenase-2 is the inducible form of the rate-limiting enzyme in prostaglandin synthesis and appears to confer its cardioprotection through two prostaglandins in particular, PGE 2 and PGI 2 (18, 119). These prostaglandins are thought to protect the myocardium through several mechanisms including activation of opioid receptors that result in the opening of sarcolemmal and mitochondrial K + ATP channels (119, 135). Therefore, iNOS and/or

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60 COX-2 may play a role in providing protection against myocardial stunning independent of, or in conjunction with antioxidant enzymes. It seems possible that exercise-induced increases in myocardial iNOS could play a contributory role in exercise-induced cardioprotection. This is an interesting area for future research. Critique of Experimental Model We selected the male Sprague Dawley (SD) rat as our experimental animal for several reasons. The invasive nature of these experiments precludes use of human subjects. Second, the SD rat does not display large inter-animal variation in coronary collateral circulation (47). Furthermore, the SD rat is a well-accepted model for studying exercise-induced myocardial adaptations (20, 62, 81, 102, 107) and is by far the best characterized for isolated perfused hearts in small mammals. Finally, to avoid the potential antioxidant effects of estrogen, we chose to study male animals (130). We chose the in vitro working heart model to investigate myocardial stunning for several reasons. First, this model is a highly reproducible preparation used to study the myocardial performance of small mammals without the confounding effects of other organ systems, the systemic circulation, and a host of peripheral complications (124). Further, the working heart model has the advantage over the Langendorff model in that cardiac pump function can be measured while controlling filling pressures and afterloads (124). For these reasons, the isolated working heart is the “model of choice” for studying post-ischemic stunning (141). To study the role of MnSOD in exercise-induced cardioprotection, we chose to use an antisense oligonuleotide to block the increase in MnSOD protein synthesis. Antisense oligonucleotides are short, single-stranded sequences of DNA designed to inhibit the expression of a specific protein (84). The antisense oligonucleotide prevents

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61 translation by hybridising to a specific mRNA and preventing the mRNA from sliding through the cytoplasmic ribosome (105). This hybridization will stimulate RNAse-H, which destroys the specific sequence of the hybridized mRNA (105). Using an antisense approach is advantageous in that it can be used like a drug, has prolonged action, is specific for the target protein, and does not induce an inflammatory reaction (105). The efficacy of our antisense oligonucleotide against MnSOD was demonstrated by the finding that injection of this antisense prevented the exercise-induced increase in myocardial activity. Importantly, treatment of exercise animals with this antisense did not lower myocardial MnSOD activity to levels below those of sedentary control animals. Further, preliminary experiments using the antisense oligonucleotide against MnSOD showed that this treatment did not lower MnSOD activity in other tissues such as the liver and soleus. To ensure that the antisense oligonucleotide was not associated with non-specific effects, we employed a control oligonucleotide (mismatch) that did not block the exercise-induced elevation in MnSOD activity. Conclusions To our knowledge, the present study is the first to critically evaluate the role of cardiac MnSOD activity and GSH levels in exercise-induced cardioprotection against an in vitro I-R insult resulting in myocardial stunning. Our results clearly indicated that as little as 3 consecutive days of exercise training provide significant protection against myocardial stunning. Furthermore, our findings indicate that increases in myocardial MnSOD activity are not essential to exercise-induced cardioprotection against myocardial stunning. Further, our experiments demonstrate that myocardial GSH content does not increase with short-term training. Hence, exercise-induced increases in cardiac GSH are not essential to exercise-induced cardioprotection against myocardial stunning.

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62 In conclusion, this study demonstrates that exercise training is cardioprotective against I-R injury resulting in stunning but the mechanisms of this protection remain elusive. Future research should be directed at investigating the role of other potential exercise-induced cardioprotective mediators such as catalase, iNOS and/or COX-2.

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BIOGRAPHICAL SKETCH Shannon Liduina Lennon grew up in Mansfield Center, Connecticut and attended the University of Connecticut (UConn) for her undergraduate education. While at UConn, she was a member of the women’s cross country and track and field teams for 4 years. She studied nutrition and graduated in 1993 with a Bachelor of Science in nutritional sciences. After graduation she did a year-long internship in dietetics at The New York Hospital-Cornell Medical Center in New York, New York. In spring 1995 she became a registered dietitian. In fall 1995 she entered the masters program in Nutritional Sciences at UConn under the mentorship of Dr. Nancy Rodriguez and graduated with her master’s degree in fall 1997. The title of her thesis was “Nutritional Supplementation and Performance in Endurance Athletes: Exploring Central Fatigue.” Because of her interest in exercise physiology, Shannon entered the exercise physiology doctoral program at the University of Florida in fall 1997. Shannon’s first advisor at UF was Dr. Michael Pollock, who passed away suddenly at the end of her first year. She then joined the exercise biochemistry laboratory of Dr. Scott Powers and became actively involved in research concerning the mechanisms behind exercise-induced cardioprotection. Her doctoral dissertation is titled “Exploring the Mechanisms of Exercise-Induced Cardioprotection against Myocardial Stunning.” She will continue to research cardiovascular disease in her future position as a postdoctoral fellow at the Whitaker Cardiovascular Institute at Boston University Medical Campus in Boston, Massachusetts. 75