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Transcript
TARGETING REACTIVE OXYGEN SPECIES PRODUCTION TO PREVENT LEFT VENTRICLE REMODELING IN VOLUME OVERLOAD by DANIELLE YANCEY LOUIS J. DELL’ITALIA SCOTT W. BALLINGER MARK O. BEVENSEE AMIT GAGGAR LUFANG ZHOU A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2014 TARGETING REACTIVE OXYGEN SPECIES PRODUCTION TO PREVENT LEFT VENTRICLE REMODELING IN VOLUME OVERLOAD DANIELLE M. YANCEY CELLULAR AND MOLECULAR PHYSIOLOGY ABSTRACT Mechanisms of left ventricular dysfunction in cardiac volume overload (VO) are not well understood and currently, no medical therapy exists to treat this condition. Cardiac VO is marked by eccentric remodeling and contractile dysfunction ultimately resulting in cardiac failure. Oxidative stress is implicated in the pathophysiology of heart failure and recent evidence suggests mitochondrially- produced reactive oxygen species play a role in VO. To study VO, we used a rat model of aortocaval fistula (ACF). ACF results in early diastolic stress on the left ventricle (LV) and recapitulates the progressive nature of heart failure with contractile function being initially maintained and then depressed by 6 weeks. To determine the role of mitochondrially-produced reactive oxygen species in the setting of VO, we utilized an established mitochondrially-targeted antioxidant, mitoubiquinone (MitoQ), in the ACF animal model. The following questions have been addressed in this dissertation: 1) What are the early events in VO that set forth a cycle of progressive remodeling and dysfunction? 2) Do mitochondrially-produced reactive oxygen species play a role in these events? 3) Are mitochondrially-produced reactive oxygen species a valid therapeutic target in cardiac VO both in a chronic setting? In testing these concepts, we have used a combined in vivo and in vitro approach to determine the cardiac response to VO with end points including ii cardiac function, cardiac remodeling, and cardiac efficiency. Our data support a causative role for mitochondrially-produced reactive oxygen species in both the acute phase of VO and in the transition to cardiac failure. These findings establish interplay between ROS production and cytoskeletal degradation that may provide a new therapeutic target to prevent progression to heart failure in VO. iii DEDICATION For my husband and parents who always believed in me. iv ACKNOWLEDGEMENTS I acknowledge the many people who have been instrumental in the completion of this work. Without the contributions of Dr. Jim Collawn, Dr. Jason Guichard, Mrs. Pam Powell, Mr. Eddie Bradley, Dr. Kevin Wei, and Dr. Ke Shi this work would not have been possible. I appreciate the effort and guidance of my graduate committee: Drs. Scott Ballinger, Mark Bevensee, Amit Gaggar, and LuFang Zhou served key roles in my training and were continually supportive. I also thank Dr. Michelle Fanucchi and Dr. Rakesh Patel, the Pathobiology and Molecular Medicine theme, the Graduate Biomedical Sciences program, the Department of Medicine Division of Cardiovascular Disease, and the Department of Cell, Integrative, and Developmental Biology for their support. Dr. Lou Dell’Italia has guided me through this process and has taught me to critically evaluate science, literature, and myself. His love of science and devotion to accuracy and ethical responsibility has taught me more than he will ever know. v TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS .................................................................................................v LIST OF TABLES ............................................................................................................ ix LIST OF FIGURES .............................................................................................................x LIST OF ABBREVIATIONS ........................................................................................... xii CHAPTER 1 INTRODUCTION ..........................................................................................................1 Introduction to Heart Failure .....................................................................................1 The Healthy Heart ......................................................................................................2 Anatomy of the Heart.......................................................................................2 Cardiac Circulation ..........................................................................................4 The Cardiac Conductance System ...................................................................5 Anatomy of the Cardiac Autonomic Nervous System .....................................6 The Cardiomyocyte..........................................................................................6 Cardiomyocyte Excitation-Contraction Coupling ...........................................9 Modulation of Contractility ...........................................................................13 Sympathetic and Parasympathetic Regulation of Contractility .....................13 Frank-Starling Law of the Heart ....................................................................14 The Failing Heart ....................................................................................................16 Systolic and Diastolic Heart Failure ..............................................................17 Assessment of Cardiac Function....................................................................18 Echocardiography .....................................................................................18 Cardiac Catheterization .............................................................................19 Electrocardiography ..................................................................................20 vi Computed Tomography ............................................................................20 Positron Emission Tomography................................................................20 Magnetic Resonance Imaging ...................................................................20 Exercise Testing ........................................................................................21 Blood Tests for Heart Failure ........................................................................22 Cardiac Functional Parameters (For Researchers) .........................................22 Description of Contractility ......................................................................22 Chamber Dimensions ................................................................................23 Pressure-Volume Relation and PV Loops ................................................23 Systolic Parameters ...................................................................................25 Diastolic Parameters .................................................................................26 Molecular Mechanisms Underlying Cardiac Hypertrophy .......................................28 Patterns of Cardiac Hypertrophy ...................................................................28 The Extracellular Matrix in Cardiac Remodeling ..........................................29 Key Components of the Extracellular Matrix ................................................30 Degradation of Matrix....................................................................................31 Inflammatory Pathways in Cardiac Hypertrophy ..........................................32 Molecular Mediators of Cardiac Inflammation ........................................32 The Renin-Angiotensin-Aldosterone System ................................................33 RAAS Components......................................................................................33 Cardiovascular Effects of the RAAS ...........................................................34 Therapeutic Inhibition of the RAAS ............................................................34 Pathological Consequences of Adrenergic Drive .......................................35 Oxidative Stress in Heart Failure ...............................................................................36 Sources of Oxidative Stress ..........................................................................37 NADPH Oxidase......................................................................................37 Xanthine Oxidase .....................................................................................37 Mitochondria ............................................................................................37 Medical Therapies for Heart Failure ...........................................................................38 Diuretics ........................................................................................................39 Vasodilators ..................................................................................................39 Calcium Channel Blockers ...........................................................................39 β-Blockers .....................................................................................................40 The Volume Overload of Mitral Regurgitation ..........................................................41 Mitral Regurgitation.......................................................................................41 Volume Overload ...........................................................................................42 Eccentric Remodeling ....................................................................................43 Aorto-Caval Fistula Model of Volume Overload ..........................................43 Strategies for Management of Mitral Regurgitation ......................................44 Summary and Hypothesis ...........................................................................................46 2 METHODS AND MATERIALS..................................................................................48 vii Animal Preparation .....................................................................................................48 Hemodynamics and Echocardiography ......................................................................49 Isolation of Cardiomyocytes .......................................................................................49 Application of Stretch to Isolated Cardiomyocytes ....................................................51 Live Cell Imaging .......................................................................................................52 Tissue Preparation .......................................................................................................53 Immunohistochemistry ...............................................................................................53 Transmission Electron Microscopy of Rat Tissue ......................................................54 Western Blot ...............................................................................................................54 Cardiomyocyte Bioenergetics .....................................................................................55 Statistical Analysis ......................................................................................................56 3 RESULTS ...................................................................................................................57 MitoQ Improves Cardiomyocyte Oxidative Stress and Mitochondrial Membrane Potential In Vitro, but Has No Effect on LV Remodeling and Function in vivo ........57 ACF Causes Mitochondrial Changes and Breakdown of Cytoskeleton .....................69 MitoQ Improves Cardiomyocyte Mitochondrial Organization and Cytoskeleton in Chronic VO Rats .........................................................................................................76 4 DISCUSSION ..............................................................................................................80 Introduction ................................................................................................................80 Interactions Between Mitochondria, ROS, and Cardiomyocyte Cytoskeleton in Acute Cardiomyocyte Mechanical Stretch and Chronic ACF ..............................................83 Difficulties in Studying Volume Overload and Cytoskeletal Degradation ................88 Future Studies .............................................................................................................90 Direct Bioenergetic Measurements in Chronic VO with MitoQ ..........................90 Determine if a Combination XO Inhibitor/MitoQ Therapy Beneficial in VO .....91 Determine if a Combination MitoQ/ β Blocker Therapy Beneficial in VO .........91 Examine the Role of Mitochondria-Sarcoplasmic Reticulum Crosstalk in VO ...92 Determine the Point of No Return for MitoQ Treatment in VO...........................93 Conclusions ...........................................................................................................94 GENERAL LIST OF REFERENCES ...............................................................................96 APPENDIX: IACUC APPROVAL .................................................................................108 viii LIST OF TABLES Table Page 1 Morphometric Data on 8 Weeks of ACF..........................................................67 2 LV Hemodynamic and Functional Parameters in Chronic 8 Week ACF.........68 ix LIST OF FIGURES 1 Basic Anatomy of the Heart ............................................................................................2 2 Transmission Electron Micrograph of Cardiomyocyte Sarcomeres ...............................7 3 Basic Molecular Structure of a Sarcomere .....................................................................8 4 Components of the Contractile Apparatus ......................................................................9 5 The Contraction Cycle of the Cardiomyocyte ..............................................................12 6 The Inner Filament Spacing Hypothesis of the Frank-Starling Mechanism.................15 7 Sample Echocardiograms of Rat Left Ventricle ...........................................................19 8 Example Pressure-Volume Loop ..................................................................................25 9 Patterns of Cardiac Hypertrophy ..................................................................................28 10 Mitral regurgitation .....................................................................................................41 11 Creation of an Aortocaval Fistula in a Rat..................................................................44 12 Mechanical Stretch of Adult Rat Cardiomyocytes Increased MitoSox Red...............58 13 Mechanical Stretch of Adult Rat Cardiomyocytes Increased TMRM ........................60 14 Basal Mitochondrial Function with MitoQ .................................................................62 15 MitoSox Red and TMRM Staining Unaffected by dTPP ...........................................63 16 Analysis of ROS in 8 Week ACF ...............................................................................65 17 TEM of LV Myocardium in Sham and ACF Hearts ..................................................70 18 IHC Analysis of Desmin and Mitochondrial Complex 4 in Sham and ACF Rats......72 19 Western Blot Analysis of Desmin...............................................................................74 20 IHC Analysis of Desmin and β2 tubulin in Sham and ACF Rats ...............................75 21 Changes in Mitochondrial Morphology by TEM in MitoQ-Treated Rats ..................77 x 22 IHC Analysis of Desmin and Mitochondrial Complex 4 in MitoQ-Treated Rats ......78 23 IHC Analysis of Desmin and β2 Tubulin in MitoQ-Treated Rats ..............................79 xi LIST OF ABBREVIATIONS ACF aortocaval fistula AI angiotensin I AII angiotensin II ANP atrial natriuretic peptide ARB angiotensin receptor blocker AT angiotensin receptor BNP brain natriuretic peptide CO cardiac output CT computed tomography dP/dt maximum rate of pressure change dV/dP ratio of volume change to pressure change ECG electrocardiography ECM extracellular matrix EDPVR end-diastolic pressure-volume relation EDV end-diastolic volume Ees elastance at end systole EF ejection fraction Emax maximum elastance ESPVR end-systolic pressure-volume relation xii FN fibronectin FSR Frank-Starling relationship GPCR G-protein coupled receptor HF heart failure HR heart rate IL interleukin IP3 inositol (1,4,5)-trisphosphate ISF interstitial fluid LA left atrium LV left ventricle LVEDD left ventricular end-diastolic dimension LVEDP left ventricular end-diastolic pressure LVESD left ventricular end-systolic dimension LVESP left ventricular end-systolic pressure MAPK mitogen-activated protein kinase MMP matrix metalloproteinase MR mitral regurgitation MRI magnetic resonance imaging NCX sodium/calcium exchanger NF-kB nuclear factor κ-B PET positron emission tomography PKA protein kinase A PKC protein kinase C xiii PO pressure overload PV pressure-volume RA right atrium RAAS renin-angiotensin-aldosterone system RV right ventricle SERCA sarco/endoplasmic reticulum calcium ATPase SR sarcoplasmic reticulum SV stroke volume TAC transaortic constriction TGF transforming growth factor TIMP tissue inhibitor of MMP TNF tumor necrosis factor TNFR tumor necrosis factor receptor VCFr velocity of circumferential fiber shortening Vmax maximum velocity of contraction VO volume overload WS wall stress xiv CHAPTER 1 Introduction to Heart Failure Heart failure is defined as the inability of a heart to meet the demands placed on it by the body. In some cases, this means the heart is unable to adequately fill with blood. In others, it means the heart is unable to pump blood with enough force. In either scenario, the heart is unable to pump enough blood to maintain regular function. Heart failure is a condition that is developed over time, as the heart weakens. Heart failure does not imply the heart has/is about to stop working; it simply means the heart is weakened and working inefficiently. Unfortunately, heart failure is a common condition, with over 5 million people suffering from some sort of heart failure in the United States. As stated above, heart failure is a condition that develops over time usually as a result of some sort of heart disease. Eighty four million people are thought to suffer from heart disease in the United States, making heart disease easily the most common cause of death1. The increases in morbidity and mortality caused by heart disease and heart failure place an enormous stress on the United States health care system, with nearly 15% of all healthcare expenditures going toward cardiovascular disease. As the current population ages, this number will only increase with projected values exceeding $800 billion by 2030. 1 Clearly, heart disease is a major hardship for patients, their families, and the economy. As such, heart disease is a primary target for developing new therapeutic strategies. However, the effectiveness of future therapies depends on understanding the molecular mechanisms underlying cardiac dysfunction and the eventual progression to heart failure. The objective of the work described in this dissertation is to better define the pathological cellular changes that occur early in the progression to heart failure and to identify a possible new pharmacological intervention. The Healthy Heart Anatomy of the Heart Figure 1: Basic anatomy of the heart. A: Layers of the myocardial wall. B: Chambers and valves. Note: Adapted from Sevier Medical Art with permission 2 The heart is located centrally in the body between the lungs and superior to the diaphragm. The heart itself is surrounded by the pericardium, or pericardial sac, which is a fibrous layer protecting the heart (Figure 1A). The pericardium contains pericardial fluid, which serves as both a cushion and lubricant for the heart. The serous pericardial fluid also prevents friction from occurring between the surface of the heart and the pericardial sac2. The heart itself is composed of an epicardium, myocardium, and endocardium. The epicardium is the surface and outermost layer of the heart, which is really a serous membrane continuous with the pericardium. The myocardium is the musculature of the heart, which is made up primarily of cardiomyocytes and fibroblasts. The fibroblasts maintain the extracellular matrix around the cardiomyocytes, while the cardiomyocytes provide the force for contraction for the heart. The cardiomyocytes are arranged in a spiral array, rotating 180° from epicardial surface to endocardial surface. The myocardium is approximately one-centimeter thick in healthy adult hearts. The endocardium is a single layer of endothelial cells lining the chambers of the heart and provides protection and support for the chambers and the valves2. The heart is divided into four chambers (Figure 1B): right and left atria, and right and left ventricles (RA, LA, RV, LV, respectively).2 The right and left atria comprise the superior portion of the heart, and the ventricles comprise the inferior portion. The ventricles are separated by the intraventricular septum. This septum helps maintains proper blood flow through the heart. The heart itself pumps blood in a forward direction, which is maintained by a series of valves which control blood flow and prevent blood from flowing in a backward direction. The valves themselves are controlled by papillary 3 muscles, which are attached to the cusps of the valves and to the inner surface of the heart chamber2. The right atrium receives deoxygenated blood from the venous system and pumps this blood into the right ventricle via the tricuspid valve. The right ventricle then pumps the deoxygenated blood to the lungs for oxygenation, via the pulmonary valve into the pulmonary artery. The pulmonary artery is the only artery that carries deoxygenated blood in the body. Once the blood enters the pulmonary artery, it is carried to the lungs where carbon dioxide is exchanged for oxygen. The pulmonary vein returns oxygenated blood from the lungs to the left atrium of the heart. The blood flows from the left atrium to the left ventricle via the mitral valve. During heart contraction, the left ventricle pushes the oxygenated blood into the aorta to be distributed throughout the body2. Cardiac Circulation The heart is responsible for delivering oxygenated blood to the body and is thus constantly consuming oxygen to produce energy. As such, the heart has its own welldeveloped vascular system. The cardiac blood supply is provided by coronary arteries, which branch directly from the aorta. The left and right main coronary arteries run along the external surface of left and right atria and ventricles. The right coronary artery branches into the posterior anterior descending artery and supplies the right ventricle. The left coronary artery, immediately following its departure from the aorta, branches into the left anterior descending artery and the left circumflex coronary artery. The left anterior descending artery supplies blood to the intraventricular septum, papillary muscles and 4 parts of the left ventricle. The left circumflex artery supplies blood to the rest of the left ventricle. The heart’s venous return is managed by small channels in the myocardial wall called thebesian veins, which allow perfusion of deoxygenated blood from the coronary artery system into coronary veins which carry the deoxygenated blood to the right atrium2. The Cardiac Conductance System The pumping action of the heart is closely regulated by coordination of contraction of the individual chambers with the contraction of papillary muscles to allow for forward flow. This careful coordination is achieved through the use of an electrical conduction system2, 3. The signal to begin heart contraction is initiated in the sinoatrial node, which is bundle of specialized muscle fibers located in the wall of the right atrium. The signal is then propagated to the atrioventricular node, located in the inferior part of the intraventricular septum. From there, the signal travels to the bundle of His, which then bifurcates within the intraventricular septum to form right and left bundle branches. These branches divide multiple times to innervate the right and left ventricles via Purkinje fibers. Impulses from the bundle of His/Purkinje fibers are transmitted first to the papillary muscles and then to the walls of the ventricles, which allows the papillary muscles to contract and open the valves prior to ventricular contraction. This coordination of heart contraction and valve opening is critical in preventing regurgitation of blood into the emptying chamber2. 5 Anatomy of the Cardiac Autonomic Nervous System The conductance system of the heart is controlled and affected by the autonomic nervous system through both sympathetic and parasympathetic innervation. Sympathetic signals originate in the spinal cord, synapse at the sympathetic ganglion or cardiac plexus, and then synapse at the sinoatrial node. These post-ganglionic neurons stimulate increases in heart rate and force of contraction by releasing epinephrine and norepinephrine. Parasympathetic signals, on the other hand, originate in the medulla oblongata and synapse at the cardiac plexus. The signals are conveyed to the heart via the vagal nerves. Parasympathetic neurons stimulate decreases in heart rate and force of contraction by release acetylcholine4, 5. The Cardiomyocyte Cardiomyocytes are the specialized cells of the heart. Cardiomyocytes are muscle fibers and are striated in appearance. Unlike other muscle fibers, cardiomyocytes are binucleated and maintain a conductance system via intercalated disks. These intercalated disks connect individual cardiomyocytes and provide a structural attachment to neighboring cells via adherens junctions and provide avenues for cellular communication via gap junctions. The cardiomyocytes themselves are divided into myofibrils, which are further divided into a series of sarcomeres2. Sarcomeres are the contractile unit of the cardiomyocyte (Figure 2). Sarcomeres are composed of contractile proteins myosin and actin as well as capping proteins which support the contractile machinery and anchor it to 6 the cell. Sarcomeres are aligned in a parallel registry, which gives the cardiomyocyte its striated appearance6. Figure 2: Transmission electron micrograph of cardiomyocyte sarcomeres. The sarcomere is comprised of actin filaments, myosin filaments, and the Z disc, which is located on either end of the sarcomere (Figure 3). Actin filaments are comprised of actin monomers dimerized into an α-helix. Tropomyosin lies within the grooves of the alpha helix along the length of the entire actin filament providing structural stability and regulating actin and myosin interaction. Myosin filaments are composed of several hundred molecules of myosin protein. Myosin is a large protein with a head region, rod region, and a hinged stalk. The head region binds the actin filament and hydrolyzes ATP, 7 the rod region forms the body of the filament, and the hinged stalk links the head and rod domains (Figure 4). The Z disc serves as an anchor for the actin and myosin cross bridges and is comprised of several proteins with both structural and regulatory functions. The actin filaments are tethered to the Z disc by the protein alpha-actinin which forms a bundled structure which is surrounded and stabilized by ring structures of desmin. The disc also links sarcomeres to the cellular cytoskeleton via polymer fibers. These fibers attach to costameres, comprised of dystrophin and focal adhesion kinase complexes, in the cell membrane. Figure 3: Basic molecular structure of a sarcomere. Note: Adapted from Sevier Medical Art with permission The costameres also connect to the extracellular matrix, thus connecting individual cardiomyocytes to the larger organ structure. While actin filaments are directly attached 8 to the Z disc, the myosin thick filaments are linked to the Z disc via the large protein titin, which provides structure and elasticity to the sarcomere. In general, each sarcomere is associated with a mitochondrion that provides ATP for cell contraction and T tubules, which are invaginations of the cell membrane that allow entry of ions essential for muscle contraction. Sarcomeres are also surrounded by sarcoplasmic reticulum to remove ions, especially calcium, from the cytoplasm and store them for subsequent contractions6-12. Figure 4: Components of the contractile apparatus. Note: Adapted from Sevier Medical Art with permission Cardiomyocyte Excitation-Contraction Coupling A heartbeat is composed of two phases: systole and diastole; systole is the contraction phase and diastole is the relaxation and refilling phase. Systole and diastole are possible because of the coordinated contraction and relaxation of sarcomeres during the contraction cycle. Cardiomyocyte contraction is controlled by cyclic increases and 9 decreases in intracellular calcium concentrations (Figure 5). Systole begins with the myosin head region hydrolyzing ATP. At the same time, the presence of calcium alters the conformation of tropomyosin and allows the binding of the myosin head region to the neighboring actin filament, forming a myosin-actin cross bridge. Once the ATP is hydrolyzed, the resulting ADP and inorganic phosphate molecules remain associated with the myosin head domain. Once the myosin head is bound to the actin filament, the inorganic phosphate molecule is released and the myosin head pulls on the actin filament, creating the power stroke, causing the entire sarcomere to contract. When the myosin head releases the actin filament, the ADP molecule is released as well, freeing space for a new ATP molecule to bind. The machinery is then reset and the contraction cycle can begin again. Following this last step of the cycle, calcium is removed from the cytoplasm and stored in the sarcoplasmic reticulum. The removal of calcium restores tropomyosin to its resting, myosin-blocking position, while the elasticity of titin restores the sarcomere to its resting length. The contraction cycle cannot begin again until an influx of calcium alters the conformation of tropomyosin that allows for myosin-actin cross bridge formation. The sarcomere remains relaxed until this time2, 8, 12. Troponin complexes assist this calcium regulation of the contraction cycle. Troponin complexes are regularly spaced along the actin filament and are composed of three subunits: T, I, and C. Troponin T adheres tropomyosin to the actin filament, Troponin I inhibits ATP hydrolysis by the myosin head domain, and Troponin C mediates the response of tropomyosin to calcium. Calcium is critical for controlling cardiomyocyte contraction and is tightly coupled to conductance pathways that transmit action potentials to the T tubules of 10 cardiomyocytes. These action potentials cause a rapid, transient increase in sodium that shifts the cells to a slightly depolarized state. This potential is then neutralized by a subsequent decrease in potassium, and increase in calcium entering through the L-type channels. This calcium entry is small but sufficient to activate ryanodine receptors on the sarcoplasmic reticulum. When the ryanodine receptors bind calcium, they release large amounts of calcium stored in the sarcoplasmic reticulum. This is often referred to as calcium-induced calcium release. This drastic increase in cytoplasmic calcium causes the tropomyosin conformation change and allows the contraction cycle to begin13-16. Removal of calcium following initiation of contraction is necessary, as large amounts of calcium are cytotoxic. This removal is accomplished by the sarco (endo)plasmic reticulum calcium ATPase 2a (SERCA 2a), which pumps cytoplasmic calcium into the sarcoplasmic reticulum for storage2. A small accessory protein, phospholamban, which inhibits SERCA when unphosphorylated, regulates the activity of SERCA. Any remaining calcium is removed by the sodium/calcium exchangers (NCX) or a secondary sarcolemmal calcium ATPase17. 11 Figure 5: The contraction cycle of the cardiomyocyte. Note: Adapted from “Muscle Fiber Contraction and Relaxation” OpenStax with permission 12 Modulation of Contractility Several pathways modulate cardiomyocyte contractility. L type calcium channels can alter entry into the cell, ryanodine receptors can alter calcium release from the sarcoplasmic reticulum, and SERCA 2a and accessory proteins can alter the rate of calcium sequestration in the sarcoplasmic reticulum. The specific point of alteration in the calcium handling process will impact specific characteristics of cardiac function including heart rate, rate of action potential conductance, rate and force of contraction, and degree of relaxation. Factors that increase calcium in the myofilaments will result in an increase in heart rate and force of contraction, while inhibitors of calcium release will reduce heart rate and force of contraction2, 18. Sympathetic and Parasympathetic Regulation of Contractility While both the parasympathetic and sympathetic arms of the autonomic nervous system innervate the heart, the sympathetic nervous system has strong influences on vascular tone and vasoconstriction giving it a greater overall effect on cardiovascular function2. Various signals can induce a sympathetic response but the most common signals are stress and indicators of poor cardiovascular performance. One such indicator is the baroreflex, which is a neural circuit beginning in arterial stretch receptors in carotid arteries and the aortic arch and links to vagal nerves via the brain stem. An increase in pulse pressure will increase stretch experienced by the receptors and stimulate vagal activity. Conversely, a reduction in blood pressure, and thus a reduction in arterial stretch, will lead to decreased vagal activity and increased sympathetic activation18, 19. 13 The cardiac sympathetic nervous system releases catecholamines, primarily norepinephrine, into the interstitium surrounding cardiomyocytes. Norepineprhine activates α- and β-adrenergic receptors on the surface of the cells, which leads to modulation of heart rate and contractility. All adrenergic receptors are G-protein coupled receptors (GCPRs) and act primarily through activating or inhibiting adenylyl cyclase18. When adenylyl cyclase is activated, cAMP levels are elevated in the surrounding cellular environment, and this activates the cAMP-sensitive Protein Kinase A (PKA).20-22 PKA activates the key calcium regulators of the cardiomyocyte through phosphorylating either the L-type calcium channel, the ryanodine receptor, phospholamban, or the sodium/calcium exchanger.21 The net result of these phosphorylations is increased calcium flux and increased rates of calcium entry and sequestration, which increases the force and frequency of contraction. On the other hand, when adenylyl cyclase is inhibited, cAMP levels are lower, PKA activity is not activated, and force of contraction is reduced22. Frank-Starling Law of the Heart Another mechanism that modulates contractility is the Frank-Starling Law of the heart, also known as length-dependent activation, in which the volume of a systolic stroke is determined by the preceding diastolic volume, or preload (Figure 6). This mechanism allows the heart to alter pump mechanics on a beat-to-beat basis and stems from stretch during diastole, which primes cardiomyocytes for a more robust contraction during the next beat.2 Functionally, this allows the heart to rapidly compensate for increases in systemic resistance23 or venous return.24 The relationship between stretch of the left 14 ventricle and force generation was recognized over a century ago by Otto Frank, Ernest Starling, and their collaborators; however the mechanisms responsible for this phenomenon were largely unknown until Allen and Kurihara discovered that force generation by a cardiomyocyte is a function of cell length at the time of stimulation.25 Since that time the molecular mechanisms underlying the Frank-Starling Relationship (FSR) have continually been studied, but are still not fully known. Figure 6: The inner filament spacing hypothesis of the Frank-Starling mechanism. Note: adapted from “Myofilament Length Dependent Activation” deTombe, et al., 2010 J. Mol Cell Cardiol 48 (5): 851-858. Copyright 2013 Elsevier B.V. Reprinted with permission. Another key finding by Allen and Kurihara is that the substantial increase in contractile force immediately following stretch is accomplished without a simultaneous increase in calcium flux. An increase in preload does eventually increase calcium flux, but this lags behind the initial onset of stretch by several minutes. This observation has led to the assumption that the immediate increase in contractile force following stretch is 15 due to increased calcium sensitivity, and that the delayed increase in calcium flux that occurs later is due to modulation of calcium handling.26 Hibberd and Jewell further elucidated the concept that force generated by cardiomyocytes at a given calcium concentration is a function of cell length.27 Several hypotheses have been put forward to explain how this phenomenon occurs at the molecular level. The first was simply that sensitivity of the troponin complex to calcium increases as the cardiomyocyte is stretched.25 This concept was expanded with the finding by Hofmann and Fuchs that increased actin-myosin binding in myofilaments leads to an increase in calcium binding.28 Importantly, sarcomeres are isovolumic. When stretched, their diameter is decreased proportionately to the increase in length. Therefore, individual filaments within a sarcomere must be pushed into closer proximity as the sarcomere is stretched. If spacing between actin and myosin is reduced, it may allow for increased cross bridge formation and greater force generation following stretch. 29 However, studies to test this hypothesis only assumed myofilament spacing without directly measuring it. Therefore, the molecular basis for the Frank-Starling relationship is not entirely clear and remains an active area of research. The Failing Heart As described previously, the heart has several mechanisms available that can compensate for changes in demand or pumping mechanics; however, these mechanisms can only compensate for a certain amount of time. Prolonged disturbances in cardiac function are accommodated through structural remodeling. While altering the size and/or 16 shape of the heart may temporarily allow for a normalization of cardiac output, this alteration in cardiac ultrastructure begins the process of pathological remodeling that eventually leads to heart failure. Heart failure is defined as an inability of the heart to pump blood at a rate that meets the body’s metabolic demand while maintaining normal filling pressures.2, 30 Patients suffering from heart failure complain of reduced ability for physical exertion, abdominal pain, rapid heart beat and shortness of breath, and fluid retention in the legs and abdomen30, 31. Systolic and Diastolic Heart Failure Heart failure is classified in several ways, but the simplest is classification based on ejection fraction. Ejection fraction is a measure of the ability of the left ventricle to completely empty during contraction. Ejection fraction is measured as the percentage of left ventricle volume at end-diastole that is ejected during systolic contraction. In a healthy heart, ejection fraction ranges from 55-75%. A reduced ejection fraction is classified as systolic heart failure, while a preserved ejection fraction is classified as diastolic heart failure31. Several conditions lead to the development of either of these forms of heart failure. Conditions that impair contractility due to fibrosis, such as ischemia of a coronary artery, myocardial infarction, aortic stenosis, or hypertension, lead to systolic dysfunction. Conditions that impair filling during diastole, such as mitral regurgitation, dilated cardiomyopathies, and volume overload, lead to diastolic dysfunction. While 17 these are all pathological conditions, the hearts are able to maintain a normal ejection fraction30. Assessment of Cardiac Function As discussed previously, ejection fraction is a key parameter in the diagnosis of heart failure, but there are others. The heart is responsible for maintaining forward blood flow, or cardiac output. Cardiac output (CO) is the product of heart rate and stroke volume so cardiac output increases when contractility and/or preload increases, and decreases when afterload increases. Heart rate is easily measured, but stroke volume requires more complex diagnostic methodologies. Clinicians have a variety of diagnostic tests to measure physical structure of the heart as well as measures of contractility and performance2, 30. Echocardiography. Echocardiography is one of the most common techniques available for studying both cardiac structure and contractility, specifically LV ejection fraction (Figure 7).2, 31, 32 Echocardiography can be performed with several degrees of complexity, depending on how the ultrasonic waves are transmitted and received by the instrument. The simplest form of echocardiography is M-mode (motion)33, which uses a straight beam of ultrasound waves and is useful for measuring chamber dimensions during systole and diastole34 and tracking the motion of valves as they open and close.35 A 2-dimensional image can be created with echocardiography by oscillating the beam through an arc.36 Because of the high rate of oscillation, these images are updated in realtime.35 Echo systems can also take advantage of the Doppler effect to determine the 18 velocity at which components of the heart are moving, as well as the speed of blood flowing through the chambers of the heart or the coronary vasculature.2, 35, 37 A more modern development of echocardiography is the 3D system, which has proven useful for both diagnostic and corrective procedures38, as it allows quantification of left ventricular remodeling.39 A B Figure 7: Sample echocardiograms of rat left ventricle. A: M mode. B: 2-dimensional. Cardiac catheterization. Another primary diagnostic tool used in both clinical and research settings is the insertion of a pressure sensor into the heart via catheterization.2 Echocardiography provides detailed information about the shape of the heart, but catheter-based measurements are used to assess hemodynamics.40 Pressure recordings can be made of the left or right side of the heart, and the latter can be broadened to include pulmonary artery measurements.41 Pressure measurements can provide critical data on systolic function, as high ejection pressures indicate elevated afterload that may be due to hypertension or aortic stenosis, and high filling pressures are a hallmark of poor ventricular compliance and diastolic dysfunction.2 19 Electrocardiography. Clinicians also use electrocardiography (ECG) to monitor electrical activity of the heart and determine irregularities in rhythm or problems with the cardiac conductance system.2, 42 ECG may also indicate functional deficits stemming from improper calcium handling. Heart failure patients with irregular rhythms typically have additional risk and require additional therapies.43-45 Conduction defects can be caused by fibrosis and are often manifested as delayed or prolonged action potentials.2 Computed tomography. Computed tomography (CT) scans are an x-ray-based technique combined with an injected contrast agent that detects pathologies in the blood vessels of the heart, as well as the walls of the heart itself.2, 46 CT scans are excellent for detecting arterial blockages due to atherosclerosis or thrombus formation, and can also be used to characterize hypertrophy and estimate cardiac output.47 Recent advances have increased the utility of CT while reducing cost and radiation exposure to the patient.2 Positron emission tomography. Positron emission tomography (PET) scans rely on injected radiolabeled tracers that are typically conjugated to metabolic substrates like glucose/ deoxyglucose2 or palmitate48. These labeled molecules assess metabolic activity of the heart. Tracers can also be used to assess blood flow and the abundance of various cell surface receptors. Magnetic resonance imaging. Magnetic resonance imaging (MRI) uses the principle of nuclear magnetic resonance.49 MRI instruments usually take advantage of the characteristic response to hydrogen nuclei in powerful magnetic fields, with water and fat 20 serving as the source of hydrogen. The primary advantage of MRI over many other imaging techniques is that it uses non-damaging radio waves instead of potentially damaging radiation.2 MRI images are thin serial slices that show soft tissue in high detail, useful in assessing valve function, hypertrophy, coronary blood flow, and extent of damage due to infarction.2, 50-52 The slices can be combined to allow for 3-dimensional modeling of the heart.53, 54 MRI techniques can also be used to generate spectra for the measurement of specific substances, such as products of metabolism.55 Exercise testing. Stress testing is frequently used in conjunction with the imaging techniques and functional assessments described above.2, 56 A typical stress test requires a patient to exercise using a stationary bike or treadmill as the resistance is slowly increased.57 As the patient exercises, heart rate and electrical conductance are measure via ECG. The test continues until the patient reaches a target heart rate, pain or discomfort occurs, or the ECG detects abnormal activity.58 Alternatively, if the patient is unable to exercise, stress testing can be performed with the administration of certain drugs. Dobutamine is often used and functions as an ionotrope to increase the work performed by the heart and thus increase metabolic demand.2 Adenosine, which affects vasodilation, and dipyridamole, which potentiates the effects of adenosine, can also be used.2 These drugs are administered to the patient and CT scans are used to assess effects on myocardial function under metabolic demand.59 21 Blood Tests for Heart Failure Heart failure prognosis can be measured via various biomarkers in blood samples. The most common biomarkers measured are atrial natriuretic peptide (ANP)60 and B-type (brain) natriuretic peptide (BNP)61, which are synthesized by myocytes in the heart and released during times of elevated wall stretch to enhance natriuresis.62 Paradoxically, these peptides are highly upregulated in heart failure patients, who often display sodium and fluid retention.63 The concentrations of these two biomarkers positively correlate with HF severity62 and thus are extremely useful clinically.64, 65 Cardiac troponin (cTnT) is detectable in blood following heart tissue damage, especially in myocardial infarction.32 The assays available to screen for this biomarker are useful for their sensitivity66 and utility in the clinic.67 Elevations in cTnT levels are predictive of poor patient outcomes.68 Cardiac Functional Parameters (For Researchers) Data from these assessments can be combined with the aid of formulas to yield parameters indicative of LV performance and structural changes. These measurements are derived from potential energy, work, and the Law of LaPlace. Performance indicators are most often derived from the pressure traces obtained during catheterization, whereas the imaging techniques provide volume measurements and detail on structural defects in various regions of the heart2. Description of contractility. Researchers determine systolic performance via contractility, preload, and afterload. Contractility enables comparison between hearts, or 22 before and after intervention in the same heart.69 Preload is the constant resistance in the heart muscle while afterload is the additional resistance required to move blood. When the heart muscle is stimulated, tension is generated as myosin and actin form cross bridges, but no motion occurs until the tension generated exceeds the resistance. At this point, the heart is undergoing isovolumic contraction.70 Once the force exceeds the resistance, the heart undergoes isotonic contraction. At this point, the resistance, or afterload, is proportional to the velocity and magnitude of contraction.69 The point of maximum contraction is determined by preload, as described by the Frank-Starling relationship. End-systole is the point of maximum contraction; once this point is reached, the heart muscle enters into isovolumic relaxation. Chamber dimensions. Echocardiography is also used in research to determine the left ventricle, right ventricle, and atria dimensions at end systole and end diastole. Stroke volume (SV) is calculated as the difference between end-systolic and end-diastolic volumes. Left ventricle ejection fraction (LVEF), a primary indicator of cardiac performance, is determined from these values as the ratio of stroke volume to enddiastolic volume. Echoes can also reveal changes in wall thickness and these values can be combined with pressure data from pressure catheters to infer increases in wall stress (WS), which may underlie the cardiac remodeling process71. Pressure-volume relation and PV loops. The relationship between pressure and volume data can be plotted to form a pressure-volume (PV) loop diagram (Figure 8). This diagram features a series of counterclockwise loops that trace the changes in pressure and 23 volume as the heart progresses through the contraction process, as shown below. The concentric loops of the diagram are generated by reducing the preload volume for each contraction through the use of pharmacologic or mechanical manipulation.70 Plotting the pressure-volume relationships of a heart under different preload conditions allows for the determination of parameters of cardiac function71. PV loop diagrams are assessed beginning at end diastole where contraction is initiated.70 The vertical phase of the loop represents isovolumic contraction, when tension is developed and pressure is increased, but the ventricle has not begun contraction. Once the ventricle begins to contract and ejection begins, the volume begins to decrease as additional pressure is developed. At end systole, ejection and force generation end. At this point, diastole beings and tension, and therefore pressure, drops rapidly, but no chamber movement occurs, which is known as isovolumic relaxation. Diastolic refilling begins and lasts until the beginning of the next contraction, resulting in an increase in volume with minimal change in pressure. 24 Figure 8: Example pressure-volume loop. Note: From “Noninvasive quantification of left ventricular elastance and ventricular-arterial coupling using three-dimensional echocardiography and arterial tonometry”, E. Gayat et al., 2011 Am J Physiol Heart Circ Physiol 301: H1916– H1923. Copyright 2011 the American Physiological Society. Reprinted with permission. Systolic parameters. As mentioned earlier, the most useful indicator clinically of cardiac function is ejection fraction. However, a variety of other measurements are used in research that has great utility in assessing cardiac performance. One of the most useful parameters obtained from PV loops is the end-systolic pressure-volume relation (ESPVR).70 The ESPVR is the line connecting the point of end systole of all the PV loops from the loop diagram. This position in each loop defines the elastance of the ventricle. Elastance is the ratio of pressure to volume normalized to the volume at zero pressure (Vo). The slope of the ESPVR line is the elastance at end systole or Emax, which can indicate changes in inotropy.69, 70 Increased inotropy shifts the slope of the ESPVR line to 25 the left and makes it more vertical while a decrease in inotropy will shift the ESPVR to the right and make the slope more horizontal71. There are several other key indicators of contractile function. One is wall stress, which is derived from the Law of LaPlace and relates the pressure exerted on a ventricle wall to its thickness.69 Increased pressure and/or decreased wall thickness increases wall stress, as is the case in eccentric hypertrophy. Reduced pressure and/or increased wall thickness decreases wall stress, as in concentric hypertrophy. Wall stress can be calculated at both end-systole and end-diastole, and the point at which wall stress is elevated or decreased is a primary determinant of the pattern of cardiac hypertrophy.71 Velocity of contraction is also a key indicator of contractile function. Velocity of contraction is related to the elasticity and function of the myofilaments and is the maximum possible velocity of contraction with no load (Vmax). On the whole heart level, the velocity of contraction is measured as the velocity of circumferential fiber shortening (VCF or VCFr).70 Similarly, the maximum rate of pressure rise during systole (dP/dt) can be a useful indicator of contractile function in certain contexts.69, 72 Diastolic parameters. Diastole is the relaxation and filling phase of heart contraction.73 Assessing diastolic function involves determining the timing, duration and degree of relaxation in conjunction with wall stress and the pressure-volume relationship. While it may seem counterintuitive, relaxation of the heart is an active process. In order for myofilaments to relax, the calcium that activated them must be removed and sequestered in the sarcoplasmic reticulum, against the concentration gradient. As described previously, this task is performed by SERCA and NCX, and requires the use of 26 ATP. How quickly calcium is sequestered, which occurs during the isovolumic relaxation phase, affects chamber relaxation and filling.73 The change in pressure per unit time (-dP/dt) is a useful measurement and is the derivative of LV pressure with respect to time. This measurement is sensitive to preload, afterload, and heart rate. The constant, tau, is the rate of pressure fall during isovolumic relaxation, and is based on a set of assumptions about the mathematical characteristics of the pressure decline, and is also sensitive to loading conditions69. Several actions occur simultaneously during the passive filling phase of diastole. During this period, the majority of filling occurs without active force generation by the heart chambers. Titin in the myofilaments stores potential energy acquired during contraction, and uses this recoil energy to pull the sarcomeres back to starting length. The left atrium also actively pumps into the ventricle. When the left atrium pumps into the left ventricle, both chambers initially experience a drop in pressure, but this is more pronounced in the left ventricle.69 The mechanical compliance of the left ventricle wall itself also influences the rate of relaxation and filling dynamics. These mechanisms all have different degrees of influence at different points during the filling process, and can be modeled with an end-diastolic pressure-volume relation (EDPVR) determined from PV loops. Unlike the linear ESPVR, EDPVR is an exponential curve. The ratio of change in volume to change in pressure (dV/dP) indicates chamber compliance, whereas the inverse of this value indicates passive stiffness.69 The ratio of chamber volume to wall thickness also contributes to the shape of the EDPVR curve. If all other factors are held constant, increases in either wall thickness or stiffness will increase the slope of the EDPVR, and vice versa. 27 Molecular Mechanisms Underlying Cardiac Hypertrophy Heart failure is typically preceded by cardiac hypertrophy, which is thought to be an attempt by the heart to compensate for hemodynamic overload.2, 74 Myocytes in the heart are terminally differentiated cells and have a limited capability for replication, so any hypertrophy is achieved through cell growth and not proliferation.72, 75 In some cases, such as exercise, hypertrophy is a beneficial physiologic response.51, 76 Hypertrophy is thought to be an adaptive response in the early development of heart disease but at some point the cardiac remodeling process becomes pathological. A pathological increase in the size of the heart has significant drawbacks including increase in energy and oxygen demand, reduced delivery of nutrients due to lengthening sarcomeres and increased wall thickness. 77 Figure 9: Patterns of cardiac hypertrophy. Note: Adapted from Sevier Medical Art with permission. Patterns of Cardiac Hypertrophy 28 There are two primary forms of cardiac hypertrophy, which develop based on the nature of the hemodynamic stress (Figure 9). Increases in afterload lead to a concentric form of hypertrophy. A resistance to forward flow causes increases in afterload; this is known as pressure overload. Concentric hypertrophy occurs due to a thickening of myocytes that leads to a thickening of the ventricle wall. Increases in preload lead to an eccentric form of hypertrophy. Conditions marked by impaired relaxation and diastolic dysfunction such as volume overload (VO), are associated with dilation of the ventricle wall. The thickness of the wall may be reduced in this case due to the stretch on the walls of the ventricle and sarcomeres being added in series. These two primary patterns of hypertrophy may not be mutually exclusive in the history of an individual. Cardiac hypertrophy may be initiated in a concentric fashion, and then give way to dilatation more characteristic of eccentric remodeling.77 This is frequently associated with decompensation and the onset of overt heart failure. The Extracellular Matrix in Cardiac Remodeling The changes in heart shape and size that occur during hypertrophy are not achieved only by altering the dimensions of cardiomyocytes. The extracellular matrix (ECM) is a complex network of proteins and proteoglycans that provides the structure of the myocardium.78 For cardiac hypertrophy to occur, the ECM must be drastically restructured, which is accomplished with finely coordinated processes of degradation and synthesis. However, disruption of this balance in either direction can have dire consequences for the structural and functional properties of the heart.79 Pathological fibrosis is of particular significance and can occur in the interstitium between myocytes 29 (interstitial fibrosis), in association with cardiac vasculature (perivascular fibrosis), or in place of dead cardiomyocytes (replacement fibrosis). Key Components of the Extracellular Matrix The core component of the ECM is collagen, a strong but flexible triple helix protein.80, 81 The cardiac ECM is composed primarily of collagen I that form thick bundles to provide strength, stiffness and support and collagen III, which is more elastic and creates a fine mesh that assists in adhering myocytes. Both collagens I and III are synthesized in cardiac fibroblasts. Other proteins also add elasticity, connectivity, and stability to the ECM. Laminin is a core component of the basement membrane of most tissues82 and provides cells a point of attachment in the ECM. This attachment is accomplished through linkages to fibronectin.83 Fibronectin can also mediate cell attachment to the matrix by binding collagens I and III and appears to be a source of elasticity in the ECM mesh that connects myocytes.84 However, fibronectin is also associated with initiation of pathological fibrosis in cardiac hypertrophy.85 Elastin also imparts stretching and recoiling properties to tissue, that have been well described in arterial vessel walls.86 The exact role of elastin in the myocardial matrix is less clear, but it is assumed to provide compliance to the myocardial wall in much the same was as it does in arteries, and it has been suggested that heart failure is associated with a loss of elastin.87 The matrix also has important regulatory roles in addition to providing structural support to heart tissue. Transforming growth factor beta (TGF-β) is one of the most 30 prominent regulatory molecules in the ECM. TGF-β is a latent growth factor sequestered in the ECM.88 TGF-β is activated via proteolysis conducted by proteases capable of activating matrix metalloproteinases,89 thus coupling the effects of TGF-β with remodeling processes. Once released from the ECM, TGF-β activates its receptor, which signals primarily through Smads, the JNK, and MAPK pathways. TGF-β signaling through these pathways can have direct hypertrophic effects on cardiomyocytes90 and promote fibrosis91. Proteoglycans are also critical regulatory molecules in the ECM. These molecules have diverse structure and function, but all are coated in glucosaminoglycans.82 Proteoglycans are important in guiding the overall organization of the matrix and mediating the effects of various growth factors. Finally, periostin is another protein with a role in myocardial ECM assembly. Periostin is virtually undetectable in the normal adult heart but is induced by cardiac hypertrophy92, where it regulates the assembly of collagen fibrils. This is critical during normal growth and development93; however, in the adult heart it contributes to pathological fibrosis.94, 95 Conversely, the absence of periostin seems to impair necessary scar formation after infarction and can lead to cardiac rupture.96 Degradation of Matrix Several families of enzymes are able to degrade ECM, but this activity is primarily performed by matrix metalloproteinases (MMPs), which are a family of zincdependent proteases activated by cleavage or modification of a critical cysteine residue through chemical reactions, especially oxidation.97 Collagen is the major target of MMPs, 31 but they also degrade other critical matrix proteins such as elastin and fibronectin 98 This activity of MMPs, primarily MMP2 and MMP999, is an essential component of the process of cardiac hypertrophy and failure.100 The action of MMPs is moderated by a family of proteins known as tissue inhibitors of MMPs (TIMPs)101, and these molecules are upregulated in concert with increases of MMP activity.99 Accordingly, a key characteristic of the remodeling process is the MMP/TIMP balance.102 Inflammatory Pathways in Cardiac Hypertrophy Inflammation is frequently associated with myocardial damage and remodeling, and involves a large variety of cell types and molecular mediators. The consequences of inflammation in the heart is also diverse, as it can directly impact cardiac function while also driving many of the molecular pathways that contribute to cardiac remodeling and failure.103 Molecular mediators of cardiac inflammation. Much of the damage and dysfunction that occurs in the heart because of inflammation is specifically caused by the release of cytokines. While infiltration of immune cells occurs, all cells found in heart tissue are capable of releasing proinflammatory cytokines.103 These cytokines include tumor necrosis factor (TNF), which is synthesized by most cells in both a membrane-bound and soluble form, and has two receptors, TNFR1 and TNFR2.104 TNF has negative inotropic effects on cardiomyocytes105 and can induce production of other proinflammatory cytokines as well as apoptosis.106 TNFR1 mediates angiotensin II-driven fibrosis107 and activates several key hypertrophic signaling 32 pathways108. Conversely, TNFR2 appears to have a protective role in the heart by opposing the induction of fibrosis by TNFR1.108 TNF activity can also have a profound influence on the balance between MMP activity and TIMP expression, and thereby contributes significantly to the remodeling of the myocardium.109 Interleukins are another class of molecules that mediate inflammatory responses, and interleukin-1 (IL-1) and IL-18 are the most common cardiac forms. These molecules activate a large variety of pathways, including JNK and MAPK, which alter gene expression. Further, IL-1 leads to activation of Nuclear Factor κ-B (NF-kB), which is the primary signaling mechanism in cardiac cellular immune responses and upregulates production of chemokine/cytokines, adhesion molecules, and colony stimulating factors.110 IL-1 mediated activation of NF-kB also alters the MMP/TIMP balance.103 The Renin-Aldosterone-Angiotensin System The renin-aldosterone-angiotensin system (RAAS) is a set of enzymes and short peptides, which effects blood pressure and cardiovascular tissue homeostasis. The RAAS has been implicated in cardiovascular pathologies, particularly hypertension and concentric remodeling, for decades and manipulation of this system has been one of the greatest medical breakthroughs of this century. However, research has uncovered additional complexities suggesting the attenuation of the pathological effects of the RAAS are not as complete as previously thought. RAAS components. A variety of stimuli can induce release of the enzyme, renin, from the juxtaglomerular cells of the kidney. These stimuli include low kidney perfusion, 33 reduced salt concentrations in blood, and elevated catecholamine concentrations.2, 111 In the classical pathway, renin cleaves angiotensinogen, which is constantly produced by the liver and circulates normally in blood.112 The cleavage product is the 10-amino acid peptide, angiotensin I. The angiotensin converting enzyme (ACE), which is found throughout the vascular system, then converts angiotensin I into the 8-amino acid peptide angiotensin II,113 which then exerts its effects through two receptors, AT1 and AT2, with AT1 being the more active receptor in the heart. Cardiovascular effects of the RAAS. AT1 activation leads to a variety of responses, including increased blood pressure due to vasoconstriction, cardiomyocyte hypertrophy and apoptosis, increased release of and sensitivity to norepinephrine, and release of aldosterone from the adrenal gland.114, 115 The AT2 receptor opposes many of the AT1 effects, as it attenuates hypertrophy and myocardial remodeling while activating nitric oxide-mediated vasodilation through kinins, though it can still activate apoptotic pathways.116 Angiotensin II also substantially contributes to fibrosis in the heart, as cardiac fibroblasts express the AT1 receptor and its activation upregulates collagen I and TGF-β.115 The aldosterone released due to AT1 stimulation also contributes significantly to the fibrosis, inflammation, and ventricular remodeling.117 Therapeutic inhibition of the RAAS. Medications have been developed which interrupt either the production of angiotensin II (ACE inhibitors) or the effects of angiotensin receptor activation (angiotensin receptor blockers, ARBs). ACE inhibitors were originally derived from snake venom with the intention of reducing systemic blood pressure.118 Since then, a multitude of ACE inhibitors have been developed including 34 captopril, enalapril and lisinopril. These drugs have great utility in a variety of cardiovascular conditions beyond hypertension, including heart failure.2 Blockade of the angiotensin II receptors has also been successful in attenuating hypertension, left ventricular remodeling, and heart failure symptoms. Aldosterone receptor antagonists have also proven useful in attenuating hypertension and left ventricular remodeling. However, renin activation of angiotensin I has been a more difficult target due to poor solubility and half-life of the agents under development.118 Other difficulties with RAAS blockade therapies include the ACE inhibitor escape phenomenon, in which aldosterone production is restored to normal after being initially depressed by ACE inhibition. This appears to be due to processing of angiotensin I to angiotensin II by non-ACE pathways, likely mediated by the mast cell protease, chymase.117 Furthermore, recent studies have recognized additional angiotensin I cleavage products, such as Ang (1-9) by ACE2, and fragments smaller than angiontensin II such as Ang (1-7), which appear to have protective effects that counter angiotensin II.113, 118, 119 Pathological consequences of adrenergic drive. As described previously, the catecholamines have a profound impact on cardiovascular function due to their ability to increase heart rate and cardiac output. Increased adrenergic drive is beneficial in an acute context however chronic activation of this system can become pathological. Sustained adrenergic activation can lead to loss of cardiomyocytes, increased hypertrophy and fibrosis, and an inability of the heart to respond to normal adrenergic signals. Some 35 evidence has also suggested a pathological increase in adrenergic drive is correlated with an increase in oxidative stress. Oxidative Stress in Heart Failure Evidence for the role of oxidative stress in the pathophysiology of heart failure has been steadily increasing over the past twenty years.120 The pathological effects of oxidative stress are reactive oxygen and nitrogen species (ROS/RNS) dependent damage to proteins, DNA, lipids, and alterations of intracellular signaling pathways. Reactive oxygen species (ROS) have high reactivity and include both free radicals and non-radical species. Free radicals have at least one unpaired electron like superoxide and hydroxyl (OH·). Non-free radical ROS such has hydrogen peroxide (H2O2) are species that are capable of generating free radicals without containing unpaired electrons themselves. ROS occur in natural metabolic pathways but become pathological when they overwhelm the innate antioxidant systems that scavenge and degrade ROS. A series of superoxide dismutase enzymes convert O2·- to H2O2, which is broken down by glutathione peroxidase and catalase to H2O. Nonenzymatic antioxidants such as vitamins E, C, beta-carotene, ubiquinone, lipotic acid and urate also remove ROS.120, 121 The balance between ROS and their breakdown defines the redox state of the cell and an imbalance of excess ROS indicates oxidative stress. 36 Sources of Oxidative Stress NADPH oxidase. NADPH oxidases are cytosolic enzymes that generate superoxide when they transfer electrons from NADPH across cellular membranes. Five isoforms of this enzyme exist, Nox2 and Nox 4 being the primary isoforms expressed in the heart. NADHPH oxidase Nox 2 and Nox 4 activity is increased in heart failure 122, 123 Xanthine oxidase. Xanthine oxidoreductase (XOR), when transformed from its parent enzyme xanthine dehydrogenase (XDH) into its oxidase form, xanthine oxidase (XO), generates superoxide and hydrogen peroxide upon conversion of xanthine to hypoxanthine and hypoxanthine to uric acid.124, 125 XOR exists as XDH and XO, both of which metabolize purines to form uric acid.124 XOR is a molybdopterin-containing flavoprotein and is comprised of two identical subunits.126 Mitochondria. The mitochondria produce ROS as a byproduct of normal metabolic processes.127 During oxidative phosphorylation, Complex IV transfers electrons to oxygen and water is produced. However, a portion of oxygen in the mitochondria is only partially reduced yielding superoxide and contributing to ROS. Also, electrons are often leaked from the electron transport chain at Complexes I, III, and IV and combine with other species to from ROS. ROS generated from mitochondria can modify or damage proteins, lipids, and DNA. Superoxide and hydrogen peroxide can negatively impact cytoskeletal structure and function, as well as multiple metabolic processes in the cardiomyocyte. 37 128, 129 In addition to cardiomyocyte myofilaments, one of the key targets for the actions of ROS/RNS in the cell is the mitochondrion itself. 130, 131 The study of mitochondrial ROS contribution to pathology is a fairly new field, but several specific inhibitors have already been studied. One of the most studied mitochondrially targeted antioxidant is 10-(6’ubiquinonyl)-decyltriphenylphosphonium (MitoQ).132-135 MitoQ is a derivative of ubiquinol, which is conjugated to a lipophilic triphenylphosphonium (TPP). TTP is a cation that is attracted to the hyperpolarized membrane potential of the mitochondria resulting in a 1000 fold accumulation in the mitochondrial matrix. 136, 137 In its quinol form, MitoQ is absorbed to the inner mitochondrial membrane and acts an antioxidant by accepting free electrons and becoming oxidized to its quinone form. Importantly, it is continually recycled to the active quinol antioxidant form by Complex II.138-140 Medical Therapies for Heart Failure Heart failure is characterized by insufficient cardiac output, elevated chamber filling pressures, or both. These conditions lead to labored breathing and edema due to pulmonary congestion. The general goal of medical intervention in heart failure is to restore CO to normal levels while maintaining normal diastolic pressures and attenuating or reversing structural changes.32, 114 The specific drugs depend on the underlying cause of dysfunction. 38 Diuretics Congestion due to sodium and fluid retention, which leads to an increase in blood volume and elevated end-diastolic pressure, is the driving force behind many of the overt symptoms of heart failure.32 Diuretics are useful for inhibiting sodium reabsorption in the early distal tubules of the kidney and sodium and chloride reabsorption in the loop of Henle.2 Spironolactone is a diuretic agent with a more favorable effect on overall potassium balance. It inhibits the actions of aldosterone instead of directly altering ion exchange, which the previously mentioned drugs do at the potential expense of potassium.114 Vasodilators As described in earlier sections, reduced cardiac output activates both the sympathetic nervous system and RAAS and results in intense vasoconstriction,2, vasodilating drugs such as nitrates, hydralazine, minoxidil, or nitroprusside31 43 31 so are used as treatment. Some vasodilators, such as the nitrates, induce vasodilation by acting as nitric oxide donors; other agents, like hydralazine, inhibit smooth muscle contraction by blocking calcium entry into these cells.31 Calcium Channel Blockers Calcium channel blockers also have a vasodilatory effect by inhibiting the flow of calcium and preventing activation of myosin and thus reducing contraction.2 Some calcium channel blockers have the added effect of slowing the activity of pacing cells in 39 the heart that further adds to the negative ionotropic effect. Reducing ionotropy reduces the workload placed on the heart and allows for more efficient functioning.73 β-blockers β-receptor blockade is one of the oldest and most successful treatment strategies for heart failure. Propranolol was introduced in the mid 1960’s, and it was rapidly proven useful for treating angina, myocardial infarction, arrhythmias, and hypertension.69 Since that time, a large family of drugs has been developed with varying receptor specificities that reduce the increases in wall stress, and metabolic alterations that are known to accompany cardiac hypertrophy and contribute to heart failure.141 A key question that has surrounded the use of β-blockers has been whether there are any differences in clinical benefit between selectively blocking only the β1 receptor, versus nonselective β1 and β2 blockade. 114 This question is complicated by the fact that many β-blockers have additional beneficial properties beyond direct inhibition of cardiomyocyte adrenergic signaling, including peripheral vasodilation, antioxidant activity, and reduction of RAAS activation.52 However, the selectivity of a particular drug appears to influence tolerability in patients, often due to effects on blood pressure.141 40 The Volume Overload of Mitral Regurgitation Mitral Regurgitation Efficient cardiac function is reliant on functioning heart valves to maintain forward blood flow, so any defect in any component of a heart valve can impair the performance by allowing reverse blood flow, or regurgitation (Figure 10). Current estimates predict 2.5% of US citizens have some form of valve disease, with 60% of those people having defective mitral valves.1 The mitral valve sits between the left atrium and left ventricle, and ensures blood is ejected from the left ventricle during systole instead of being pumped back into the left atrium. Mitral regurgitation results when the mitral valve does not function correctly. This dysfunction can be due to structural defects, fibrosis in the valve leaflets, improper length or rupture of the chordae tendae or papillary muscles, which control the valve, or calcification or shape distortion of the mitral annulus, upon which the valve is seated.2 Figure 10: Mitral regurgitation. Note: Adapted from Sevier Medical Art with permission. 41 Mitral regurgitation (MR) can result from acute damage or can develop chronically over the course of years or decades.2 Coronary artery disease or myocardial infarction can cause MR secondarily by limiting perfusion of papillary muscles, or causing scarring which distorts the shape of the valve. Conversely, calcification, fibrosis, or degeneration can all contribute to a very slowly developing mitral regurgitation.52 Specifically, calcification of the mitral annulus can be associated with hypertension, diabetes, kidney disease, or Marfan’s syndrome. In myxomatous degeneration, fibrous components of the valve leaflets are broken down and a shift from stiff type I collagen to flexible type III collagen renders them “floppy”, resulting in prolapse and incomplete valve closure during systole. Patients with chronic forms of MR can often be asymptomatic for many years, as the structural issues at the core of the condition develop slowly.2 Volume Overload MR causes significant alterations in hemodynamics resulting in a low-pressure volume overload.52 The backward blood flow from the left ventricle to the left atrium during systole increases the blood volume in the left atrium during systole and in the left ventricle during diastole. This increased blood volume in the left ventricle causes an increase in stretch in the left ventricle during diastole, which causes an enhanced contraction, due to the Frank-Starling mechanism. This adaptation can compensate for the fraction of forward flow lost to regurgitation, but only temporarily. In the long term, the fraction of blood regurgitated into the left atrium gradually increases, resulting in a drop in forward cardiac output and the development of heart failure symptoms.2 42 Eccentric Remodeling In order to accommodate the increased diastolic volume associated with volume overload, the left ventricle remodels in an eccentric fashion. Eccentric cardiac hypertrophy is so named in reference to the eccentricity index, which correlates the short and long axes of the heart, indicating a more spherical shape.117 The left ventricle wall expands and becomes more compliant and the ratio of chamber diameter to wall thickness increases.2 Further, the ratio of LV mass to volume decreases.52 These changes are generally associated with the earlier, more adaptive phase of remodeling; as the heart begins to fail, chamber stiffness increases.117 Aorto-Caval Fistula Model of Volume Overload Many of the hemodynamic and structural changes associated with chronic volume overload in humans can be recapitulated in rodents with the aorto-caval fistula (ACF) model.92 In this model, a fistula is created between the abdominal aorta and inferior vena cava by a needle puncture between the two vessels (Figure 11). The fistula is maintained by the application of cyanoacrylate adhesive, and can be visually verified be the observation of red arterial blood mixing with dark venous blood. The ACF model demonstrates eccentric remodeling, neurohormonal activation, and changes in cardiac function highly analogous to those found in patients with volume overload.92 43 Figure 11: Creation of an aortocaval fistula in a rat. Note: From “Aortocaval Fistula in Rat: A Unique Model of Volume-Overload Congestive Heart Failure and Cardiac Hypertrophy” by Z. Abassi et al., 2011, J Biomed Biotechnol 2011:729497. Copyright 2011 Zaid Abassi. Reprinted with permission. Strategies for Management of Mitral Regurgitation The treatments discussed previously have seen little to no success in MR patients. Findings from our laboratory demonstrate, in a dog MR model, that despite high levels of RAAS activation, ACE inhibition fails to attenuate LV remodeling1, 94 leading to the conclusion that the RAAS is not a viable target for therapeutic intervention in MR. Currently, clinicians are forced to rely on surgical treatment for MR, yet the timing of corrective surgery is still under debate.1 Recent studies from our laboratory point out an early onset of cytoskeletal damage in both animal models and human patients suffering from MR. 44 The studies in animal models of VO have identified extensive loss of extracellular matrix in the pathophysiology of this adverse eccentric remodeling.92, 142-144 Recently, we have demonstrated increased cardiomyocyte oxidative stress in patients with VO of isolated chronic mitral regurgitation (MR)145 and in rats with VO of aortocaval fistula (ACF).143, 146 Specifically, there is extensive myofibrillar degeneration and mitochondrial dysfunction and damage with LV dilatation, despite a preserved LV ejection fraction.145 The preservation of LV shortening has been shown to belie underlying cardiomyocyte dysfunction due to ejection into the low pressure left atrium52 145 in MR and into the arteriovenous fistula in ACF.146 Novel drug targets that attenuate oxidative stress and maintain normal cardiomyocyte morphology is an unmet need in arresting the progressive LV dilatation in a pure VO. In both chronic145 and acute VO,146 there is evidence of increased cardiomyocyte xanthine oxidase (XO) expression and activity. We have demonstrated that cyclical stretch of adult rat cardiomyocytes results in increased XO activity, mitochondrial swelling and disorganization, and myofibrillar degeneration that is similar to the changes identified in the human LV with isolated MR145 and is prevented by XO inhibitor allopurinol in the rat with ACF.146 However, recent studies evaluating the effects of chronic XO inhibition with 8 weeks of VO of ACF in the rat do not attenuate LV dilatation and LV wall thinning despite an improvement in LV contractile function.147 In our previous in vitro studies of stretched cardiomyocytes, the mitochondrial targeted antioxidant mitoubiquinone (MitoQ) prevented myofibrillar degeneration, mitochondrial damage, and the increase in XO activity.146 In testing the effectiveness of MitoQ in blocking the effects of VO in vivo, we find that MitoQ attenuates mitochondrial reactive 45 oxygen species (ROS) production, prevents the depolarization of the mitochondrial transmembrane, protects mitochondrial ultrastructure, and attenuates the extensive breakdown of desmin and tubulin in the ACF animals. In the sham animals, MitoQ results in what appears to be mitochondrial proliferation, not observed in the ACF MitoQ treatment, with an assocated impact on the cytoskeletal ultrastructure but with no effect on desmin breakdown. Summary and Hypothesis The studies described in this work utilize the ACF model in rats to generate a state of cardiac volume overload studied at 8 weeks post-induction. These studies were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. Previous work from our laboratory demonstrated ACF was associated with left ventricle remodeling and dysfunction as well as increased oxidative stress. Studies inhibiting xanthine oxidase showed some success, but were unable to inhibit cardiac remodeling. Thus, we decided to test the hypothesis that MitoQ would be an effective agent in the prevention of the progression to heart failure by quenching mitochondrial ROS production. Preliminary studies in isolated cardiomyocytes exposed to mechanical stress indicated a decrease in reactive oxygen species and maintenance of a healthy mitochondrial membrane potential, which led to the overall hypothesis of this work: Volume overload results in an increase in ROS derived from the mitochondria that lead to cytoskeletal breakdown and overall cardiac remodeling and dilatation and can be 46 inhibited by treatment with MitoQ. This hypothesis was critically evaluated with a combined in vitro and in vivo approach. To establish a cause and effect role between myocardial stretch in VO and mitochondrial ROS production, we mechanically stretched isolated cardiomyocytes in vitro and measured mitochondrially produced ROS using fluorescent dyes and microscopy. These studies were confirmed by transmission electron microscopy of stretched cardiomyocytes with and without MitoQ to determine the ultrastructural changes undergone by the cardimyocyte under this stress. The data from these studies led us to examine the effects of MitoQ on the cytoskeletal disruption, increased ROS production, and progressive decline in cardiac function and transition to heart failure associated with volume overload in vivo. Biweekly serial echocardiography characterized the temporal response of the left ventricle to VO throughout the eight-week study. High fidelity pressure catheritization was used to assess hemodynamic parameters, and fluorescence microscopy and biochemical analyses of left ventricle tissue were used to investigate the effects of MitoQ on the cytoskeletal disruption, specifically the intermediate filament desmin. 47 CHAPTER 2 Methods and Materials Animal Preparation Adult male Sprague-Dawley rats (200-250g) at 12 weeks of age were subjected to either sham or ACF with and without mitochondrial antioxidant MitoQ treatment (5 mg/kg) for 8 weeks. ACF was induced by opening the abdominal cavity of the rat, moving the abdominal viscera, and exposing the abdominal aorta and inferior vena cava. Once the abdominal aorta and inferior vena cava were exposed, an 18-gauge needle was inserted into the abdominal aorta, piercing the wall of the aorta. The needle was then inserted through the medial wall of the inferior vena cava, creating a fistula between the two blood vessels. A patch of glue was placed over the fistula to keep the fistula in tact and to keep the fistula from opening any further. Sham rats were treated in the same way, with the exception of creating the fistula. The abdominal cavity of the rats was opened, visceral organs shifted, and abdominal aorta and inferior vena cava exposed. MitoQ treatment was initiated during the week of surgery and delivered in the drinking water (5 mg per kg per day) in dark bottles. Separate sets of sham and ACF rats were sacrificed 8 weeks after surgery for studies of isolated cardiomyocytes for live cell imaging (N=6 per group). Another set of sham and ACF rats were studied for in vivo hemodynamic and echocardiographic measurements prior to sacrifice and this tissue was used for protein analysis and immunohistochemistry (N=5 per group). The animal use in these studies was approved by the University of Alabama at Birmingham Animal Resource Program (Protocol 130409070). 48 Hemodynamics and Echocardiography Echocardiography and hemodynamics were performed prior to sacrifice using the Visualsonics imaging system (Vivo 770, Toronto, Canada) combined with simultaneous high-fidelity LV pressure catheter recordings (Millar Inst. Houston, TX). With the rats under isoflurane anesthesia, a high fidelity LV pressure catheter was advanced into the LV cavity via a right carotid artery cut-down. LV pressure and echocardiography dimensions (wall thickness and chamber diameter) were obtained simultaneously using software included in the Visualsonics system. LV volume was calculated from traced mmode LV dimensions using the Teicholz formula: V = [7/(2.4 + LVID)]·[LVID]3, where V = volume, LVID = LV internal dimension. LV wall stress was calculated from traced m-mode LV dimensions and simultaneous LV pressure data using the equation described below: LV σ = [LVP · r]/[2·LVwt], where LV σ = LV wall stress, LVP = LV pressure, r = LV chamber radius, LVwt = LV wall thickness. These LV pressure-volume data were analyzed for LV PVA and stroke work using the Labscribe2 (iWorx System Dover, NH) software package. Isolation of Cardiomyocytes Cardiomyocytes were isolated from sham and ACF rats by removing the hearts from the rats under isofluorane anesthesia. Hearts were removed by making an incision into the abdomen of the rats to ensure the ACF was still intact. An intact ACF will demonstrate a visible mixing of arterial and venous flow at the level of the fistula. Once ACF or sham was verified, the heart was exposed by opening the chest cavity by incising 49 both sides of the rib cage, grasping the zyphoid process, and folding the sternum anteriorly. Once the heart was exposed, the aorta was clamped to restrict blood flow, the heart was lifted superiorly, and an incision was made under the heart to excise it. The aorta was then cut, leaving approximately 3 cm of aorta attached to the heart. Once the heart was excised, it was placed in a dish of perfusion buffer (120 mmol/L NaCl, 15 mmol/L KCl, 0.5 mmol/L KH2PO4, 5 mmol/L NaHCO3, 10 mmol/L HEPES, and 5 mmol/L glucose, at pH 7.0), a cannula was inserted into the aorta and down into the left atrium of the heart. The cannula was then attached to a pump that pumped perfusion buffer warmed to 37°C through the heart in a reverse Langendorff method. This type of perfusion is a non-working heart perfusion. The hearts were perfused with perfusion buffer for 5 min and then perfused with digestion buffer (perfusion buffer containing 2% collagenase II (Invitrogen, Carlsbad, CA)) for 30 min at 37°C. The heart was then removed from the perfusion apparatus and the right ventricle, atria and apex were removed before the perfused-heart was minced. Mincing involved cutting into the perfused tissue with surgical scissors to release the cardiomyocytes. Cellular material was collected and passed through a cell filter, and allowed to settle. Several passes of this procedure were performed to collect cardiomyocytes. Myocytes have the most mass of the cell types of the heart, so the pellet that accumulates after approximately five minutes contains predominantly myocytes. These cells were counted and then used for a variety of procedures depending on the study. Only samples with viability (rod-shaped) > 80% were used. 50 Application of Stretch to Isolated Adult Rat Cardiomyocytes Isolated cardiomyocytes were plated (50,000/well) and allowed to adhere to laminin-coated Flexcell plates (Flexcell International Corp., Hillsborough, NC, USA) in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 nM glutamine, 10 U/mL penicillin, and 100 mg/mL streptomycin for 2 hours before use. During this time, the cells were left undisturbed in a 37°C incubator. After the 2 hour plating time, cells were subjected to cyclical strain (60 cycles/ min) for 3 hours on the Flexcell Strain apparatus (Model FX-4000; Flexcell International Corp.) at a level of distension sufficient to promote an increment of approximately 20% in surface area at the point of maximal distension on the culture surface.148-151 Flexcell plates are cell culture plates with a vinyl membrane surface for plating. Cells are adhered to this membrane, and then exposed to the Flexcell appartus which applies a negative pressure below the membrane surface. The cells are maintained at 37°C for the duration of the stretch experiment. This negative pressure pulls the membrane down, and then the negative pressure is released so the membrane returns to its starting position. The level of stretch is pre-determined. For this study, we chose 20% stretch, which indicates a 20% distension in the surface area of the membrane. This process is repeated at a frequency of 1 Hz, or 1 stretch per second. We chose this level of stretch as it most closely resembles the strain cardiomyocytes experience in a volume-overloaded heart. We also exposed a group of cells to this level of stretch following pre-treatment with MitoQ. 30 minutes before stretching the cells, we added media containing 50 nM MitoQ. We then subjected these cells to stretch in the same manner discribed above. Control cells were prepared on 51 indentical culture plates following the procedure listed above, but were not exposed to stretch. Live Cell Imaging The cationic potentiometric fluorescent dye tetramethyl rhodamine methyl (TMRM) ester (50 nM) was used to monitor changes in mitochondrial membrane potential. This dye fluoresces red. A bright red signal indicates a hyperpolarized mitochondrial membrane. A more faint red signal indicates a depolarized mitochondrial membrane. ROS production was monitored with either MitoSox red (10 µmol/L), a mitochondria-specific superoxide fluorescent indicator, or CM-DCF (5 µmol/L), an H2O2-sensitive fluorescent indicator.151 These dyes also fluoresce red. A bright red signal in this case indicates a high level of ROS production, while a fainter signal indicates a lower level of ROS production. For in vitro studies, plates containing fluorescent dyeloaded cells (either MitoSox red or TMRM) were equilibrated at 37°C for 10 minutes with unrestricted access to atmospheric oxygen in a cell incubator. MitoSox and TMRM images were recorded using a Leica DM6000 epifluorescence microscope (100X objective, 4000X video-screen magnification) with Simple PCI software (Compix, Cranberry Township, PA, USA). For in vivo studies, the dish containing fluorescent dyeloaded cardiomyocytes was equilibrated at 37°C with unrestricted access to atmospheric oxygen on the stage of an Olympus microscope. CM-DCF and TMRM images were recorded using an Olympus FV1000 confocal laser-scanning microscope with excitation at 488nm and 543nm, respectively. Images were analyzed offline using Simple PCI software or ImageJ software (Wayne Rasband, National Institutes of Health), respectively. 52 Tissue Preparation Hearts from in vivo functional studies were used for histology purposes. Hearts were removed, as described above, and soaked in saline for approximately five minutes. During this time, the heart was gently massaged to remove any remaining blood. The apex, right and left atria, and right ventricle were then removed, leaving the left ventricle. The left ventricle was then sliced coronally along the long axis of the heart. This slice results in a ring of tissue representative of the endocardium, myocardium, and epicardium of both the septal and left ventricle free wall. These slices were then prepared for specific histological techniques. Immunohistochemistry Left ventricle slices were immersion-fixed in 10% neutral-buffered formalin for two hours and then soaked in ethanol overnight. The slices were then embedded in paraffin. Sections (5 µm) were then mounted on slides, deparaffinized in xylene, and rehydrated in a gradient series of ethanol. After being blocked with 5% goat serum in 1% bovine serum in phosphate buffered saline, they were incubated overnight at 4°C with either desmin antibody (1:100; Abcam, rabbit, Cambridge, UK), mitochondrial complex IV subunit IV (1:200; Abcam, mouse, Cambridge, UK), β2 tubulin (1:500; Abcam, rabbit, Cambridge, UK). Alexa Fluor-conjugated secondary antibodies (1:500 each; Molecular Probes, Eugene, OR, USA) were applied to visualize desmin (green), β2 tubulin (red), and complex IV (red) in the tissue. Nuclei were stained (blue) with DAPI (1.5 µg/mL; Vector Laboratories, Burlingame, CA, USA). Image acquisition (100X objective, 4000X video-screen magnification) was performed on a Leica DM6000 53 epifluorescence microscope with SimplePCI software (Compix, Cranberry Township, PA, USA). Images were adjusted appropriately to remove background fluorescence. Transmission Electron Microscopy of Rat Tissue Left ventricle slices, as prepared above, were cut into 5 mm cubes for transmission electron microscopy. Tissue was fixed overnight in 25% glutaraldehyde and mounted for transmission electron microscopy (TEM), and analyzed by EmLabs Inc. (Birmingham, AL, USA). The glutaraldehyde fixative reacts with amines in the tissue, especially lysine and arginine, to generate cross-linkages. This cross-linking stabilizes the cytosol, which preserves the cellular structures. Fifteen images per sample were acquired and analyzed for cellular morphology and structural changes. Western Blot Left ventricle tissue slices, as prepared above, were cut into pieces of approximately 100 mg and snap frozen in liquid nitrogen. 100-200 mg of tissue was homogenized using a tissue homogenizer (Fisher PowerGen Model 500, Pittsburgh, PA) in two volumes of RIPA (Radio-immunopreciptation assay) buffer. Tissue was homogenized to the point of solid-liquid dispersion. The homogenate was then centrifuged at 3000 RPM for 10 minutes and the supernatant was retained for protein content analysis via the standard Bradford colormetric protein assay. LV tissue lysates (30 µg protein) were prepared for western blot analysis per the Invitrogen western blotting kit protocol. The samples were prepared in a solution of LDS sample buffer, reducing agent, and water to produce a sample containing 30 µg of protein. The samples were separated by electrophoresis (180 V) on a 4-12% Bis-Tris gradient gel (Invitrogen), 54 transferred (30 V) to a polyvinylidene difluoride membrane, blocked with 5% non-fat milk, then incubated with an antibody to desmin (1:1000; Abcam, rabbit, Cambridge, UK) overnight at 4°C. Desmin antibody solution was made in blocking solution. Following overnight desmin incubation, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for one hour. Then membranes were developed by incubating them with chemiluminescent substrate (Pierce, Rockford, IL) and then imaged using a CCD camera. Membranes were incubated with three different desmin antibodies in order to optimize the western blot conditions. Abcam antibodies raised against mouse and rabbit as well as a Sigma antibody raised against mouse were used. The most effective antibody was the Abcam rabbit antibody and the results reported here were obtained using the rabbit antibody. Cardiomyocyte Bioenergetics To determine the effects of MitoQ on cardiomyocyte bioenergetics, the Seahorse Bioscience XF24 extracellular flux analyzer was used to measure the O2 consumption of adult cardiomyocytes in culture.152 The O2 consumption rate (OCR) was related to mitochondrial function using the mitochondrial stress test. Isolated primary adult rat cardiomyocytes were attached to specialized V28 plates (Seahorse Bioscience) coated with laminin at 7,500 cells/well.152 Cells were then allowed to attach for 2 hours, after which the culture media was changed to unbuffered Dulbecco’s minimal essential medium supplemented with 1% FBS, 4 mM L-glutamine, and 5 mM glucose for the XF24 assays. The basal O2 consumption of the cardiomyocytes was determined by measuring the OCR of the cells over time without any treatment. Next, FCCP (1 µM) was injected to uncouple mitochondrial respiration and stimulate the maximal OCR of the 55 cells. Finally, antimycin A (10 µM) and rotenone (1 µM) were injected simultaneously to completely inhibit the mitochondrial electron transport chain, thus yielding the nonmitochondrial OCR of the cardiomyocytes. The OCR of each well was normalized to the total protein in that well, as measured by the DC protein assay (BioRad, Hercules, CA). Statistical Analysis Data are expressed as mean ± SEM. A two-way ANOVA with Student-NewmanKeuls post hoc test was used for all comparisons among sham, ACF, sham MQ, and ACF MQ except those data expressed in Table 2. For the data presented in Table 2, all hypotheses tests were performed with the type III sums of squares from the SAS GLM (general linear model) procedure (SAS, Cary, N.C.) for all comparisons among sham, ACF, sham MQ and ACF MQ. This statistical method was chosen due to the variability between animal parameters in the study. All statistical analyses were two-sided and p < 0.05 was considered statistically significant. Adjustments for multiple comparisons were not made because there were no repeated comparisons. 56 CHAPTER 3 Results MitoQ Improves Cardiomyocyte Oxidative Stress and Mitochondrial Membrane Potential In Vitro, But Has No Effect on LV Remodeling and Function In Vivo Cyclical stretch of isolated cardiomyocytes from normal rats caused a significant increase in oxidative stress measured by MitoSox Red, which was significantly decreased by pre-treatment with MitoQ (Figure 12). 57 Figure 12. Mechanical stretch of adult rat cardiomyocytes demonstrates increased MitoSox red staining that is significantly decreased by treatment with MitoQ (MQ). Left. Live cell MitoSox red staining of stretched and control non-stretched cardiomyocytes. Right. Intensity quantification of live cell imaging. (n= 50 cells per group) *P< 0.05 vs sham. 58 Mitochondrial membrane potential of isolated cardiomyocytes from normal rats was also measured using live cell imaging with the dye TMRM (Figure 13). A healthy mitochondrial membrane is hyperpolarized, as evidenced by the bright red signal of the unstretched control cells. Three hours of cyclical stretch caused a significant depolarization of the mitochondrial membrane that was significantly improved by pretreatment with MitoQ. 59 Figure 13. Mechanical stretch of adult rat cardiomyocytes shows decreased TMRM staining, indicating a depolarized mitochondrial membrane and mitochondrial damage that is significantly improved by treatment with MitoQ (MQ). Left. Live cell TMRM staining of stretched and control non-stretched cardiomyocytes. Right. Intensity quantification of live cell imaging. (n= 50 cells per group) *P<0.05 vs sham. ^P<0.05 vs ACF. 60 MitoSox red and TMRM fluorescent staining in stretched rat cardiomyocytes treated with decyl-triphenylphosphonium (dTPP) which contains the linker group but not the pharmacologically active redox component of MitoQ. High concentrations of MitoQ or dTPP can disrupt mitochondrial function in cell culture. Neither dTPP or MitoQ (50nM), at the concentration used in this study (50nM), had any effect on cellular bioenergetics (Figure 14). The dTPP did not prevent the stretch-dependent loss of mitochondrial membrane potential or increased MitoSOX staining indicating that the redox active component of MitoQ was essential for its protective effects (Figure 15). 61 Figure 14. Neither MitoQ nor the triphenylphosponium (dTPP) control changed basal or mitochondrial function in control rat cardiomyocytes. n=6-7 per group. 62 Figure 15. MitoSox red and TMRM fluorescent staining in stretched rat cardiomyocytes treated with decyl-triphenylphosponium (dTPP) control compound were not significantly different from control untreated stretched cells indicating no effect of dTPP on the assay. 63 The beneficial effects of MitoQ on cardiomyocyte stretch led us to test its effects in chronic VO. Using the same live cell imaging, cardiomyocytes isolated from 8 week animals with or without MitoQ treatment were examined. Sham hearts demonstrated a punctate CM-DCF signal pattern (Figure 16). In contrast, ACF resulted in both an increased CM-DCF signal with a diffuse pattern. Interestingly, MitoQ had no effect on the basal mitochondrial punctate CM-DCF signal in the sham animals treated with MitoQ and the diffuse pattern in response to the ACF was prevented by treatment with MitoQ. Next, the TMRM signal was assessed and in the sham rats the characteristic pattern of cardiac mitochondrial networks is evident and unaffected by MitoQ. As we found in the stretch studies, ACF results in a mitochondrial depolarization which is preserved by MitoQ (Figure 16). 64 Figure 16. Analysis of ROS by CM-DCF in cardiomyocytes isolated from 8 week ACF rat hearts indicates a significant increase in ROS in ACF rats that is significantly attenuated by treatment with MitoQ (top panel). Mitochondrial membrane potential, as measured by TMRM, is significantly depolarized in ACF animals and significantly improved by treatment with MitoQ (bottom panel). (n= 50 cells per group) *P<0.05 vs sham. 65 However, these beneficial effects on ACF cardiomyocytes in vitro did not translate into improved LV remodeling or function after 8 weeks of VO in vivo. Heart, LV, RV and lung weights, as well as the heart weight/body weight ratios were increased in ACF compared to sham rats and did not improve in ACF rats with MitoQ treatment (Table 1). Mean arterial pressure (MAP) did not differ between ACF groups (Table 2). LV end-diastolic dimension (LVEDD), LV end-diastolic volume (LVEDV), and LV enddiastolic wall stress were increased as LV end-systolic pressure volume relationship (LVESPVR) was decreased in both ACF and ACF MQ groups vs. sham (Table 2). We previously showed extensive cardiomyocyte ultrastructural breakdown with cell stretch that was improved by MitoQ.146 Thus, we next examined the cardiomyocyte cytoskeleton in chronic VO. 66 Table 1. Morphometric Data on 8 Weeks of ACF Sham ACF Sham+ MitoQ ACF+ MitoQ Body weight (g) 475 ± 16 517 ± 11 470 ± 29 489 ± 17 Heart weight (g) 1.15 ± 0.04 2.11 ± 0.06* 1.20 ± 0.04 2.33 ± 0.14* LV weight (g) 0.80 ± 0.02 1.37 ± 0.03* 0.81 ± 0.04 1.37 ± 0.09* RV weight (g) 0.23 ± 0.01 0.46 ± 0.03* 0.22 ± 0.01 0.43 ± 0.04* Lung weight (g) 1.6 ± 0.06 2.2 ± 0.06* 1.6 ± 0.09 2.2 ± 0.17* Tibia Length (mm) 49 ± 0.6 50 ± 0.3 49 ± 0.4 50 ± 0.5 Heart/TL (%) 22 ± 0.4 42 ± 1* 23 ± 1 41 ± 3* Heart/BW(%) 2.4 ± 0.07 4.09 ± 0.09* 2.47 ± 0.07 4.26 ± 0.20* 7 7 8 8 n *p<0.05 vs. Sham 67 Table 2. LV Hemodynamic and Functional Parameters in Chronic 8 Week ACF Sham ACF Sham+ MitoQ ACF+ MitoQ Heart Rate 343 ± 19 338 ± 12 312 ± 11 330 ± 4 MAP 106 ± 6 92 ± 2 100 ± 4 95 ± 2 +LVdP/dtmax 7437 ± 327 7287 ± 248 6900 ± 282 7458 ± 339 -LVdP/dtmax -7901 ±405 -6654 ± 295* -6880 ± 394* -6494 ± 238 LVESP 79 ± 5 75 ± 2 80 ± 4 75 ± 2 LVEDP 6±2 11 ± 1* 6±1 10 ± 1* LVES σ 76 ± 5 102 ± 14* 89 ± 11 104 ± 8* ESPVR 0.36± 0.05 0.18± 0.02 0.35±0.07 0.29± 0.08 LVEDD (mm) LVESD (mm) LV EF (%) LV FS (%) VCFr (%) 8.19 ± 0.18 5.61 ± 0.16 57 ± 3 33 ± 1 6.6 ± 0.4 11.33 ± 0.34* 7.69 ± 0.53* 57 ± 2 34 ± 3 6.2 ± 0.8 8.41 ± 0.21 5.74 ± 0.28 54 ± 3 34 ± 2 6.7 ± 0.4 11.09 ± 0.34* 7.82 ± 0.30* 59 ± 2 32 ± 1 5.7 ± 0.2 7 7 8 8 n LVESP=LV end systolic pressure, LVEDP=LV end diastolic pressure, LVES σ=LV End systolic stress, LVED σ= LV end diastolic stress, ESPVR= end systolic pressure volume relationship, LVEDD=LV end diastolic dimension, LVESD=LV end systolic dimension, LV EF=LV ejection fraction, LV FS%= LV Fractional Shortening, VCFr= Velocity of circumfrential shortening *p<0.05 vs. Sham 68 ACF Causes Mitochondrial Changes and Breakdown of Cytoskeleton Normal mitochondrial distribution, as demonstrated by TEM in sham rats (Figure 17), demonstrates the typical orderly linear array of one mitochondrion per sarcomere. These mitochondria are electron dense with tightly packed cristae. The VO of ACF caused a complete disruption of this highly organized structure of interfibrillar mitochondria such that mitochondria lost their linear register and were no longer in close proximity to the sarcomere with large spaces between the mitochondria and individual sarcomeric units. In addition, mitochondria became crowded with several small, round mitochondria per sarcomere compared to sham hearts. These mitochondria demonstrated a loss of electron density as well as a disruption and loss of cristae. 69 Figure 17. TEM of LV myocardium in sham and ACF hearts demonstrates pathological changes in mitochondrial morphology in ACF hearts compared to sham. ACF mitochondria exhibit a loss of linear registry, clustering, disassociation with sarcomeres, and decreased electron density in ACF. ACF myocardium also shows a breakdown in myofibrils, a decrease in Z line electron density, as well as large gaps between sarcomeric units (see ACF inset). 70 Evidence of mitochondrial structural disruption was also evident in mitochondrial complex IV (subunit IV) staining by immunohistochemistry (Figure 18). Complex IV staining indicates a marked decrease in mitochondrial staining in ACF, which in conjunction with the TEM data in Figure 17, indicates a loss of mitochondrial connectivity with the cellular structure. The mitochondrial disorganization in ACF was directly paralleled by disruption and breakdown of the cytoskeletal intermediate filament desmin as demonstrated in Figure 18. Desmin is the major intermediate filament in the heart providing a structural framework that extends from the subsarcolemma to the nuclear membrane. Desmin is an important structural protein that is essential in the mitochondrial-sarcomere connection, especially along the Z-lines between adjacent sarcomeres. In sham rats, the desmin is clearly seen along the Z-lines of the cardiomyocytes in a highly regular linear array, which is extensively disrupted in ACF rats, but to a variable extent throughout (Figure 18). 71 Figure 18. IHC analysis of desmin (green) and mitochondrial complex 4 (COX4) (red) in sham and ACF rats demonstrates that the normal desmin distribution along Z lines is completely disrupted along with a loss and disruption of COX4 staining indicates mitochondrial distribution in ACF. DAPI (blue) indicates nuclei. Left: Desmin and DAPI. Middle: COX4 and DAPI. Right: Merge. 72 The representative desmin Western blot (Figure 19) for the immunohistochemistry demonstrates a pattern of desmin loss and breakdown manifested by a single whole-length desmin band in sham rats which is decreased in ACF rats along with the appearance of a lower molecular weight band, which has previously been reported as a product of desmin breakdown in ACF.153 Preservation of the whole-length desmin and substantially lower levels of the breakdown product in ACF MQ rats is consistent with a protective effect of MitoQ. 73 Figure 19. Western blot analysis of desmin indicates a significant decrease in desmin whole length protein in ACF, as well as the appearance of a lower molecular weight breakdown product, which corresponds to the IHC in Figure 5. The decrease in desmin protein in ACF is rescued by treatment with MitoQ, leading to a decrease in the breakdown product band. 74 β-2 tubulin is also disrupted in the ACF rat. β-2 tubulin is a cytoskeletal support protein involved in anchoring the mitochondria to the sarcomeres. In sham rats, β-2 tubulin is located in a linear array along the Z-lines, but does not co-localize with desmin. The loss of β-2 tubulin in ACF, however, coincides with the loss of desmin in ACF (Figure 20) and with the overall loss and disruption of mitochondria revealed by complex IV staining. Figure 20. IHC analysis of desmin (green) and β2 tubulin (red) indicates disruption of β2 tubulin in ACF. β2 tubulin is located in a linear array along the Z lines, but does not colocalize with desmin. DAPI (blue) indicates nuclei. Left: Desmin and DAPI. Middle: β2 tubulin and DAPI. Right: Merge. 75 MitoQ Improves Cardiomyocyte Mitochondrial Organization and Cytoskeleton in Chronic VO Rats MitoQ treatment had an ameliorating effect on the mitochondrial organization and structure by TEM analysis following ACF. MitoQ-treated cardiomyocytes had a more electron dense mitochondria with visibly more dense cristae and a somewhat improved linear registry of mitochondria (Figure 21) as well as restored complex IV staining (Figure 22). In spite of these improvements, the mitochondrial distribution is not completely normalized and remains less organized compared to the untreated sham. Treatment with MitoQ in ACF rats also attenuates the desmin and β-2 tubulin degradation (Figures 22 and 23, respectively) consistent with the western blot analysis (Figure 19) indicating that MitoQ treatment attenuates the desmin loss and breakdown in ACF rats. Treatment of sham rats with MitoQ caused a proliferative response with many more mitochondria in the interfibrillar spaces evident by TEM (Figure 21). Immunohistochemistry of the MitoQ-treated sham rats exhibited minor disruption of desmin and β-2 tubulin along the Z lines, and in some regions decreased complex IV staining (Figures 22 and 23). 76 Figure 21. The changes in mitochondrial morphology by TEM in the ACF rat (Figure 17) are attenuated in ACF LV myocardium with MitoQ treatment. Mitochondrial electron density is restored, but mitochondria remain clustered in groups and lack their characteristic one mitochondrion per sarcomere segment between Z lines. However, sham rats treated with MitoQ demonstrate clustering of those mitochondria between sarcomeric units but as opposed to the ACF TEM there is a preservation of the Z-line. 77 Figure 22. IHC analysis of desmin (green) and mitochondrial complex 4 (COX4) (red) in sham and ACF rats treated with MitoQ. In Sham + MitoQ rats there is a loss of the normal desmin distribution along the Z lines (see Sham Figure 5) that is paralleled by a similar disruption of COX4 staining. In ACF + MitoQ rats, the desmin and COX4 loss and disruption is improved compared to ACF rats in Figure 5. In sham MitoQ treated rats, there was a disruption of desmin and distribution of mitochondria within cardiomyocytes. Left: Desmin and DAPI. Middle: COX4 and DAPI. Right: Merge. 78 Figure 23. IHC analysis of desmin (green) and β2 tubulin (red) indicates attenuation of the disruption of β2 tubulin in ACF by treatment with MitoQ (see Figure 8). Treatment with MitoQ in sham rats caused a disruption in the distribution β2 tubulin. Left: Desmin and DAPI. Middle: β2 tubulin and DAPI. Right: Merge. 79 CHAPTER 4 Discussion Introduction When exposed to a volume stress, a set of changes occurs within the myocardium involving its shape, size, and tissue composition. Typically, the myocardium begins an eccentric remodeling pattern with increases in LV chamber diameter that outpace changes in LV wall thickness. These changes are thought to be made in an effort to normalize the stress placed on the ventricle. However, they are never able to fully compensate for the increased work load and result in a feed forward cycle of remodeling that ends with a dilated heart failure phenotype. Treatment strategies have largely been borrowed from treatments that have been successful in pressure overload. However, pressure overload and volume overload are fundamentally different pathologies, so these treatments have met with little to no success. To date, no standard medical therapy exists that stops or even slows the progression of the eccentric remodeling of volume overload. Volume overload is clearly associated with increased myocardial energy demand and therapies directed toward protection or improvements in bioenergetics are gaining interest. The goal of these types of therapies is maintaining a certain amount of cardiac work produced per unit of oxygen consumed. This relationship depends on normal production of ATP by the mitochondria 80 and factors that regulate cardiac contractility such as cardiac geometry, sensitivity to calcium and myofibrillar structural integrity. Oxidative stress is an integral part of this system due to the high sensitivity of mitochondria to ROS and also the mitochondrial production of ROS. As energy demand, and thus ATP consumption, increase due to volume overload the mitochondrial production of ROS becomes more and more important. The case for mitochondrial ROS involvement in volume overload is supported by three major concepts: As myocardial energy demand increases, ATP is consumed requiring the mitochondria to produce ATP quickly and efficiently. Due to the increased demand for energy, the mitochondria must work harder and faster, leading to an increase in electron leak from the electron transport chain and causing an increase in ROS, specifically superoxide, in the mitochondria. The progression of volume overload is thought to be due to the stretching forces placed on the myocardium due to the increased volume load. Previous studies154 and data from this work indicate an increase in mitochondrially-produced ROS following an increase in mechanical stretch. The increased stretching forces due to an increased volume load cause mitochondrial damage which causes an increase in ROS production and a decrease in endogenous ROS scavenging mechanisms. Both animal models and patients suffering from volume overload show evidence of oxidative stress suggesting a pro-oxidant pathway in this disorder.143, 145 Previous studies of pro-oxidant pathways xanthine oxidase and NADPH oxidase did not inhibit the 81 progression to heart failure caused by VO so the evidence points to the mitochondria as the source of oxidative stress in cardiomyocytes in VO. These findings are of particular importance in the case of mitochondriallyproduced ROS because mitochondria themselves are both a source and target of ROS, leading to a feed-forward cycle of ROS generation and damage, which leads to further ROS generation by the mitochondria. As a progressive disease, theories involving VO pathogenesis must consider processes that are feed-forward in nature. The work presented here examines the role of mitochondrially produced reactive oxygen species in both in vitro and in vivo models of volume overload and their effects on cardiac function. Mitochondrial ROS inhibition with MitoQ was utilized in both settings and this combination of in vitro and in vivo experiments was used to address both biochemical parameters as well as whole-body effects. Well-defined end-points were measured with advanced technological approaches to assess cardiac function and the cardiomyocyte’s bioenergetics state. The first set of experiments focused on the early events in VO and uncovered a potential role for mitochondrial ROS in the acute setting of 3 hours of mechanical stretch. These data describe the cardiomyocyte response to mechanical stretch and the beneficial effects of MitoQ treatment. Additional studies were conducted to determine the temporal response of MitoQ treatment in chronic VO using serial echocardiography through 8 weeks of ACF. Further, we utilized in vitro extracellular flux technology in combination with in vivo cardiac 82 functional analysis to examine the role of cardiac efficiency and its interaction with mitochondrial ROS in disease progression. The implications and relevance of the data presented in this dissertation will be discussed with a focus on mitochondrial ROS as a central factor in VO pathogenesis. The validity of mitochondrially produced ROS as a therapeutic target in VO will be examined along with possible future directions of these projects and their application to other pathologies. Interactions Between Mitochondria, ROS, and Cardiomyocyte Cytoskeleton in Acute Cardiomyocyte Mechanical Stretch and Chronic ACF Initially, we characterized the response of the myocardium to acute mechanical stretch utilizing isolated adult rat cardiomyocytes. Fluorescent microscopy analysis determined ROS production and mitochondrial membrane potential. Transmission electron microscopy revealed intracellular morphology. The data from these experiments demonstrated an increase in mitochondrially-produced ROS and a depolarized mitochondrial membrane present after only 3 hours of mechanical stretch. These increases in mitochondrially produced ROS were accompanied by cytoskeletal damage and large spaces between myofibrillar structure and mitochondria. MitoQ treatment attenuated ROS production and returned mitochondrial membrane potential to normal. TEM of these cells indicate normal mitochondrial morphology, as well. Taken together, these data demonstrate a novel interaction between mitochondrially produced reactive oxygen species and cytoskeletal damage in cardiomyocytes in the setting of acute 83 mechanical stretch. The damage along cardiomyocyte Z lines is of particular interest due to the function of the Z line and its connection to large structural sarcomeric proteins. The Z-line defines the lateral boundaries of the sarcomere and is responsible for force transmission between cardiomyocytes.155 Along with providing an anchor point for structural proteins, recent evidence suggests proteins that form the Z-line are involved in nuclear signaling. For example, myopalladin is highly expressed near actin anchorage points and its overexpression results in disruption of both the Z-line and sarcomere.156 The Z line is an important part of the cytoskeletal organization of the cardiomyocyte. The cytoskeleton itself is composed of three components: actin microfilaments, tubulin microtubules, and intermediate filaments.157 Desmin is the main intermediate filament protein expressed in the heart and interacts with other structural proteins at the Z-disc, extending from the subsarcolemmal membrane to the nucleus. Desmin thus forms a continuous cytoskeletal network that maintains the spatial relationship between the sarcomeric contractile apparatus and the mitochondria that provides cellular integrity, mechano-chemical signaling, and force transmission in the cardiomyocyte.158 Currently, 49 mutations have been identified in the desmin gene159 which largely alter the desmin filament assembly process and interactions with its protein partners. This results in the disorganization of the desmin network with cardiac and/or skeletal myopathies that are characterized by disruption of the Z-line and the accumulation of desmin-containing aggregates in the cells. A transgenic mouse model inducing protein aggregation of αβcrystallin, a heart-specific chaperone for desmin, has recapitulated the desmin cardiomyopathy disease phenotypes observed in humans.153 In this model, there is formation of perinuclear aggregates and progression to a dilated cardiomyopathy 84 resulting in death from heart failure in the transgenic mice by 5-7 months of age.153 In the current study, the loss of desmin by immunohistochemistry and its decrease and degradation by western blot taken together are consistent with the loss of electron density of the Z-line in the TEM of the ACF rats. The data presented here shows in a pure cardiac VO, where there is a wellestablished increase in cardiomyocyte oxidative stress, MitoQ improves cytoskeletal breakdown and mitochondrial damage, but has no effect on LV dilatation and systolic function. These studies suggest the existence of an important balance between mitochondria-derived oxidants and cytoskeletal-mitochondrial structure in the cardiomyocyte. In rats with VO, desmin pathology along with swelling and disorganization of the interfibrillar mitochondria are improved by treatment with MitoQ and suggests that mitochondrial oxidative stress and subsequent activation of intracellular proteases may be the causative factor. TNFα overexpression in transgenic mice is associated with desmin cleavage by caspase 6, resulting in sarcomere degeneration, formation of desmin protein aggregates and a dilated cardiomyopathy.150 In the current study, loss of desmin with VO is associated with decreased mitochondrial complex IV staining. In fact, some cardiomyocytes exhibit a near complete loss of desmin in conjunction with decreased mitochondrial complex IV staining. A patchy, near total loss of desmin in cardiomyocytes has also been reported in patients with idiopathic dilated cardiomyopathy.160 It is of interest that the intracellular arrangement of desmin and mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum have been shown to be present prior to the onset of muscle failure.150 The relation of desmin 85 breakdown to long-term survival and to the severity of LV dysfunction in human heart failure provides further evidence for a non-genetic cause of its pathological degradation and LV functional deterioration160. Similar to desmin, tubulin is a critical cytoskeletal protein. The most common members of the tubulin family are α and β tubulin, which dimerize to form the microtubular cytoskeletal network. There are many isoforms of β tubulin, including β1, 2, 3, 4, and 5. In the cardiomyocyte, β2 tubulin is involved in interactions between mitochondria and the cytoskeleton and participates in regulation of mitochondrial respiration. Saks and coworkers have shown that different isotypes of β tubulin have varied intracellular distribution and organization and play different roles in mitochondrial respiration.161 β2 tubulin has a regular arrangement in rows along the long axis of the cell and co-localizes with the mitochondria. In VO, we find a degradation of β2 tubulin associated with decreased mitochondrial density. As with desmin, MitoQ preserves β2 tubulin in ACF, but also causes a minor degree of β2 tubulin disruption in sham animals, possibly due to the apparent proliferation in mitochondrial number. An important finding of the current study is that the breakdown of desmin and β2 tubulin with VO is in direct contrast to their increase in either compensated or decompensated pressure overload in multiple animal species including humans.159 This result is consistent with the divergent hemodynamic loads of pressure versus volume overload. The finding that treatment with MitoQ attenuates the breakdown of cytoskeletal proteins in vitro and in vivo suggests that oxidative stress activates intracellular proteases and provides an important underpinning mechanism in the intracellular remodeling in VO. To further support this contention, we have demonstrated that increased superoxide 86 production, hydrogen peroxide formation, and xanthine oxidase activity in cardiomyocytes is associated with cardiomyocyte matrix metalloproteinase activation after 24 hours of VO of ACF in the rat.143, 146 In the data presented here, MitoQ treatment in sham rats is associated with mitochondrial clustering along with a disruption of tubulin and desmin, which does not appear to involve desmin proteolytic degradation or loss of Z-line integrity by TEM. These findings suggest a different mechanism than occurs in the ACF left ventricles where MitoQ has a beneficial effect on desmin and tubulin preservation. One possibility is that the delivery at the same dosage of MitoQ to the mitochondria is higher in the normal mitochondria but lower in the ACF animals due to the lower mitochondrial membrane potential in response to ACF.136 The resulting effect of what seems to be a mitochondrial proliferative response has a secondary reorganization effect on cardiomyocyte intermediate filaments as demonstrated by immunohistochemistry but with Z-line preservation. Nevertheless, there was no effect of MitoQ on LV morphology and function in the sham rats even at 12 weeks of treatment (data not shown). Pure VO has been characterized by extracellular matrix loss142, 143 and the current study demonstrates a matching cytoskeletal loss and breakdown associated with loss of mitochondrial connection to sarcomeric units. The critical relevance of this finding in the rat is underscored by a similar pathology in the human with isolated MR and preserved LV ejection fraction.145 Understanding the pathological imbalance of oxidative stress and other signaling pathways that cause cytoskeletal breakdown in a pure VO will lead to the development of novel drug targets for early intervention to prevent the progression to heart failure. 87 Difficulties in Studying Volume Overload and Cytoskeletal Degradation Studying the cytoskeletal degradation, in particular desmin, proved extremely difficult throughout the work presented here. Desmin is estimated to be posttranslationally modified on more than 50 sites on the protein. Only one of these phosphorylated forms of the protein has an antibody developed against it, and this antibody is under patent for the formation of an assay kit. Therefore, this antibody is currently unavailable for purchase. Because of these post-translational modifications, desmin is extremely difficult to blot. Antibodies developed against desmin are mostly directed toward the full-length protein, and thus results vary based on the availability of the epitope for binding. Adding to the difficulty is the cleavage of desmin by, as of yet, an unknown enzyme. The cleavage product is difficult to detect due to the unavailability of antibodies to recognize it. Thus, the loss of desmin we see in these studies, may be less of a loss and more of a cleavage or degradation of the whole protein. Unfortunately, we are unable to detect these products of degradation. Similarly, the extent of phosphorylation varies between desmin proteins, so the results of our Western blots are extremely variable leading an inability to achieve statistically significant differences between groups. Five separate desmin antibodies were used to try to determine desmin levels in the studies presented here. Literature concerning desmin phosphorylation and experimental methods to measure it are almost nonexistent, but it is an emerging field of research. As such, much trial and error went into determining the amount of desmin present in cardiac tissue from our studies. 88 Antibodies against desmin are targeted to either the whole protein or to one of three areas of the protein: the head, body, or tail region. The small amount of desmin literature indicates most phosphorylation occurs at the head (N terminal region) or the tail (C terminal region). We were able to use an antibody targeted toward the body (rod region) of the protein to determine desmin differences between groups. However, the differences we see in these studies cannot totally be attributed to desmin loss, because we many not be recognizing desmin fragments with the antibodies currently available. Because of these struggles our lab is currently investigating creating our own antibody against desmin in order to recognize desmin more specifically and more reliably. Similarly, we saw a large range of variability in the animals used in this study. While we try to control the creation of the ACF, the process is fallible. The fistulas are created by the same-sized needle, but the size of the fistulas seems to vary at the time of sacrifice. This could simply be due to the way each animal responds to the surgery, or it could be due to human error in the generation of the fistula. This amount of variability in the animals in the study is actually useful in that the patient population will demonstrate similar levels of variability in disease progression and phenotype. However, this variability makes reaching statistical significance in many parameters very difficult. We also discovered, through the course of this study, the effect of a carotid cut down on echocardiographic measurements. We use echocardiography in conjunction with pressure volume analysis to determine hemodynamic parameters. However, the carotid cut down, which is necessary to determine pressure-volume relationships, caused many echocardiography parameters to be lower than expected. The ejection fraction in particular was affected, and was extremely low even in the sham animals. Therefore, 89 moving forward we will determine echocardiographic parameters prior to beginning the carotid cut down to eliminate any confounding variables. Future Studies Direct Bioenergetic Measurements in Chronic VO with MitoQ. The studies presented in this dissertation heavily implicate pathological energetic involvement in cardiac VO. While these studies provide novel insight into possible therapeutic approaches involving mitochondrial ROS quenching in VO, a direct measurement of cardiac energetics in vivo would be preferable. Such studies have been conducted in HF patients treated with acute allopurinol administration.124 However, it is as yet unknown if chronic MitoQ administration affects cardiac efficiency and cytoskeletal organization in patients. Due to small vessel size and limited experimental equipment, this is a technically complicated task to achieve in rodents. To produce these data, the O2 concentration of blood going into the heart muscle and the concentration of the heart’s venous return must be determined in the context of how much contractile work the heart is producing per amount of O2 consumed. Utilizing a donor rat to perfuse the heart of the studied rat, it is possible to make these measurements in rodents. Indeed, this experimental design has demonstrated a decrease in cardiac efficiency in the 12-week ACF.162 Future studies utilizing this technique could provide direct evidence that MitoQ, in chronic ACF, improves cardiac efficiency. 90 Determine if a Combination XO Inhibitior/MitoQ Therapy is Beneficial in VO As previously described in our lab, MitoQ prevents XO activation in response to mechanical stretch. In addition, stretched-induced myofibrillar integrity and mitochondrial swelling are prevented with MitoQ. This suggests mitochondrial ROS are at least the initial event responsible for stretch-induced XO activation. Therefore, combination therapies utilizing both MitoQ and XO inhibitor allopurinol may result in a synergistic effect that maintains or prolongs cardioprotection. Indeed, multiple studies suggest a role for MitoQ therapy in cardiovascular disease.136, 139 Determine if a Combination MitoQ/ Beta Blocker Therapy is Beneficial in VO Also previously described in our lab, β blocker therapy is a somewhat effective treatment in patients suffering from mitral regurgitation. As described here, β blocker therapy is useful in decreasing adrenergic drive experienced by the heart during times of increased volume. This suggests a decrease in adrenergic drive would be beneficial in preventing the progression to heart failure. MitoQ protects against cytoskeletal damage and degradation of the mitochondrial registry within the sarcomeres, but does not protect against myocardial remodeling and progression to heart failure. The combination of a decreased adrenergic drive and inhibition of cytoskeletal degradation may result in a synergistic cardioprotective effect. 91 Understanding the sequence of events that leads to cardiac remodeling will also be helpful in future studies of volume overload. We hypothesized mitochondrial dysfunction was the initial insult that began the progression to heart failure. However, the data presented here indicate the mitochondria are functional through mechanical stretch insults as well as in vivo hemodynamic insults. Indicating that, while the framework that holds the mitochondria within the sarcomere is broken down, the mitochondria continue to function properly and produce ATP. These data led us to revise our hypothesis that the cytoskeletal damage precedes the mitochondrial dysfunction. The initial insult that begins the process is still a mystery, as we seem to have a “chicken and egg” situation in which cytoskeletal damage seems to lead to an increase in mitochondrial ROS production, while an increase in mitochondrial ROS production seems to lead to cytoskeletal damage. This feed-forward system seems to indicate not a single initiating event; rather a series of events happening in tandem via various sources creates an unfavorable hemodynamic environment leading to eccentric remodeling and the eventual development of heart failure. This complicated progression of dysfunction lends more evidence to the idea of combination therapy. No “magic bullet” seems capable of addressing all of the vast and complicated changes occurring in the myocardium. Therefore, addressing several arms of the disease concurrently may have the best chance of success. 92 Examine the Role of Mitochondria-Sarcoplasmic Reticulum Crosstalk in VO Bioenergetics and the mitochondria are implicated here in both acute cardiomyocte stretch and chronic VO with possible associations with sarcomeric proteins. The spatial organization of the mitochondria is of interest due to its proximity to the sarcoplasmic reticulum. This organization allows for a possibility of Ca2+ -mediated crosstalk between the mitochondria and the sarcoplasmic reticulum. Ca2+ reuptake during diastole is regulated by the sarcoplasmic protein SERCA2, which requires large amounts of ATP provided by the mitochondria. In addition, mitochondrial Ca2+ signaling plays an integral role in autophagy, apoptosis, cell death, and regulation of the permeability transition pore. Many studies have linked heart failure, aging, and other cardiovascular insults to apoptosis and recent studies are beginning to explore the connection between autophagy and heart failure. Therefore the regulation of Ca2+ in connection with effective ATP production could be interconnected with cardiovascular pathologies, especially in the setting of increased energy demand inherent to VO. Future studies should explore this possible interplay that may regulate cardiomyocyte function and death in HF. Determine the Point of No Return for MitoQ Treatment in VO This work demonstrated that MitoQ therapy started at the onset of VO in the rat preserved cytoskeletal structure and mitochondrial registry. To expand the application of this therapy, studies that define the point at which MitoQ treatment confers no benefit should be conducted. Relevant questions that should be addressed are: 93 Is MitoQ beneficial if started later in disease progression? What is the role of MitoQ following removal of VO by either repair of the ACF in rats or the mitral valve in patients? Studies that begin MitoQ treatment during the chronic compensated state and before overt failure ensues would provide valuable information that has a translational benefit. In addition, MitoQ’s effectiveness in protecting against cytoskeletal degradation and mitochondrial morphological changes should be determined after removal of VO. MR patients operated on under current guidelines demonstrate cardiovascular abnormalities and dysfunction after mitral valve repair. Therefore, it is possible that MitoQ may provide a cardioprotective effect for VO patients prior to surgical repair. These studies could be conducted in the ACF model utilizing MitoQ prior to repair of the fistula vs untreated animals following repair. Conclusions The work presented here investigates pathogenic mechanisms mediated by oxidative stress in both isolated cardiomyocytes and chronic volume overload in a combined in vitro and in vivo approach. This work contributes important information to the understanding of the progression and treatment of VO in patients. The beneficial effects of MitoQ on cytoskeletal remodeling help determine critical time points in the progression to heart failure. The data presented here suggests mitochondrial ROS are activated by a myocardial stretch-dependent pathway. These findings establish a possible feed-forward mechanism present in ACF that may drive the progressive cardiac 94 remodeling and dysfunction in VO. This hypothesis is supported as MitoQ treatment prevented cytoskeletal damage and mitochondrial morphological changes during a prolonged, 8 week study. 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