James N. Weiss, M.D. - US grants

The goal of this project is to define how changes in the extracellular environment, particularly ion accumulation, influence the electrophysiological properties of heart during myocardial ischemia and hypoxia. An experimental model, the arterially perfused rabbit ventricle, has been developed in which it is feasible to study these relationships in a quantitative and comprehensive manner. K+ and pH sensitive extracellular electrodes will be used in conjunction within tracellular electrodes and multiple extracellular electrodes to monitor [K]o and [H]o accumulation, intracellular potential, threshold of excitability, refractory periods, conduction intervals and tension during ischemia and hypoxia in the isolated arterially perfused rabbit ventricle. Changes in [K]o, pHo and electrophysiological parameters during the first 10-15 minutes of global ischemia are reversible, reproducible, and reasonably homogeneous, making it possible to study the contributions of various components of ischemia to electrophysiological alterations by altering the perfusate to simulate the ischemic environment. In addition to ion accumulation, other components of ischemia can be simulated, including catecholamine release, hypoxia, lactate accumulation, hypoglycemia, free fatty acids, and lysophosphoglycerides. The principle hypotheses which will be tested are: 1) During early global ischemia most electrophysiological changes can be attributed to extracellular K+ accumulation, acidosis, and catecholamine release. The other factors listed above may modify these electrophysiological alterations. 2) The electrophysiological actions of drugs known to influence arrhythmogenesis during ischemia may modify or be modified by [K]o accumulation, acidosis, or other components of ischemia. 3) During early hypoxia, electrophysiological alterations cannot be attributed to changes in [K]o or pH, and most probably result directly from the depression of metabolism. 4) The electrophysiological alterations during early global ischemia predispose this preparation to reentrant arrhythmias in the setting of regional ischemia. Regional perfusion of the preparation with perfusate modified to simulate the ischemic environment produces similar arrhythmias.

The broad objective of this renewal application is to continue to investigate relationships between cardiac metabolism and function. The major areas to be studied include: 1). the metabolic regulation of cardiac ATP-sensitive K+ (K.ATP) channels and further assessment of their role in cellular K+ loss during hypoxia and ischemia. Cellular K+ loss during acute myocardial ischemia is a major arrhythmogenic factor. One goal of these studies is to resolve how K.ATP channels, which are suppressed by [ATP]i in the micromolar range in excised patches, are activated sufficiently to account for cellular K+ loss during myocardial hypoxia and ischemia when [ATP]i remains well in the millimolar range. We will continue to investigate how the sensitivity of K.ATP channels to [ATP]i is extrinsically and possibly intrinsically modified by various components of the ischemic environment. We will also evaluate interactions between sulfonylurea antagonists and agonists of K.ATP channels and various components of the ischemic environment in order to understand the mechanisms of their effects on cellular K+ loss during hypoxia and ischemia, and to evaluate their potential as cardioprotective agents. 2). the mechanisms underlying functional compartmentation of glycolytic and oxidative metabolism in heart. Compartmentation of cardiac metabolism if it exists has important implications for strategies to prevent irreversible cardiac injury. Studies arising from the previous application demonstrated that anaerobic glycolysis preferentially suppressed cardiac K.ATP channels due to the association of key glycolytic enzymes with K.ATP channels in cardiac sarcolemma. We will further explore the mechanisms of the preferential dependence of K,ATP channels on glycolysis, specifically testing the hypothesis that the major function of glycolytic enzymes associated with K.ATP channels is to lower the free [ADP]i. We will also further explore the hypothesis that contractile function is preferentially supported by oxidative metabolism in heart. Finally we will further investigate the mechanisms underlying our observation that exogenous glucose utilization is superior to glycogenolysis at preserving cardiac function during hypoxia and reoxygenation in intact rabbit ventricle. 3). mechanisms of transsarcolemmal lactate movement in heart. We will quantitate the extent to which electrogenic transsarcolemmal lactate and K+ movement are coupled in heart, in order to evaluate the hypothesis that lactate-coupled K+ efflux is an important cause of increased cellular K+ efflux during early myocardial ischemia and hypoxia. To accomplish these goals the experimental approach will utilize standard patch clamp techniques in isolated intact and permeabilized ventricular myocytes, fluorescent pH indicators in patch-clamped myocytes, and ion-selective electrode and radioisotopic techniques in isolated intact arterially perfused interventricular septa. These studies will evaluate at a fundamental level how various aspects of cardiac function are regulated by cardiac metabolism. Knowledge of these interactions is essential to understand the response of heart muscle to ischemia and other metabolically impaired states and to provide new insights relevant to the protection of ischemic injury, currently the leading cause of death in our society.

Despite the key role of intracellular Ca (Ca) in the pathogenesis of ischemic and reperfusion injury to the myocardium and coronary vasculature, the effects of metabolic inhibition and other components of the ischemic environment on the complex interaction between the multiple cellular processes responsible for regulating Ca is not well-understood. The objectives of this continuation proposal are to further characterize the subcellular mechanisms by which metabolic inhibition and components of the ischemic environment alter Ca regulation in ventricular myocytes and vascular endothelial cells. In ventricular myocytes, Ca, (using fura-2), membrane current and voltage and cell shortening will be monitored simultaneously under whole cell patch clamp conditions during exposure to combined or selective inhibition of glycolysis and oxidative metabolism, and to various components of the selective inhibition of glycolysis and oxidative metabolism, and to various components of the ischemic environment. Using appropriate pharmacologic interventions and ionic substitutions, the effects of these interventions and ionic substitutions, the effects of these interventions on individual components of cardiac excitation-contraction coupling will be characterized in detail. Studies will also be performed in giant excised membrane patches to assess Na-Ca exchange and Na-K pump function, and in permeabilized myocytes to test whether glycolysis plays a preferential role in supporting SR function. In vascular endothelial cell monolayers, Ca (Using fura-2 imaging), membrane potential and membrane resistance will be monitored simultaneously under whole cell patch clamp conditions to characterize: 1) cell signalling pathways which couple receptor binding of endothelium-dependent vasodilators (e.g. bradykinin, ACh, histamine, etc.) to increases in Ca associated with release of EDRF, ii) the effects of combined and selective metabolic inhibition, components of ischemia and cytokines on these cell signalling pathways, and iii) the effects of these interventions on cell-to-cell propagation of Ca wavers in response to locally-applied endothelium-dependent vasodilator agonists. Experiments in single endothelial cells using whole cell voltage clamp and single channel recordings will be used to elucidate underlying ionic currents involved. The proposed experiments offer a uniquely comprehensive approach to understanding how metabolic factor alter Ca regulation in myocardium and vascular endothelium.

NO PARENT ABSTRACT AVAILABLE

Objective. The longterm goal of this project is to investigate further the role of the sympathetic nervous system in determining sudden cardiovascular death during ventricular tachycardia in humans, and thereby improve survival. Background. In humans, sympathetic nerve activation during ventricular tachycardia is an important determinant of hemodynamic stability during ventricular tachycardia, independent of ventricular function and tachycardia rate. In animal models, the arterial baroreflex and cardiopulmonary baroreflex have important, yet opposing, effects on sympathetic activation during ventricular tachycardia. The relative contributions of these control mechanisms in humans is unknown. Specific Aims. The immediate aim o this study is to determine the roles of the arterial and cardiopulmonary baroreflexes, and the additional contributions of ventricular dysfunction and orthostatic stress, in determining sympathetic activation and hemodynamic stability during ventricular tachycardia in humans. Design. Using microneurography of the peroneal nerve, sympathetic nerve activity directed to muscle and to skin will be measured and compared in patients with hemodynamically stable and unstable ventricular tachycardia. A series of interventions which selectively activate arterial and cardiopulmonary baroreceptors will be performed to determine the contribution of each to sympathetic responses observed during induced ventricular tachycardia or rapid ventricular pacing. Patients with a wide range of ventricular function will be studied, including those with advanced heart failure and patients who have undergone orthotopic heart transplant (which denervates cardiopulmonary baroreceptors). Chaos theory will be used to analyze sympathetic recordings in the different patient groups, based on preliminary results suggesting qualitative differences between normal subjects and heart failure patients. Significance. The knowledge gained from these studies may serve as a basis for the development of medical and surgical therapies directed at the correction of the underlying abnormalities that predispose the patient to hemodynamically unstable ventricular tachycardia degenerating into ventricular fibrillation and sudden cardiovascular death.

During acute myocardial ischemia, extracellular K accumulation is a major factor predisposing the heart to the development of reentrant arrhythmias and VF. The mechanism, however, remains controversial. Two major hypotheses are: activation of metabolically-sensitive K channels such as ATP-sensitive K(KATP) channels, and K loss coupled to anion efflux (lactate and/or Pi) as a charge-balancing mechanism. The major goals of this project are to further elucidate the role of KATP channels in cellular K loss during hypoxia and ischemia, to characterize the biophysical, regulatory and pharmacologic properties of KATP channels in greater detail, and to evaluate mechanisms of transsarcolemmal lactate movement and its relationship to cation fluxes in heart. The effects of activation of KATP channels on cellular K loss will be studied in isolated arterially perfused rabbit interventricular septa loaded with 43K to measure unidirectional K efflux rate of tissue K content during exposure to KATP channel agonists. Our preliminary findings indicate that selective activation of KATP channels with cromakalim caused action potential shortening and an increase in unidirectional K efflux rate similar to hypoxia, but did not cause net K loss. In the proposed experiments we will activate the hypothesis that in addition to activation of KATP channels, enhancement of inward currents is required for net K loss to occur. Experimental findings in the rabbit septum will also be simulated in a computer model of the ventricular action potential to provide further insights. We will contribute to investigate the biophysical, regulatory and pharmacologic properties of KATP channels, using patch clamp techniques in isolated ventricular myocytes. We will attempt to delineate the mechanism by which glycolysis preferentially regulates KATP channel activity. We will test a novel hypothesis that surface charge plays an important physiologic role in regulating the ATP- sensitivity of KATP channels. We will explore our observation that c Ca- dependent process during severe metabolic inhibition irreversibly modified the ATP-sensitivity of KATP channels, and that treatment of the cytosolic surface of excised inside-out membrane patches with trypsin and other agents mimicked this effect. These observations will be investigated further to provide insight into channel regulation under pathophysiological conditions, and to gain insight into how proteolysis and chemical modification of KATP channels alters function. The final major goal is to evaluate the mechanisms of transmembrane lactate movement in heart and its relationship to cation (particularly K) fluxes. We have developed a novel method for studying transmembrane lactate movement in isolated patch-clamped cardiac myocytes using fluorescent indicators to monitor intracellular H, K and Na in response to lactate influx for this purpose. These studies should provide important new insights into the mechanisms of a major arrhythmogenic factor, extracellular K accumulation, contributing to sudden death during acute myocardial ischemia.

DESCRIPTION (provided by applicant): The training program described in this renewal application represents a new paradigm for training academic cardiologists as cardiovascular scientists, based on the UCLA STAR (Specialty Training and Advanced Research) Program combining clinical subspecialty training with research training leading to a Ph.D. degree or equivalent. M.D. trainees must complete 2 years of subspecialty cardiology training and 3 years (on average) of research training fulfilling requirements for a Ph.D. degree, including formal course work, qualifying examinations, and research leading to successful thesis defense. M.D./Ph.D. trainees undergo 2 years of post-doctoral research training, including elective course work. Eight positions are requested, to provide support for the research training component only. The training faculty consists of 36 senior preceptors and 16 supporting faculty. Only senior preceptors may serve as primary research mentors, and they come from 5 groups: the UCLA Cardiovascular Research Laboratory, the UCLA Atherosclerosis Research Unit, the UCLA ACCESS Program (a multi-departmental program administering Ph.D. training in life sciences), the School of Engineering, and, for health services research, the UCLA School of Public Health and RAND Graduate School. Almost all senior preceptors have joint or primary appointments in degree-granting basic sciences departments including Biological Chemistry, Microbiology and Molecular Genetics, Neurobiology, Pathology, Pharmacology, Physiology, Physiological Science, various Engineering disciplines, or in the RAND Graduate School. Supporting faculty do not act as primary mentors, but play a key role in enhancing the overall research environment and fostering translational research from the basic to the clinical arena. The research programs of the faculty are supported by over 24 million dollars in direct costs annually from extramural sources. By integrating cardiology subspecialty training with the formal recruitment of Ph.D. training (or equivalent post-doctoral training for those already having a Ph.D.), the UCLA Cardiology STAR Program provides graduates with the rigorous research background essential to translate advances in molecular health sciences into modern molecular medicine.

Inward rectifier K (Kir) channels are essential for the normal function of both excitable and nonexcitable cells. The specific aims of this proposal are two-fold: to better understand the mechanisms of inward strong rectification in Kir2.1 (IRK1), the major component of the high resting K conductance in many cell types, and to investigate the basis of mechanosensitivity of the G-protein regulated K channel Kir3.4 (GIRK4), a major component of the resting K conductance in atrial muscle and brain. We will apply electrophysiological (patch-clamp), molecular biological and biochemical techniques to various cloned Kir channels expressed in Xenopus oocytes or mammalian cell lines. In the first specific aim, we will determine how an intrinsic gating mechanism, which we have recently identified and postulate to be a tethered gating particle, interacts with polyamines and Mg to contribute to strong inward reactivation in Kir channels. We will test the novel hypothesis that the tethered gating particle contains binding sites for polyamines and Mg which enhances its ability to cause inward rectification, providing further insight into the molecular basis of strong inward rectification. In the second specific aim, we will characterize the molecular mechanisms underlying stretch-induced inactivation of Kir3.x channels, a property which we have recently identified in Kir3.4 and native cardiac KACh channels. We will determine: whether mechanosensitivity is also a property of other members of the Kir3.x family, the regions of the Kir3.4 channel required for mechanosensitivity, using chimeric constructs and site-directed mutagenesis; the role of G proteins; and the cytoskeletal and/or extracellular matrix elements responsible for transducing mechanosensitivity. The mechanosensitivity of Kir3.x channels may contribute to a variety of stretch-induced responses, including stretch- induced arrhythmias, atrial natriuretic peptide (ANP) release, and/or hypertrophic gene programming. Together, these studies in Kir channels will provide important insights into the regulation of excitability in ventricular and atrial cardiac muscle, as well as in other excitable tissues.

bioimaging /biomedical imaging; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): A central mechanism leading to necrosis and apoptosis during ischemia/reperfusion is believed to be the mitochondrial permeability transition (MPT), due to permeability transition pore (PTP) opening in the inner mitochondrial membrane. Based on our recent work, we hypothesize. In this revised application that two separable components predispose mitochondria to injury during anoxia/reoxygenation. The MPT threshold component is most relevant to the anoxic or ischemic period, and sets the threshold for MPT during reperfusion. It is manifested as progressive MPT-independent cytochrome c loss and inner membrane leakiness, which can be attributed to accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to reperfusion. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers/inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (deltapsim),which in turn depends on cytochrome c content and inner membrane leak. Consistent with its known cardioprotective role, we find that mitoKATP channel agonist diazoxide protects against both the MPT threshold and MPT trigger components, and that this protection is blocked by mitoKATP antagonist 5-HD. In addition, PKC epsilon, a key signaling component in cardioprotection, protects against the MPT trigger component. The objective of this proposal is to further explore the signal transduction pathways protecting mitochondria from the MPT threshold and MPT trigger components under conditions generally relevant to ischemia/reperfusion. Our strategy is to integrate functional studies with proteomics analysis. Functional studies will use spectrofluorometric, imaging (fluorescent, confocal and high voltage electron microscopy), and adenoviral gene transfer techniques to study mitochondria and cardioprotection at three levels: isolated mitochondria, in situ mitochondria in permeabilized myocytes, and isolated myocytes. Proteomic analysis will dissect mitochondrial protein complexes associated with PKCepsilon and PTP components in protected and unprotected intact hearts. Using this integrated approach, we will 1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT threshold and trigger components, and how mitoKATP channel agonists are protective; 2) define the roles of isoform-specific PKC signaling in protection against the MPT threshold and trigger components; 3) examine whether other signaling pathways implicated in cardioprotection modulate susceptibility to the MPT threshold and MPT trigger components. 4) identify, using functional proteomics, the proteins forming multiprotein signaling complexes with PKCepsilon and known PTP components in unprotected and protected hearts, and 5) characterize deltapsim depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsive to mitoKATP activation and cardioprotective signaling pathways. [unreadable] [unreadable]

Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Weiss, Project 1) The theme of this Program Project application is to understand the signal transduction pathways mediating cardioprotection using a multidisciplinary approach that combines biophysics, physiology, proteomics and genetics. Project 1 focuses on a central tenet of this theme, that cardioprotective signaling converges on protection of mitohchondria by preventing the mitochondrial permeability transition (MPT). Project 1 will characterize the role of two distinct components of MPT to predispose mitochondria to injury during anoxia/reoxygenation. The MPT priming component is most relevant to the anoxic, or ischemic, period and primes the mitochondria to undergo MPT during reperfusion. The MPT priming component manifests as progressive MPT-independent inner mitochondrial membrane (IMM) proton leak, matrix condensation and remodeling, and cytochrome c mobilization and release and is promoted by accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to the reoxygenation, or reperfusion, phase. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers and inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (Av|/m), which in turn depends on cytochrome c content and IMM proton leak. Building upon previous studies demonstrating a role of mitoKATp channel and PKCe in modulating these components, Project 1 will further explore the signal transduction pathways protecting mitochondria from the MPT priming and trigger components under conditions generally relevant to ischemia/reperfusion by integrating functional studies with proteomic analyses. In collaboration with the Heart Biology Core, the functional studies will use spectrofluorometric, imaging (fluorescent, confocal and high voltage electron microscopy), and adenoviral gene transfer techniques to study mitochondria and cardioprotection at three levels: isolated mitochondria, in situ mitochondria in permeabilized myocytes, and isolated myocytes. In collaboration with Project 2, Project 3, the Proteomic Core, and the Heart Biology Core, the proteomic analyses will dissect mitochondrial protein complexes associated with the voltage-dependent anion channel (VDAC, an MPT pore component), PKCe and the novel mitochondrial protein phosphatase PP2CK in protected and unprotected intact hearts. Using this integrated approach, Project 1 will (1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT priming and trigger components, and how mitoKATp channel agonists are protective in this setting;(2) define the roles of key mitochondrial subproteomes in protection against the MPT priming and trigger components; (3) characterize Avym depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsiveness to mitoKATp activation and cardioprotective signaling pathways.

disease /disorder model; disease /disorder prevention /control; sudden cardiac death; therapy design /development; tissue /cell culture; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Ventricular fibrillation (VF) is the most common cause of sudden death, accounting for over 300,000 deaths annually in the US alone. The objective of this proposed Program Project is to develop a rational approach to the therapy of sudden cardiac death (SCO) through understanding the pathogenesis of VF at the mechanistic level. The proposal represents a continuation of our efforts, which began with our SCOR in Sudden Cardiac Death in 1995, to address this objective by combining mathematical biology with experimental biology to integrate information at the molecular, cellular, tissue and systems levels. The theme of this proposed Program Project is that dynamic wave instability (regulated by electrical restitution, intracellular Cai cycling, cardiac memory and diffusive currents) interacts synergistically with increased electrical and structural tissue heterogeneity in the diseased heart to increase the risk of VF and SCO, and that by developing molecular interventions designed to decrease dynamic wave instability, VF and SCO can be prevented. The four projects and three cores will address this theme using experimental approaches including patch clamping, optical mapping with fluorescent dyes, molecular biology, adenoviral gene transfer, and theoretical approaches including mathematical modeling, computer simulations and nonlinear dynamics. Studies ranging from isolated myocytes to intact normal and diseased ventricles, both in vivo and in silico, will be conducted interactively through this Program Project to evaluate comprehensively dynamic factors controlling wave stability as therapeutic targets for prevention of VF and SCO.

DESCRIPTION (provided by applicant): Efficient coupling of energy production to energy needs is critical for the beating heart, and factors disrupting this relationship are likely to promote injury. Glycolytic oscillations have been characterized extensively in yeast and pancreatic ?-cells, but conditions promoting their occurrence in cardiac myocytes are less established, compared to reactive oxygen species (ROS)-induced metabolic oscillations arising from the mitochondrial network. Our preliminary studies in rabbit ventricular myocytes show that the normally tight buffering of the ATP/ADP ratio by oxidative phosphorylation and the creatine kinase (CK) shuttle prevents glycolysis from oscillating, but under conditions in which this buffering is disrupted, glycolytic oscillations, manifested as large scale action potential duration (APD) oscillations via activation of ATP-sensitive K channels, develop in 90% of cardiac myocytes, as predicted by theoretical predictions. In this multi-PI proposal, our goal is to combine experimental and mathematical biology approaches to address two questions: 1) how are glycolytic oscillations regulated/modulated by physiological factors such as Cai cycling, autonomic tone, insulin, and signaling pathways relevant to cardioprotection? 2) do glycolytic oscillations occur during acute myocardial ischemia, in which the ability of oxidative phosphorylation and the CK shuttle to buffer cellular ATP/ADP ratio is markedly compromised? These questions will be addressed in three Specific Aims which integrate computer simulations and nonlinear dynamics with experimental patch-clamp and imaging studies in isolated myocytes from rabbits, wild-type and genetically-altered mice and neonatal rat ventricular myocyte monolayers subjected to coverslip ischemia/reperfusion. In addition, a novel fluorescent bioprobe designed for subcellular imaging of ATP will be further developed to study metabolism dynamics. These studies will increase our understanding of how cardiac metabolism functions at the systems level during metabolic stress, which may lead to new therapeutic insights towards preventing ischemia/reperfusion injury. PUBLIC HEALTH RELEVANCE: Since heart disease is the major cause of death in industrialized societies, understanding how to protect the heart from injury has major implications for the health care mission of the NIH/NHLBI and for society as a whole. To facilitate this understanding, this interdisciplinary project will integrate experimental physiology and mathematical biology to study the pathophysiology of oscillations in glycolysis, the key metabolic pathway for energy production during ischemia, and how they may contribute to ischemic cardiac injury by uncoupling energy production from energy needs in this critical situation. These insights may suggest novel therapies to protect the heart from injury during metabolic stresses such as heart attacks.

DESCRIPTION (provided by applicant): Investigations in the past decades have significantly advanced our understanding of signaling mechanisms underlying the protection and pathogenesis of myocardial ischemic injury. It is increasingly recognized that preservation of mitochondrial function plays a pivotal role in cardioprotection against ischemia reperfusion injury (I/R). However, it remains virtually unknown as to who the molecular targets of cardioprotection are in the mitochondria; what specific molecular events led to the protection of mitochondria; and whether there is a systems integration of cardioprotective signaling at the mitochondria to support the manifestation of a protected phenotype. Using a murine model of nitric oxide (NO) induced late phase of cardioprotection, we elect to examine the plausible intrinsic signaling properties of mitochondria using a novel experimental strategy enabling a parallel examination of mitochondrial signaling, mitochondrial proteomes, and mitochondrial behavior by computational modeling. The proposed studies are based upon preliminary evidence by others and our own demonstrating that activation of PKC?-Src module occurs in the NO donor treated mice and that both are localized to mitochondrial membranes. In this proposal we will test the innovative hypothesis that the PKC?-Src module interacts with the brief mPTP openings to protect cardiomyocytes from Ca++ overload induced jury. The working hypothesis is that NO activates PKC?-Src module, leading to brief mPTP openings which results in transients Ca++ releases and reactive oxygen species (ROS) bursts, and consequently inactivates Ca++ reuptake and further activates PKC?-Src to form a feed- forward loop. When the homeostasis is interrupted (e.g., calcium overload or elevated ROS), brief mPTP openings transits into irreversible, long-lasting mPTP openings, which instead induce cardiac injury. In this application we propose to delineate the functional effects of brief openings of mPTP on Ca++ handling and ROS production; and to elucidate mPTP regulation by the mitochondrial PKC?-Src module in the setting of NO-induced late phase of cardioprotection (Aim 1). In the specific Aim 2 we will conclusively establish the activation of a PKC?-Src signaling module in the mitochondria as a mandatory signaling element of NO-induced cardioprotection against myocardial ischemic injury. At last we will systematically define the molecular targets of mitochondrial Src-kinase in NO- induced late phase of cardioprotection (Aim 3). The proposed studies will advance our understanding of cardiac biology by providing novel mechanistic insights into how interactions of brief mPTP openings with mitochondrial PKC?-Src module can be beneficial in mediating NO-induced late phase of cardioprotection. (End of Abstract)

DESCRIPTION (provided by applicant): The overall objective of this research project is to achieve a better understanding of the mechanisms of arrhythmias causing sudden cardiac death by tackling the ionic and cellular mechanisms of early (EADs) and delayed (DADs) after depolarizations. EADs are classically attributed to reactivation of the L-type Ca current or to spontaneous sarcoplasmic reticulum (SR) Ca release (i.e. SR Ca release not directly gated by the L-type Ca current) in the setting of reduced repolarization reserve. DADs are attributed to spontaneous SR Ca release in the form of Ca waves stimulating Ca-sensitive inward currents such as Na-Ca exchange. Recently, we have presented evidence for a mechanism (chaos synchronization) by which EADs simultaneously create triggers and enhance tissue substrate vulnerability to promote lethal arrhythmias. A comparable theory does not yet exist, but is being developed, for DADs. The goals of this project are: i) to explore the cellular basis of EADs that set the process of chaos synchronization in motion;ii) to test whether theoretically-predicted rotors mediated by the L-type Ca current (related to the biexcitability of cardiac tissue) can be detected experimentally in cardiac tissue as a mechanism of Torsades de pointes;iii) to explore the cellular basis of DADs, specifically how the microscopic behavior of Ca release units in the sub cellular Ca cycling network integrates to generate Ca alternans, Ca waves, DADs and EADs at the whole cell level;iii) to explore the interactions between EADs and DADs that together generate triggers and modify substrate by increasing tissue electrical dispersion predisposing to VF. To accomplish these goals, we will combine patch clamp (including a dynamic patch clamp technique) and fluorescent dye studies at the cellular level with optical mapping studies at the tissue level. Improved understanding of the cellular mechanisms of after depolarizations which cause lethal arrhythmias is essential for developing novel therapy. PUBLIC HEALTH RELEVANCE: The proposed research will study the mechanisms of sudden cardiac death due to ventricular arrhythmias, which prematurely takes the lives of more than 300,000 U.S. citizens each year. The goal is to use this information to develop novel therapies to prevent this deadly manifestation of heart disease.

The overall objective of this Program Project is to achieve a better understanding of the mechanisms of arrhythmias causing sudden cardiac death. Project 2 tackles the ionic and cellular mechanisms of early (EADs) and delayed (DADs) afterdepolarizations. EADs are classically attributed to reactivation of the L-type Ca current or to spontaneous SR Ca release (i.e. SR Ca release not directly gated by the L-type Ca current) in the setting of reduced repolarization reserve. DADs are attributed to spontaneous SR Ca release in the form of Ca waves stimulating Ca-sensitive inward currents such as Na-Ca exchange. Recently, we have presented evidence for a mechanism (chaos synchronization) by which EADs simultaneously create triggers and enhance tissue substrate vulnerability to promote lethal arrhythmias. A comparable theory does not yet exist for DADs. The goals of this project are: i) to explore the cellular basis of EADs that set the process of chaos synchronization in motion; ii) to test whether theoretically-predicted rotors mediated by the L-type Ca current (related to the biexcitability of cardiac tissue) can be detected experimentally in cardiac tissue as a mechanism of Torsades de pointes; iii) to explore the cellular basis of DADs, specifically how the microscopic behavior of Ca release units in the subcellular Ca cycling network integrates to generate Ca alternans, Ca waves and DADs at the whole cell level; iii) to explore the interactions between EADs and DADs that together generate triggers and modify tissue substrate by increasing tissue electrical dispersion predisposing to VF. To accomplish these goals, we will combine patch clamp (including a new dynamic clamp technique) and fluorescent dye studies at the cellular level with optical mapping studies at the tissue level. These studies will be performed in close collaboration with the mathematical modeling studies in Project 1, the tissue level studies in Project 3, and the therapeutic development in Project 4, and will utlize both Core A and B for support.

CORE C DESCRIPTION This core will provide administrative support to all the projects and cores. This will include: [unreadable] Receiving and recording orders from the component projects and cores, placing these orders with the appropriate vendors, tracking and recording delivery of the orders, returning those that are incorrect or damaged, etc. [unreadable] Maintenance of all personnel records. [unreadable] Preparation and electronic submission of abstracts and manuscripts. [unreadable] Coordinating and arranging the scheduling of conferences, including the weekly PPG research meeting, and site visits by internal and extemal reviewers of the PPG. [unreadable] General secretarial duties [unreadable] Coordination of videoconferencing between UCLA and Indiana University

DESCRIPTION (provided by applicant): Cardiac arrhythmias are a major cause of mortality and morbidity in the developed world. Yet, the mechanisms of deadly arrhythmias remain to be elucidated. The main goal of this biannual Gordon Research Conference is to explore the fundamental mechanisms of the most complex arrhythmias, including atrial and ventricular fibrillation. New approaches to diagnosis, treatment, and prevention of cardiac arrhythmias will require the use of highly sophisticated tools that combine multiple technologies including novel imaging modalities, genetics, molecular and structural biology, tissue engineering, immunochemistry, patch-clamping, optical and high density electrode mapping of electrical wave propagation, and computer modeling. This field is represented by outstanding investigators at the forefront of research and experts in molecular biology, cellular electrophysiology, biophysics, biomedical engineering, mathematical biology, systems biology and clinical cardiology. This conference provides a forum to interact and share ideas about the role of numerous factors in the mechanisms of complex cardiac arrhythmias and sudden cardiac death, towards the goal of developing novel therapeutics.

DESCRIPTION (provided by applicant): Ischemic and pharmacologic preconditioning (PC) constitute the most powerful protection of the heart from ischemia/reperfusion (I/R) injury; however, the detailed molecular mechanisms underlying cardioprotection are still being defined. There is a general consensus that mitochondria are the final effectors of cardioprotective signaling regimes, and hexokinase (HK) has been suggested by multiple groups to regulate the mitochondrial permeability transition (MPT). Though the association of HK with voltage-dependent anion channels (VDAC) was elucidated over 10 years ago, two fundamental questions regarding the physiologic consequences of this interaction have remained unanswered, and consequently, have stalled the progression of the cardioprotection field. First, is the dissociation of HK from cardiac mitochondria a molecular trigger of cell death? That is, does HK dissociation from mitochondria precede all other cell death events [e.g., MPT, ?? loss, and cytochrome C (cyto C) release]? Second, what are the unknown molecular players that stabilize the HK-VDAC interaction and impart its unique cardioprotective properties? Unequivocal answers to these questions have been unattainable due to the lack of technologies for (i) temporal profiling of the spatial distribution of HK in relation to MPT, ?? loss, and cyto release in live cardiomyocytes, and (ii) quantifying the molecular constituents of the HK-VDAC complex and deciphering their stoichiometry. In view of these challenges, our program has tailored state-of-the-art live-cell imaging and quantitative proteomic innovations to comprehensively delineate the dynamics of HK-induced cardioprotection on a biological timescale. We hypothesize that HK is a core regulator of cardioprotection, common to multiple models of injury and preconditioning. We will employ real-time imaging in live myocytes to define the temporal profile of the molecular events during injury (Aim 1); we will use an extensive biochemical and genetic toolbox to delineate the molecular paradigm of HK interaction with mitochondria as well as its physiological consequences mediating cardioprotection (Aim 2); we will quantitatively define the proteome dynamics and molecular stoichiometry of HK interaction with VDAC; characterize isoform-selective changes in assembly of the HK- VDAC interactome; and identify candidate proteins essential to stabilize the HK interaction with VDAC during cardioprotection (Aim 3); and we will use cardiac gene delivery of HK constructs or other molecular candidates identified in Aims 1-3, to test an in vivo gene therapy strategy to protect adult rats from I/R injury (Aim 4). The proposed investigations promise conceptual, technological, and methodological innovations. We will leverage close collaborations with the UCLA NHLBI Proteomics Center for immediate and efficient translation of knowledge obtained in cell/animal models to clinical studies. The success of the proposed investigations will undoubtedly propel the field of cardioprotection forward.

DESCRIPTION (provided by applicant): Unraveling the genetic basis of common polygenic diseases, such as hypertension, diabetes and heart failure, will require fresh approaches to view how genes work together in groups rather than singly. In this proposal, we investigate gene network analysis as a promising new approach. Our goal is to identify specific expression patterns of gene modules, rather than single genes, which predict susceptibility to heart failure (HF). A network analysis of DNA microarray data typically groups 20,000 genes into 20-30 modules, each containing 10's to 100's of gene, drastically reducing number of possible candidates required to perform a gene network- based Gene Module Association Study (GMAS), which will be complementary to GWAS. To test the GMAS concept, we will use a systems genetics approach integrating DNA microarray analysis with physiological studies and computational modeling, to examine whether gene module expression patterns predict susceptibility to heart failure (HF) induced by cardiac stress. For this purpose, we will utilize a novel resource developed at UCLA, the Hybrid Mouse Diversity Panel (HMDP), consisting of 102 strains of inbred mice from which a common mouse cardiac modular gene network comprised of 20 gene modules has been constructed. Our preliminary findings reveal that different HMDP strains show considerable variability in both gene module expression patterns and phenotypic response to chronic cardiac stress (isoproterenol). Using biological and computational experiments, we will test the hypothesis that gene module expression patterns among HMDP strains represent different good enough solutions, all of which are adequate for normal excitation-contraction- metabolism coupling, but have different abilities to adapt to chronic cardiac stress. Three Specific Aims integrating experimental and computational biology and combining discovery-driven, hypothesis-driven, and translational elements are proposed, towards the goal of relating HMDP results directly to human heart failure.