Stephen F. Vatner - US grants

The overall goal of this study is to examine modifications in ventricular performance, coronary perfusion, myocardial energy metabolism and cardiac and coronary vascular adrenergic receptor activity in experimental models of chronic pressure overload right ventricular (RV) and left ventricular (LV) hypertrophy and failure. Measurements of cardiac pressures and dimensions, aortic pressure and coronary blood flow and diameter will be radiotelemetered from conscious, chronically instrumented dogs. Three models will be studied: RV pressure overload hypertrophy and failure; LV pressure overload hypertrophy (subcoronary and supracoronary banding). The following specific goals will be pursued. 1) To determine the changes in myocardial function that occur with the development of stable cardiac hypertrophy and progression to failure. 2) To determine the accompanying coronary vascular adaptations, and particuarly the difference in vasodilator capacity of the hypertrophied left ventricle with subcoronary vs supracoronary banding. 3) To determine if abnormalities in cardiac and coronary vascular control mechanisms can be elicited with stress, e.g., severe spontaneous exercise, pacing, sympathomimetic amines. 4) To determine the extent to which the alterations in coronary and cardiac control mechanisms are associated with changes in a) adrenergic receptor binding and affinity, b) myocardial energy metabolism, c) diastolic mechanical properties of the hypertrophied hearts. 5) To determine the extent to which alpha adrenergic control of the coronary circulation is depressed, and specifically, the extent to which carotid chemoreceptor reflex induced coronary constriction is altered in the presence of RV hypertrophy and failure. 6) To determine whether the depressed coronary vascular response to chemoreceptor stimulation is specific for the coronary vessels, or whether it also extends to derangements in responses of cardiac contractility, cardiac cycle length, and iliac vascular resistance. These studies are designed to elucidate alterations in fundamental myocardial coronary vascular and cellular control mechanisms with the development of myocardial hypertrophy and progression to cardiac failure.

heart revascularization; heart pharmacology; heart function; heart circulation; myocardial ischemia /hypoxia; coronary occlusion /thrombosis; physiologic stressor; catecholamines; adenylate cyclase; sympathetic nervous system; intracardiac pressure; heart contraction; edema; calcium channel blockers; cardiotonic agents; collateral circulation; heart dimension /size; heart innervation; beta antiadrenergic agent; radiotracer; blood flow measurement; heart catheterization; electrocardiography; myocardial infarct sizing; disease /disorder model; echocardiography;

DESCRIPTION (provided by applicant): This application represents the request for extension of continuously funded work in mechanisms mediating cardiovascular control under normal conditions and in the presence of hypertrophy and heart failure in chronically instrumented animals since 1972. One proposed model that persists is the pressure-overload model of left ventricular hypertrophy LVH), however, with one major modification. The superimposition of rapid ventricular pacing or spontaneous decompensation permits the study of the transition from stable, severe LVH to LVH/Heart Failure (HF), a critically important clinical problem, physiologically in a well characterized large mammalian model. A multidisciplinary team has been assembled to investigate the overall hypotheses and specific aims related to the regulation Ca2+ handling and EC coupling during the development of LVH and transition to HF. One hypothesis is that the transition from stable VH to LVHIHF involves a change in Ca2+ handling, and excitation - contraction (E-C) coupling at the cellular or molecular level, including alterations in L- and T-type Ca2+ currents, and the action potential, which could increase Susceptibility to arrhythmias. Furthermore, the T-type Ca2+ currents in LVH may play a compensatory role. We will examine the regulation of L- and T-type Ca2+ channels electrophysiologically at the myocyte level and also examine the mechanical correlates both in vivo and in isolated myocytes in vitro. In order to understand the mechanisms involved in he alterations in physiology and E-C coupling, it will be necessary to understand the corresponding changes in genomics and proteomics and vice versa. We will investigate genes known to be involved in Ca2+ regulation and also Novel genes, related to Ca2+ regulation, whose function in the heart is less established. In addition, utilizing a unique model in nature, the hibernating woodchuck, which is characterized by elevated Ca2+ stores and responses of phospholamban and phospholamban phosphorylation diametrically opposed to that observed heart failure, allows us to test the hypothesis that elevated SR Ca2+ is protective in response to pressure overload. These aims and hypotheses will be tested by investigators with expertise in physiology in intact animals, isolated myocytes, Ca2+ channels, proteomics and genomics.

The overall goal of this study is to examine modifications in ventricular performance, coronary perfusion, myocardial energy metabolism and cardiac and coronary vascular adrenergic receptor activity in experimental models of chronic pressure overload right ventricular (RV) and left ventricular (LV) hypertrophy and failure. Measurements of cardiac pressures and dimensions, aortic pressure and coronary blood flow and diameter will be radiotelemetered from conscious, chronically instrumented dogs. Three models will be studied: RV pressure overload hypertrophy and failure; LV pressure overload hypertrophy (subcoronary and supracoronary banding). The following specific goals will be pursued. 1) To determine the changes in myocardial function that occur with the development of stable cardiac hypertrophy and progression to failure. 2) To determine the accompanying coronary vascular adaptations, and particuarly the difference in vasodilator capacity of the hypertrophied left ventricle with subcoronary vs supracoronary banding. 3) To determine if abnormalities in cardiac and coronary vascular control mechanisms can be elicited with stress, e.g., severe spontaneous exercise, pacing, sympathomimetic amines. 4) To determine the extent to which the alterations in coronary and cardiac control mechanisms are associated with changes in a) adrenergic receptor binding and affinity, b) myocardial energy metabolism, c) diastolic mechanical properties of the hypertrophied hearts. 5) To determine the extent to which alpha adrenergic control of the coronary circulation is depressed, and specifically, the extent to which carotid chemoreceptor reflex induced coronary constriction is altered in the presence of RV hypertrophy and failure. 6) To determine whether the depressed coronary vascular response to chemoreceptor stimulation is specific for the coronary vessels, or whether it also extends to derangements in responses of cardiac contractility, cardiac cycle length, and iliac vascular resistance. These studies are designed to elucidate alterations in fundamental myocardial coronary vascular and cellular control mechanisms with the development of myocardial hypertrophy and progression to cardiac failure.

This conference is the seventh organized by the Circulation Council of the American Heart Association, and the second co-sponsored by the Basic Science Council. The theme of the present conference is Autonomic Cardiovascular Control, with sessions designed to cover mechanisms at the molecular and cellular level as well as experiments in animals and man. The conference deals with components involving a) the cellular mechanisms and molecular biology of the calcium channel, b) the cellular and molecular biology of the beta adrenergic receptor, c) neural recordings in conscious animals and man, and d) autonomic control of the microcirculation and of the cerebral circulation. One of the unique aspects of the conference is that it provides support for young investigators to attend the meeting and discuss in depth their projects with the senior faculty. While an important component of the prior meetings of the Council of Circulation involved the personal interaction among cloistered senior scientists, the feature of sponsoring junior investigators is new and represents the major fraction of requested support.

Despite recent advances in the treatment of human heart failure, the overall impact on morbidity and mortality has been limited, and heart failure remains the pre-eminent cardiovascular health problem in the country. Limitations in our understanding can be attributed, in part, to the inability to examine basic mechanisms in appropriate animal models in which the progressive pathogenesis of hypertrophy and heart failure can be studied. Thus, the overall aim of this Program Project is to identify physiological, biochemical and molecular mechanisms which are fundamental to the progression from imposition of the abnormal load to development of compensated hypertrophy to heart failure. A secondary goal is to understand the mechanism of potential therapeutic agents within the context of the pathogenesis of hypertrophy and heart failure, focusing on beta-adreriergic blockade therapy. The latter is central to one of the major themes of this Program Project, i.e., alterations in beta-adrenergic and G-protein signaling in hypertrophy and heart failure. To achieve these goals, we will focus primarily on the study of transgenic mice and a novel canine model of heart failure superimposed on chronic, severe cardiac hypertrophy. The organization of the Program Project includes 4 projects and 3 cores. The first Project will examine genomic and proteomic mechanisms of hypertrophy and heart failure utilizing the novel canine model of severe, chronic cardiac hypertrophy with heart failure superimposed. The next Project will emphasizes altered beta-adrenergic receptor signaling and signaling mechanisms involving stress-activated protein kinases in hypertrophy and heart failure. The overall goals of this projectare to elucidate cellular and molecular mechanisms, which may explain the adverse action of enhanced beta-adrenergic receptor signaling in heart failure and conversely, the salutary action of beta-adrenergic receptor blockade therapy. The next Project is designed to provide novel information on molecular mechanisms of adenylyl Cyclase regulation, also, using transgenic models. The final Project will focuses on molecular mechanisms involving Gprotein coupled receptor signaling, e.g., beta-adrenergic and angiotensin receptors in the development of hypertrophy and heart failure. This Program Project will provide significant new information on molecular mechanisms involved in mediating hypertrophy and heart failure, which will also have relevance for understanding corresponding mechanisms in myocardial ischemic disease as well.

cardiovascular function; biomedical facility; laboratory mouse;

Despite recent advances in the treatment of human heart failure, the overall impact on morbidity and mortality has been limited, and heart failure remains the pre-eminent cardiovascular health problem in the country. Limitations in our understanding can be attributed, in part, to the inability to examine basic mechanisms in appropriate animal models in which the progressive pathogenesis of hypertrophy and heart failure can be studied. Thus, the overall aim of this Program Project is to identify physiological, biochemical and molecular mechanisms which are fundamental to the progression from imposition of the abnormal load to development of compensated hypertrophy to heart failure. A secondary goal is to understand the mechanism of potential therapeutic agents within the context of the pathogenesis of hypertrophy and heart failure. To achieve these goals, we will focus primarily on the study of transgenic mice and pigs. The organization of the Program Project includes 4 projects and 4 cores. The physiological studies contained in Project 1 examine mechanisms of altered myocardial function in ventricular hypertrophy and failure. An additional component of Project 1 is to understand the mechanisms of cardiac dysfunction in terms of energy metabolism. Accordingly, NMR technology will complement the work on integrative physiology. Project 2 emphasizes altered beta-adrenergic receptor signalling and signalling mechanisms involving stress-activated protein kinases in hypertrophy and heart failure. Both of these projects will study transgenic animal models, including that of cardiac Gsalpha overexpression, which develops cardiomyopathy later in life. Project 3 is designed to provide novel information on molecular mechanisms of adenylyl cyclase regulation, also using transgenic models. Project 4 focuses on novel sarcomeric protein mutation-induced cardiomyopathies. Thus, this Program Project represents a multi-disciplinary approach to the problems of myocardial hypertrophy and heart failure combining expertise in integrative physiology and cellular and molecular biology.

It is well known that vascular stiffness increases with aging, yet the mechanisms involved are poorly understood, potentially due, in part, to lack of appropriate models. Indeed, the majority of research in aging has been conducted in rodent models or in humans with associated diseases of aging, e.g., diabetes or atherosclerosis. The primate model is unique because it is phylogenetically closer to humans, yet does not have associated diseases of aging. Over the past funding period, we have developed this primate model and have uncovered novel preliminary data supporting the renewal application. Our preliminary data indicate that female monkeys appear relatively protected from the vascular changes associated with aging compared with males. There are major gender differences observed in the composition of the vascular wall with aging and also in response to sympathomimetic amines. Accordingly, one important theme in this proposal includes examination of gender differences during aging. We will examine three hypotheses 1) Vascular stiffness increases in old male monkeys, but is relatively protected in old female monkeys. However, importantly, our hypothesis is that increases in vascular stiffness cannot be ascribed entirely to changes in collagen and elastin. Therefore, we propose two novel hypotheses: a.) that one mechanism of increased vascular stiffness with age involves caveolin and microtubules polymerization; and B.) that in part the increase in vascular stiffness with age resides at the level of the smooth muscle cell. For these studies, vascular smooth muscle stiffness will be assessed using an atomic force microscope; 2) Differences in the pattern of the expression of genes could explain the gender differences involved in the development of vascular stiffness; and 3) Gender differences must exist in the pattern of expression of proteins that could explain the differences involved in the development of vascular stiffness. These hypotheses and aims will be investigated using a multidisciplinary approach to maximally utilize this novel primate model.

Despite recent advances in the treatment of human heart failure, the overall impact on morbidity and mortality has been limited, and heart failure remains the pre-eminent cardiovascular health problem in the country. Limitations in our understanding can be attributed, in part, to the inability to examine basic mechanisms in appropriate animal models in which the progressive pathogenesis of hypertrophy and heart failure can be studied. Thus, the overall aim of this Program Project is to identify physiological, biochemical and molecular mechanisms which are fundamental to the progression from imposition of the abnormal load to development of compensated hypertrophy to heart failure. A secondary goal is to understand the mechanism of potential therapeutic agents within the context of the pathogenesis of hypertrophy and heart failure. To achieve these goals, we will focus primarily on the study of transgenic mice and pigs. The organization of the Program Project includes 4 projects and 4 cores. The physiological studies contained in Project 1 examine mechanisms of altered myocardial function in ventricular hypertrophy and failure. An additional component of Project 1 is to understand the mechanisms of cardiac dysfunction in terms of energy metabolism. Accordingly, NMR technology will complement the work on integrative physiology. Project 2 emphasizes altered beta-adrenergic receptor signalling and signalling mechanisms involving stress-activated protein kinases in hypertrophy and heart failure. Both of these projects will study transgenic animal models, including that of cardiac Gsalpha overexpression, which develops cardiomyopathy later in life. Project 3 is designed to provide novel information on molecular mechanisms of adenylyl cyclase regulation, also using transgenic models. Project 4 focuses on novel sarcomeric protein mutation-induced cardiomyopathies. Thus, this Program Project represents a multi-disciplinary approach to the problems of myocardial hypertrophy and heart failure combining expertise in integrative physiology and cellular and molecular biology.

The mechanisms responsible for myocardial hibernation have yet to be elucidated, despite numerous investigation. Two road types of animal models have been utilized: 1.) Acute or short-term hibernation, which involves a coronary stenosis for 90 minute duration 2.) chronic hibernation with coronary stenosis > 2-3 weeks. Recent studies from Dr. S Vatner's laboratory found that 1.) Chronic coronary stenosis induced by ameroid coronary constriction in swine results in several features of hibernating myocardium in humans, but does not results in reduced blood flow at rest despite significant reduction in function, 2.) short-term coronary stenosis (1.5 hr) results in a protective effect mediated by up- regulation of nitric oxide (NO) function, and 3.) myocardial blood flow in woodchucks during true hibernation is also maintained despite reduction of apparent metabolic demand, i.e., temperature and heart rate, and visceral flow reductions ranging from 80-90%. Specifically, this application will address the following hypotheses related to the maintenance of myocardial blood flow and myocardial performance in models of "short-term", "chronic" and "true" hibernating myocardium: 1.) Coronary stenosis induces an up-regulation of NO production that is protective during a sustained coronary stenosis in a model of acute hibernating myocardium, and that this protective mechanism is mediated by cardiac nerves. 2.) Hibernating myocardium can result from chronic stunning. 3.) Coronary blood flow is maintained in a model of chronic hibernating myocardium, i.e., during long-term coronary stenosis with ameroid coronary constriction in swine, potentially due to NO up- regulation of NO. 4.) Coronary blood flow is maintained in true hibernating myocardium in woodchucks, potentially due to up-regulation of NO. The work in this project is closely related to work in the other projects, which utilize similar models, and to all of the Cores.

DESCRIPTION (provided by applicant): The NIH and Academic Community in this country recognize the seriousness of the lack of MD's remaining in academic medicine in general and having careers in clinical investigation in particular. This cardiovascular research training program is designed to address this critical problem and produce research scientists and clinician/scientists prepared to meet current and future challenges in the arena of cardiovascular function and disease. As the title of the program indicates, one unique aspect of this program is an integrative approach beginning with genomics, proteomics, molecular biology, and cellular and molecular signaling - integrated with whole animal physiology, with emphasis on genetically engineered animals. The integrative approach not only involves the scientific disciplines of the faculty of this training program, but also provides the direction for training as well. For example, we will expose graduate students and postdoctoral fellows with a primary interest in cellular/molecular mechanisms to physiology, so that they understand the target for research is ultimately cardiovascular disease such as heart failure and myocardial ischemia. Conversely, an important component of the postdoctoral program is to include M.D. and M.D./Ph.D. students who have finished, or are in the midst of their clinical training with plans for a clinical cardiology fellowship, and expose them to an in-depth two to three year training program in molecular/cellular biology. The goal of this part of the program is to train academic, clinician/scientists cardiologists to remain in the University setting to conduct full time, clinical care, research and teaching To these ends, we have a group of well funded mentors, with additional training faculty from the New Jersey Medical School (NJMS) and New Jersey Institute of Technology (NJIT), who will work together to make this program successful. This program has relevance to Public Health at several levels. First of all, it must be appreciated that cardiovascular disease is a major health problem, as it is the major cause of disability and death in the U.S. To gain knowledge regarding the pathogenesis of these diseases and their therapy it will be necessary to have research scientists trained in cardiovascular research for the 21^'century. This training program will help provide the next generation of trained cardiovascular scientists and clinician scientists.

The most common form of heart disease is myocardial ischemia, which is characterized by an insufficient supply of blood, substrates and oxygen to the heart due to coronary artery obstruction. If not treated, irreversible damage ensues in the form of myocardial infarction (heart attack). The overall aim of the Project is to identify mechanisms which are fundamental to the understanding of ischemic heart disease, which will be accomplished by utilizing an integrative approach including cellular and molecular studies as well as integrative whole animal physiology. This Project is based on a model of repetitive stunning in the swine, developed in the current funding period, that reproduces the chronic myocardial dysfunction with maintained viability that characterizes the human hibernating myocardium. We show in the Preliminary Data that the well defined cardioprotective mechanisms attributed to the first and second window of preconditioning are not activated in the model of repetitive stunning. Rather, in this model, cardiac protection results from the activation of a different gene/protein program of cell survival, and also from the regulation of specific intracellular pathways, including autophagy. Accordingly, this may represent a third window of protection. The goal of this proposal is to better define the mechanisms of cardioprotection activated in this model of repetitive stunning, to determine their durability, to compare those mechanisms with those activated during preconditioning, and to determine whether the repetition of ischemia extends this cardioprotection to the remote, normal myocardium. Importantly, the swine model of repetitive stunning resembles pathophysiology in humans more closely than rodents, lacks preformed coronary collateral vessels, and the heart is sufficiently large to provide measurements of regional function, blood flow, biochemistry, molecular biology and pathology from the same animals in both the ischemic zone and a contralateral, remote, non-ischemic zone. This project is tied closely to the other projects and cores, as well as to the major themes of the Program Project: 1)Mechanisms of myocardial ischemia and reperfusion;2)Molecular signaling; 3)Myocardial protection and cell survival vs. cell death;4)lntegrative cardiovascular research. This project is linked closely to Project 1, which also studies the chronically instrumented swine model, but in Project 1 the model is one of regional cardiac denervation. Indeed, several of the aims are shared by Projects 1 and 2, using two different models. It will be critical to compare the cellular/molecular alterations in Projects 1 and 2 to derive an understanding of the differences between the second and potentially, third window of protection. Project 2 interacts with Project 3 in terms of molecular signaling and mechanisms of apoptosis, and with Project 4 particularly related to H11 kinase and its role in the protection afforded by chronic, repetitive stunning. Project 2 also utilizes all of the Cores.

DESCRIPTION (provided by applicant) This Core is designed to support all of the four projects in the Program Project for work on gene expression and proteomics. We combined the Proteomics and Genomics Core under one umbrella, because each part of the Core will need the other in terms of determining whether gene expression is accompanied by protein expression and vice versa. It also conserves costs, since the technical help at the Newark campus can be shared. This Core supports all the Projects with the following distribution: Project 1"40%; Project 2: 30%; Project 3: 10%; Project 4: 20%. The tissues used by the Core will be provided by the individual projects and Core B. Therefore, the function of this Core does not involve tissue harvesting and processing.

DESCRIPTION (provided by applicant): The overall theme of this project is to test the hypothesis that life span extension, longevity and stress resistance are mediated by common mechanisms. Growing lines of evidence suggest that the longevity of a wide variety of organisms, from yeast to worms and flies to mammals, is regulated by defined molecular mechanisms, including Sir2, an NAD-dependent histone deacetylase, and the adenylyl cyclase-protein kinase A pathway. A major limitation to understanding the key regulatory mechanisms responsible for causing the adverse effects of aging, or conversely, those that extend longevity, is the lack of animal models which exhibit prolonged lifespan and which do not develop cardiomyopathy or osteoporosis or other end- points, which are normally observed with aging. The model, which is accepted best for increasing longevity from yeast to primates, is caloric restriction. Relatively few other models for longevity are available. In this connection, we have recently identified a novel, genetically engineered animal model, which lives longer than wild type animals and does not exhibit many of the cardiac and osteoporotic features of old age, i.e., mice with the adenylyl cyclase (AC) type 5 "knocked out" (ACS KO). It is our contention that examining mechanisms that are unique to the ACS KO model will provide important insight into the aging process and, potentially, mechanisms which might be utilized to reverse this process. Projects 1 and 2 examine these mechanisms in this mouse model. In addition, Project 3 has developed and will study other mouse models of aging and stress resistance, related to Sir2alpha. The central hypothesis in that project is that Sir2alpha mediates anti-aging as well as cell protective effects in the heart in vivo. These 3 projects are supported by 5 cores: Administration/Physiology;Animal Care;Genomics/Proteomics;Bioinformatics/Biostatistics;Pathology. This Program Project has major implications for public health. The disability associated with aging has a major impact on the public health and the U.S. economy. Finding molecular switches, such as the ones described in this project, could ameliorate disability with aging and would be a major step forward.

Heart failure represents the end-point of the most common forms of heart disease, and its current therapy is at best palliative. The decornpensated stage of heart failure is usually preceded by a chronic, compensatory cardiac hypertrophy. The mechanisms that precipitate the transition from hypertrophy to failure remain largely unknown. This is due in large part to the lack of relevant chronic models that reproduce the clinical conditions. It is our hypothesis that a better understanding of the molecular adaptations to heart failure, both at the gene and protein level, will lead to the development of improved therapy. Our first Aim is to use genomic and proteomic approaches to better define the alterations of the beta-adrenergic signaling pathway during the transition from cardiac hypertrophy to heart failure. We will compare these data to those obtained in our murine models of cardiomyopathy and in myocardial samples from patients with congestive heart failure. Our second Aim will determine whether protein synthesis and degradation is qualitatively and quantitatively altered in heart failure. Specifically, we will measure the activity of ubiquitin and of the proteasome, and we will determine the identity of the proteins degraded by these mechanisms. Our third Aim will be to define the alterations in the expression of novel or unexpected genes in the failing heart, in an attempt to unravel potential novel physiological mechanisms and therapeutic strategies. In summary, the overall theme of this Project is to better understand the pathophysiology of heart failure in a novel model of heart failure superposed on chronic, severe hypertrophy, using a combination of both genomic and proteomic methodologies.

DESCRIPTION (provided by applicant): The Cardiovascular Research Institute (CVRI) and the Department of Cell Biology and Molecular Medicine (CBMM) of the New Jersey Medical School (NJMS), University of Medicine and Dentistry of New Jersey (UMDNJ) are dedicated to understanding the cellular and molecular mechanisms involved in cardiovascular disease. The main research foci are related to myocardial ischemia, hypertrophy, heart failure (HF) and aging. As the Department and NIH funded research continue to expand, the need for improved echocardiographic (echo) imaging in genetically altered mouse models has become acute. The CVRI and the Department of CBMM are now housing over 110 genetically altered mouse models in the animal facility at NJMS. Our current equipment cannot sustain the needs of the NIH funded research, even with excessive overtime work. The Justification of Need for purchase of the VisualSonics Vevo 770 Ultrasound Imaging System falls into 6 categories: 1. Increased NIH funding 2. Increased number of investigators 3. Increased need for mouse model studies 4. Need for a dedicated mouse echo facility 5. Need for higher quality data and additional echo studies 6. Need to pursue new directions in research without the new instrument, the NIH funded cardiovascular research programs at the New Jersey Medical School will be in jeopardy. In this proposal, we present organizational/management plans for increasing technical help, which in concert with the new instrument, will alleviate the needs listed above. The main research foci are related to myocardial ischemia, hypertrophy, heart failure and aging, which are among the most important health problems facing our country. PUBLIC HEALTH RELEVANCE: The Cardiovascular Research Institute (CVRI) and the Department of Cell Biology and Molecular Medicine (CBMM) of the New Jersey Medical School (NJMS), University of Medicine and Dentistry of New Jersey (UMDNJ) are dedicated to understanding the cellular and molecular mechanisms involved in cardiovascular disease. The main research foci are related to myocardial ischemia, hypertrophy, heart failure and aging, which are among the most important health problems facing our country.

DESCRIPTION (provided by applicant): Heart failure (HF) remains pre-eminent as a cause of mortality and morbidity in the U.S. Over the past half century several advances in the treatment of patients with HF (e.g., 2-adrenergic receptor blockade therapy) have improved survival, but only modestly. Since HF remains the most prominent cause of morbidity and mortality in this country, it follows that current therapeutic techniques are not adequate. This application is based on the concept that an increased understanding of adenylyl cyclase (AC) regulation in the heart will lead to novel therapeutic approaches. We are testing the hypothesis that the cardiac AC isoforms, AC5 and AC6, behave differently in the pathogenesis of HF leading to the concept that inhibition of AC5 may be a novel approach to the treatment of HF. Inhibition of AC5 may be superior to inhibition of 2-adrenergic receptor (2-AR) stimulation, since it does not impair left ventricular (LV) function, yet may lead to more effective 2-AR desensitization. The apparent different actions of AC5 and AC6 are the cornerstones of this application. There are 3 major hypotheses: Hypothesis A: In the AC5 Tg mice, cardiac stress leads rapidly to LV decompensation, LV hypertrophy (LVH), and HF, whereas AC5 KO mice are protected. AC6 Tg are also thought to be protected, but the effects of chronic pressure overload and chronic catecholamine stress have never been examined in AC6 Tg mice. Our hypothesis is that AC6 Tg are relatively protected from chronic pressure overload and catecholamine stress compared with AC5 Tg. Hypothesis B: There are four mechanisms, which can reconcile the differences between AC5 Tg and AC6 Tg in response to stress (1) Although AC5 contributes roughly a third of cyclase activity normally, the AC5 Tg generates more AC activity than AC6 Tg for a given increase in transgene expression;(2) AC5 and AC6 generate differences in intracellular microdomains of cAMP, which are regulated by different subtypes of phosphodiesterase (PDE), e.g. PDE3 and PDE4;(3) differences in protein-protein interactions;and (4) differences in intracellular localizations. Hypothesis C: Oxidative stress is a major mechanism by which chronic pressure overload or catecholamine stress induces apoptosis and LV decompensation. Overexpression of AC5, but not AC6, increases oxidative stress and apoptosis, which can explain the differences in response to chronic pressure overload and chronic catecholamine stress in AC5 KO and AC6 Tg mice. The implications for Public Health are self-evident, considering that heart disease and HF are the disease processes which have the greatest impact on Public Health in the US, in terms of finances and task- force, and using similar logic, finding new therapies will be crucial to minimize the impact of these disease states on Public Health. PUBLIC HEALTH RELEVANCE: Heart failure (HF) remains pre-eminent as a cause of mortality and morbidity in the U.S. Over the past half century several advances in the treatment of patients with HF have improved survival, but only modestly. Since HF remains the most prominent cause of morbidity and mortality in this country, it follows that current therapeutic techniques are not adequate. The implications for Public Health are self-evident, considering that heart disease and HF are the disease processes which have the greatest impact on Public Health in the US, in terms of finances and task-force, and using similar logic, finding new therapies will be crucial to minimize the impact of these disease states on Public Health.

PROJECT SUMMARY: It is well known that vascular stiffness increases with aging, and that the effects of aging on arterial stiffness are relatively protected in older women. Although most prior mechanistic work on the effects of aging on vascular regulation and stiffness has been conducted in rodent models, the extent to which these data can be extrapolated to humans is limited by the marked differences in lifespan over which changes in vascular stiffness develop. Studies of gender differences with aging are even more limited in rodents, due to the fact that the estrogen levels never decline even in very old rodents, and they do not go through menopause. It is generally agreed that non-humans primates are the best models to study gender differences with aging, since the changes in hormones and menstruation in old female (OF) monkeys parallel those in older human females. Our previous studies and preliminary data in aging monkeys have demonstrated that the stiffness of the aorta increases with aging and this aging alteration is greater in males than females, and also much greater in the abdominal aorta (AA) vs. the thoracic aorta (TA), which is only partially explained by variance in extracellular matrix (ECM). Here, we will test the novel Hypothesis that intrinsic mechanisms in the vascular smooth muscle cells (VSMCs) as well as alterations in VSMC-ECM interaction also contribute to the increased stiffness of the aorta in older males, particularly the AA, and conversely, contribute to the protection in pre-menopausal females. This Hypothesis is supported by Preliminary Data demonstrating enhanced stiffness of VSMC in culture from old male (OM) aortas and showing that the number of senescent VSMC increases in OM compared to young males (YM), particularly in AA. Specifically, we will test our Hypothesis through two approaches. In the first approach, we will determine how VSMC stiffness and senescence are affected by age and gender using atomic force microscopy (AFM) and also an artificial tissue model. In the second approach, we will determine both in vivo and in vitro how these factors may explain the regional differences in aortic stiffness between TA and AA.

DESCRIPTION (provided by applicant): Congestive heart failure (HF) in the U.S is the leading cause of death, and the major cause of HF is myocardial ischemic disease. Therefore, improvement of therapy post myocardial infarction (Ml) is extremely important, and the development of a new class of medicine that prevents the progression of HF would have a large market opportunity, representing a significant clinical advance. Additionally, most of these patients have elevated blood lipids and many have impaired glucose metabolism and are either diabetic or pre-diabetic. Therefore, a HF drug which can exert a favorable effect on diabetes control will have a unique niche in the HF market. The goal of this grant proposal is to demonstrate the efficacy of a new class of HF drugs, with a mechanism of inhibition of type 5 adenylyl cyclase (ACS), to improve the adverse effects of remodeling following chronic Ml and to simultaneously improve disorders of glucose metabolism,. This proposal is based on our prior work in a mouse model with disruption of the ACS gene, i.e., ACS knockout mice (ACS KO), and based on the utilization of a specific pharmacological ACS inhibitor. ACS KO mice have prolonged lifespan and are protected from the cardiomyopathy of aging. They are also protected against the development of HF induced by either chronic pressure overload, or by excessive sympathetic stimulation. Another unique feature of the ACS KO mouse model is its ability to increase coronary reserve, which should be particularly useful in preventing the development of HF. Importantly for this proposal these mice eat more than wild type, but weigh less, which points to a favorable metabolic profile, confirmed by our preliminary data using a specific pharmacological ACS inhibitor. In our preliminary screening for ACS inhibitors, adenine 9-?-D-arabinofuranoside (AraAde, also known as Vidarabine or Vira-A(r)), which was used in the clinic for a different indication, i.e., treating viral infections, showed potent and selective inhibition of ACS. Furthermore, our preliminary data demonstrate that the pharmacological AC 5 inhibitor protects against HF following chronic Ml and also protects against development of hyperglycemia induced by a high-fat diet in mice, suggesting that inhibition of ACS improves glucose metabolism in addition to its salutary effects in protecting against HF. Accordingly, in this preclinical study, we will examine the effects of AraAde on post-MI HF with or without impaired glucose metabolism. In addition we will examine the efficacy of this drug in the best pre-clinical model for HF, the chronically instrumented, conscious monkey post-MI. RELEVANCE (See instructions): Almost 5.5 million patients are diagnosed with congestive heart failure in the U.S and the major cause of HF is myocardial ischemic disease. However, no effective therapy has been established to treat congestive HF. The current investigation is aimed at generating a new class of medicine that will prevent progression of HF. Due to the large number of patients with this condition, this may have a significant impact on public health.

DESCRIPTION (provided by applicant): Improving exercise tolerance, one of the major goals of physicians treating patients with cardiovascular disease, can be achieved either by improving cardiac output and blood flow to skeletal muscle, or by improving energy metabolism in skeletal muscle, or both. However, less is known regarding specific molecular pathways that might be approached therapeutically to improve exercise tolerance. In the past, we have demonstrated that the adenylyl cyclase type 5 (AC5) knockout (KO) mouse lives one third longer than wild type and is protected against aging induced cardiomyopathy, and the development of heart failure induced by either chronic catecholamine or pressure overload stress. The current project is based on these studies and our preliminary data demonstrating that AC5 KO mice exhibit increased exercise capacity. Our overall hypothesis is that AC5 is a critical enzyme affecting stress resistance and exercise capacity. The goal of this project is to examine mechanisms involved in AC5 inhibition, which could lead to a novel approach to improve exercise performance. There are two major hypotheses: HYPOTHESIS A: AC5 inhibition permits enhanced exercise performance due to improved limb blood flow through enhanced vasodilator mechanisms or angiogenesis, whereas improved cardiac function and cardiac output are less likely mechanisms. HYPOTHESIS B: AC5 inhibition permits enhanced exercise performance due to improved mitochondrial function and/or resistance to oxidative stress and/or improved glucose utilization. The implications for Public Health are clear: improving exercise tolerance will have broad significance for aging, heart disease, most other diseases and even for the young, healthy population. PUBLIC HEALTH RELEVANCE: Improving exercise tolerance, one of major goals of physicians treating patients with cardiovascular disease, can be achieved either by improving cardiac output and blood flow to skeletal muscle, or energy metabolism in skeletal muscle, or both. However, less is known regarding specific molecular pathways that might be approached therapeutically to improve exercise tolerance. The goal of this project is to examine mechanisms involved in AC5 inhibition, which could lead to a novel approach to improve exercise performance. The implications for Public Health are clear: improving exercise tolerance will have broad significance for aging, heart disease, most other diseases and even for the young, healthy population.

? DESCRIPTION (provided by applicant): The role of the regulator of G protein signaling 14 (RGS14) in the heart has never been studied, and we found that the RGS14 KO mice are protected from the adverse effects of acute and chronic ischemia, through angiogenesis/arteriogenesis, which protects from myocardial remodeling and development of heart failure. To accomplish the aims of this proposal, we will examine the following hypotheses: Our first hypothesis is that disruption of RGS14 is a novel mechanism to protect the heart against chronic myocardial ischemia through angiogenesis and arteriogenesis. Our second hypothesis is that the mechanism of acute and chronic ischemic protection involves Gi AC/cAMP and Ras-mediated activation of the MEK/ERK pathway and consequently nitric oxide (NO)/VEGF activation, as well as blocking oxidative stress. A particularly novel feature of the RGS14 Knockout (KO) mouse is its ability to protect against both acute and chronic myocardial ischemia and to induce arteriogenesis/angiogenesis. A second novel feature, that underlies the importance of studying inhibition of a gene with multiple effects, such as RGS14, is that it elicit these unusual protective effects mediated by several distal signaling pathways, which in their combination are likely more salutary than any one of the individual mechanisms.

? DESCRIPTION (provided by applicant): The current submission is a revised application for our grant on effects of exercise in the receptor of G protein signaling 14 (RGS14) knock out (KO) mouse. The RGS14 KO model, demonstrates enhanced exercise tolerance and energy utilization, resulting not only in longevity, but more significantly, healthful aging. The RGS14 KO reduces beta adrenergic receptor signaling, which might be consistent with longevity, but novel and unexpected as a mechanism for improved exercise, the focus of this application, since enhanced beta adrenergic receptor signaling has always been associated with improved exercise performance. The RGS14 KO model has the additional novel, salutary attribute of increased brown adipose tissue, which is known to increase energy utilization and protect against diabetes, but is not known to mediate enhanced exercise performance, which will be examined in this application. Another key feature of the RGS14 KO is its ability to increase angiogenesis, which would also enhance exercise performance, since limitation of blood flow to exercising muscle causes exercise to cease. The major focus of the current application is to examine the physiological and molecular mechanisms mediating the brown adipose tissue and angiogenesis induced enhanced exercise capacity in the RGS14 KO model. This is important because reduced exercise tolerance is central to all patients with cardiovascular and other diseases, impairing a healthy life style and aging, and conversely enhanced exercise protects against disease and extends longevity. The RGS14 KO also mimics the beneficial features of exercise training. Accordingly, developing an RGS14 inhibitor that can be given to patients would recapitulate the beneficial effects of exercise training without the burden placed on patients to undergo daily exercise. This last point is the focus of Aim C1 in this application.

? DESCRIPTION (provided by applicant): Mammalian hibernation is a unique and potent strategy for survival in winter when food and water are not available. Our hypothesis is that some of the mechanisms utilized for protection against the stress in winter, might also be used to protect ischemic myocardium, even though hibernating mammals do not have coronary artery disease or myocardial ischemia. The focus of this proposal is to examine the woodchucks' protection in winter against complete coronary artery occlusion and its consequences of remodeling and the development of heart failure. After a complete occlusion of a coronary artery the major mechanism that can ameliorate the effects of ischemia relate to the coronary vessels and development of angiogenesis, which is supported by the preliminary data in this application. Our preliminary data also indicate that vascular stiffness and the composition of vessels are altered in winter thereby permitting enhanced blood flow. Potential cellular mechanisms include cAMP response element-binding protein (CREB) and nitric oxide synthase (NOS). It is important to keep in mind that these studies will be conducted for the first time in a natural model of cardioprotection, quite different from traditional studies in experimental animal models or genetically altered mice. Our Hypothesis: Woodchucks prepare for winter by developing mechanisms that extensively modify their blood vessels resulting in reduced vascular stiffness and induction of new coronary vessels, which provide blood flow to the ischemic heart and attenuate heart failure development and remodeling after chronic, complete coronary artery occlusion.

Project Summary Over the past half century there have been hundreds, if not thousands, of studies identifying cardioprotective agents, but relatively few have progressed to the clinics. There are probably several reasons for this lack of translational success: 1. That models identifying these potential cardioprotective mechanisms are based on acute experiments, not similar to chronic ischemia in patients; 2. many prior protective mechanisms cannot induce angiogenesis/arteriogenesis, which is required for patients with chronic myocardial ischemia and limited coronary blood flow; and 3. the data were derived solely from rodent models. The current application is based on the discovery of a novel cardioprotective agent, sFRP3, which was found in pigs with chronic preconditioning. Although relatively little is known about this gene in heart disease, it has been also found to be upregulated in patients with heart disease, which stimulated prior studies to conclude that sFRP3 exerted an adverse effect in heart failure, a conclusion diametrically opposed to our hypothesis and preliminary data. One of the major limitations to clinical translation of prior cardioprotective agents, is the inability to improve myocardial blood flow to the chronically ischemic heart by inducing angiogenesis/arteriogenesis. Our preliminary data indicate that sFRP3 induces both angiogenesis and arteriogenesis, which makes it an important new mechanism designed for not only acute cardioprotection, but also protects against chronic myocardial ischemic disease and finally will be of use to protect other organs where compromised blood flow induces disease, e.g., peripheral arterial and cerebral vascular disease. We will study 2 major hypotheses: Hypothesis A: sFRP3, when overexpressed either by injection into the heart, or genetically, exerts an important protective effect on coronary vessels, by induction of angiogenesis/arteriogenesis. Hypothesis B: sFRP3 induced protection against acute coronary artery occlusion is mediated by novel signaling mechanisms rather than angiogenesis/arteriogenesis.

Project Summary: We have generated a mouse knockout (KO) of the regulator of G protein signaling 14 (RGS14). The RGS14 KO mice live longer than their wild type (WT) littermates and are protected against stress, type 2 diabetes and obesity, all features related to cardiovascular risk protection. The mechanisms mediating these salutary effects of the RGS14 KO involve the striated muscle. The RGS14 KO also has increased brown adipose tissue, which is likely involved in the mechanism of its protective effects. The goal of this proposal is to determine the mechanisms mediating these salutary actions. We plan to establish the RGS14 KO as a novel model for longevity and determine to what extent striated muscle specific loss of RGS 14 and brown adipose tissue contribute to longevity, and protection against obesity and diabetes. In this proposal ?diabetes? is used to reflect protection of glucose utilization and insulin resistance, rather than the clinical disease. Our long term goal is to develop a clinically useful pharmacological inhibitor of RGS14. The proposal is based on the following hypotheses: Hypothesis A: Striated muscle specificity of RGS14 KO protects against the development of insulin resistance, glucose intolerance and obesity and extends lifespan mediated by increased energy metabolism through mitochondrial oxidation and protection against oxidative stress through the NAD+/SIRT3/MnSOD pathway. Hypothesis B: The RGS14 KO has the additional novel feature of increased brown adipose tissue, which also protects against diabetes, obesity and prolongs lifespan.

PROJECT SUMMARY The Cardiovascular Research Institute (CVRI) and the Department of Cell Biology and Molecular Medicine (CBMM) of the Rutgers University ? New Jersey Medical School (NJMS) are dedicated to understanding the cellular and molecular mechanisms involved in cardiovascular disease, and its risk factors. These include diabetes, exercise, obesity, and other factors that impair healthful aging. Accordingly, the main research foci are related to myocardial ischemia, hypertrophy, heart failure and cardiovascular risk factors. As the Department and CVRI continue to expand, the need for improved cardiac and vascular imaging in genetically altered mouse models and other animal models has become acute. There is also a need for expanding collaboration into the fields of development, neurology, and cancer. The Justification of Need for purchase of the VisualSonics Vevo 3100 Ultrasound Imaging System falls into 11 categories: 1. Need for Increased NIH funding 2. Allow the investigators on this grant to conduct their NIH funded research in the best possible manner and help insure the continued funding and research of the members of CVRI and CBMM. 3. Need to pursue new directions in research 4. Provide better data for young investigators and new faculty recruits for their first RO1 application 5. Enhance training of post-doctoral and graduate students supported on NIH grants, for future research. 6. Expand collaboration and uses to investigators with interest in development, neurology, and cancer. 7. Need to support collaborations with NIH funded investigators at other institutions 8. Increased need for mouse and other animal model studies 9. Reduce costs for animals due to ability to make multiple measurements over time. 10. Improve scheduling problems and reduce the need for overtime 11. Enhance the quality and quantity of research supported by the NIH grants and need for additional functions to measure cardiac function, regional blood flow and vascular dimensions and for higher quality data for LV echo Without the new instrument, the NIH funded cardiovascular research programs at the Rutgers-NJMS will be in jeopardy and future grants will be limited. In this proposal, we present organizational/management plans for increasing technical help, which in concert with the new instrument, will alleviate the needs listed above. The main research foci are related to myocardial ischemia, hypertrophy, heart failure and aging, which are among the most important health problems facing our country.