YIBIN WANG - US grants

Heart failure (HF) is one of the leading causes of mortality and immobility. The development of HF involves persistence of various stresses on cardiomyocytes that will lead to cardiac hypertrophy (CH), loss of contractile function and chamber dilation at advanced stage. Stress-activated MAP kinases (SAPKs), mainly consisted of cJun N-terminal kinases (JNK) and p38 kinases, have been implicated as important signaling molecules in a variety of stress responses. We demonstrated in neonatal cardiac myocytes that activation of JNK and p38 beta activities induced hypertrophy and activation of p38 alpha led to cell death. However, the in vivo function of JNK and p38s in the induction of CH and HF has not been established. The focus of this study, therefore, is to determine the in vivo function of JNK and p38 pathways in cardiac hypertrophy and development of HF, and test the hypothesis that activation of JNK and p38 isoforms contributes to specific features of cardiac hypertrophy, dysfunction and failure. The overall strategy for the study is to use efficient gene transfer technique and transgenic approach to specifically manipulate individual JNK and p38 activities in mouse heart, and to determine the effects of such manipulation on the development of CH and CF through comprehensive molecular, cellular and physiological analysis. Specifically, the proposed study will accomplish the following aims: 1). To determine the effects of specific activation of JNK and p38 MAP kinases on cardiac function and morphology in vivo. 2). To determine whether JNK and p38 activation is required in the development of CH and HF under physiological (pressure-overload) and genetic manipulations (Ras activation). 3). To determine the in vivo function of p38 alpha and beta isoforms in cardiac myocytes. The proposed study will provide a broad and comprehensive analysis for the functional roles of JNK and p38s in the development of CH and HF, covering levels from molecular and single cell to whole organ physiology. The expected results will contribute to the overall understanding of the underlined mechanisms of heart failure and may ultimately lead to identification of novel approaches for better treatment of this disease.

DESCRIPTION (provided by applicant): Knowledge of the signaling mechanisms involved in the pathological remodeling in heart failure holds great promise in finding new therapies to treat the disease. Activation of the stressactivated c-Jun N-terminal kinase pathway (JNK) has been implicated in the development of heart failure that results from hemodynamic stress or myocardial injury, yet its specific functions and the underlying mechanisms are still unclear. Although JNK is associated with global stress responses in heart, preliminary studies by the Project Leader indicate that JNK activation leads to a specific and localized pathology at the gap junction, including dramatic disruption of local cytoskeletal/ membrane organization, significant loss of connexin43 (Cx43) and mis-localization of Cx43-associated cytoskeletal protein which accompany diminished cell-cell coupling. These observations motivate an exciting hypothesis that JNK mediated signaling is responsible for the local pathological changes at the gap junction, as part of common manifestations in heart failure. How does JNK, as a global stress-induced signaling pathway, lead to such a very specific and localized pathology at the gap junction? Thus, global activation becomes a problem of local signaling and cytoskeletal structures, the major themes of this PPG proposal. Taking advantage of the synergy among the participating projects, the Project Leader proposes to accomplish the following specific aims: 1) to determine the changes of membrane/cytoskeletal structure induced by JNK activation and the impact on Cx43 expression and function at the gap junction. (Collaboration with Project 2); 2) to determine the role of protein phosphatase in downstream signaling events of JNK in regulating gap junction protein organization and expression (Collaboration with Project 1); 3) to complement the in vitro studies by characterizing the process of JNK mediated cardiac remodeling in a newly established transgenic model with temporally regulated JNK induction; and 4) to determine the impact of JNK mediated signaling on contractile function in heart cells (Collaboration with Project 4). Thus, the state-of-the-art plans are well integrated with those of the other projects. This project is designed to provide new insight into fundamental molecular pathways that link cardiac stress to local signaling and cytoskeletal disruption that underlie human heart disease.

DESCRIPTION (provided by applicant): Heart failure (HF) is the leading cause of mortality and immobility in the US, and is associated with contractile dysfunction and life-threatening arrhythmia. Signaling mechanisms involved in the pathological remodeling in the failing heart are not yet fully understood. One of the stress-activated MAP kinases (SAPKs) pathways, the cJun N-terminal kinases (JNK), has been implicated as an important signaling component mediating a variety of stress responses in the development of HF. In our preliminary study, we discovered that specific activation of JNK in transgenic hearts caused pathological remodeling in heart with significant downregulation of cardiac connexin43 (Cx43) and animals died from premature sudden death. While in cultured cells, activation of JNK also resulted in hypertrophy and the loss of Cx43 expression and cell-cell coupling, an effect that can be attenuated by blocking JNK activity. Our findings implicated, for the first time, a stress-related cellular signaling pathway in the negative regulation of Cx43 expression in heart, and led to us to hypothesize that JNK mediated signaling is an important pathway for pathological remodeling in failing heart, involving down-regulation of Cx43. Accordingly, the main focus of the current proposal is to determine the molecular and cellular mechanisms of JNK mediated down-regulation of Cx43 expression in cardiac myocytes and to establish the physiological significance of JNK signaling pathway in pathological remodeling involving inter-cellular communication. Specifically, the proposed study will accomplish the following aims: 1). To establish the specific role of JNK pathway in the regulation of Cx43 expression in cardiomyocytes. 2). To determine the molecular mechanism underlying JNK mediated Cx43 regulation in myocytes. 3). To establish the temporal correlation between JNK activation and cardiac remodeling in vivo at molecular, cellular and whole heart levels using a newly established inducible transgenic model. 4). To determine the physiological basis and functional significance of JNK induced pathological remodeling in the development of heart failure and premature death. The proposed study may provide better understanding to stress-mediated signaling mechanisms in the patholgenic process of heart failure and leads to potential new therapeutic avenues for the disease.

Principal Investigator/Program Director (Last, First. Middle): Ping, Peipei (Wang, Project 3) PROJECT 3: ROLE OF P38a MARK AND PP2CK IN ISCHEMIC INJURY AND PROTECTION UCLA Project Leader: Yibin Wang, Ph.D. Co-Project Leader: Enrico Stefani, M.D., Ph.D. PHS 398 (Rev. 05/01) Page 181 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Wang, Project 3) Project 3 will address the central theme of ischemic injury and protection from a perspective of cardiac remodeling and heart failure. An important deleterious consequence of myocardial infarction is the induction of pathological remodeling, which recent reports shown is characterized by mitochondrial dysfunction, including altered energy metabolism and apoptotic cell death. The intracellular signaling events that mediate stress-induced pathological remodeling and myocyte apoptosis involve intricate regulation by protein kinases and phosphatases. In this regard, ischemic injury potently induces a highly conserved Ser/Thr protein kinase, p38 MARK, that regulates mitochondrial energy metabolism and apoptosis. In our previous studies, targeted induction of p38 activity in the heart was sufficient to induce pathological remodeling and heart failure, whereas genetic inactivation of the p38a isoform significantly protects the heart against ischemic injury. Despite significant progress in this field, very little is known regarding stress signaling at mitochondria. To this end, the discovery of a novel protein phosphatase-2C isoform (PP2Cic) by Project 3 represents important new insight into signaling at this organelle. Preliminary data indicate that PP2dc is highly expressed in the heart with targeted localization to the mitochondria. Moreover, the findings demonstrate that PP2CK is down-regulated in the failing heart and indicate that overexpression of PP2CK protects cardiac cells against oxidative stress-induced injury. These exciting findings led to the central hypothesis of Project 3: The stress-activated protein kinase p38a MAPK and the mitochondrial protein phosphatase PP2CK are two important signaling components in ischemia-reperfusion injury and they contribute to the genesis of cardiac phenotype by modulating mitochondria function and myocyte apoptosis during ischemic injury. In collaboration with Projects 1, 2, and 4, the Heart Biology Core and the Proteomic Core, Project 3 will undertake a comprehensive analysis of the role of individual p38 isoforms and PP2dc in ischemic injury. Four specific aims are proposed: In collaboration with Project 1 and the Heart Biology Core, Aim 1 will investigate the role of p38 MAPKs in regulating mitochondrial function and modulating susceptibility to MPT. In collaboration with Project 2 and the Heart Biology Core, Aim 2 will investigate the in vivo role of individual p38 MAP kinase isoforms in modulating susceptibility to MPT in ischemia/reperfusion injury. In collaboration with the Proteomic Core, Aim 3 will utilize a functional proteomic approach to identify p38a- associated proteins in the heart to elucidate the subproteome of molecules involved in p38a signaling during ischemic injury and protection. Finally, in collaboration with Projects 1 and 4, Aim 4 will fully characterize the newly- discovered mitochondria-specific phosphatase, PP2dc, and explore its role in modulation of stress signaling and cardioprotection. The proposed studies will provide novel insights into the role of stress proteins in regulation of mitochondrial dysfunction during ischemic injury and can lead to new therapeutic approaches to heart failure.

DESCRIPTION (provided by applicant): Heart failure is one (1) of the most significant health issues in the US with ischemia/myocardial infarct as one (1) of its leading causes. Pro-inflammatory genes, including TNFalpha, IL-6 and COX2 are highly induced and thought to contribute to pathological decompensation in heart failure following ischemia/reperfusion injury. However, the signaling mechanism mediating their induction in cardiomyocytes is not yet well established. In our preliminary studies, we find that activation of stress-activated mitogen activated protein (MAP) kinase, p38 is highly correlated with ischemia/reperfusion. Targeted activation of p38 activities leads to induction of pro-inflammatory genes in cardiomyocytes and pathological remodeling in intact heart. These findings lead to our current hypothesis that p38 MAP kinase activation contributes to specific aspects of cardiac pathology during ischemia/myocardial infarction via targeted regulation of stress-response genes and inflammatory cytokine in cardiomyocytes. In this proposal, we will rigorously test this hypothesis by achieving the following 3 specific aims: 1) To determine the functional role of p38 in regulating inflammatory gene induction and cardiac remodeling in vivo. 2).To determine the physiological role of p38 pathway in inflammatory gene induction and cardiac injury following myocardial infarction. 3) To determine the molecular basis of p38 function and regulation in cardiomyocytes. Accomplishing these aims will establish the functional significance of p38 pathway in ischemic heart failure and add important new insight to the molecular mechanisms of pathological remodeling in failing heart. More importantly, it will provide critical information about p38 pathway as a potential new therapeutic target to treat myocardial infarction and ischemic heart failure.

DESCRIPTION (provided by applicant): ER stress, also known as unfolded protein response (UPR) is a critical signaling mechanism that has been implicated in neuron degeneration, cancer, diabetics and other diseases. Recent evidence suggests that UPR is also induced in cardiac myocytes following hemodynamic overload and ischemia/reperfusion insults. Activation of UPR can provide cellular protection against cytoxicity from protein aggregate as in the cases of amyloidosis, or oxidative and ischemic injury. However, prolonged stimulation of UPR can also trigger apoptosis. Therefore, calibrated regulation of UPR may have significant implication in cardiac protection and injury. IRE1 (a or b isoform) is an ER membrane targeted ser/thr protein kinase with specific RNase activity that is critical to UPR as well as ER stress induced JNK activation and cell death, and is essential for normal embryonic development. IRE1 activity is induced during UPR via dimerization and ser/thr trans-phosphorylation. However, the molecular mechanism involved in its dephosphorylation is unknown. Through genome mining, we found a novel Ser/Thr protein phosphatase (PP2Ce) that is highly enriched in brain and heart, and is exclusively targeted on ER membrane and possesses a remarkable selectivity to dephosphorylate IRE1 in vitro and in vivo. Preliminary studies demonstrate that PP2Ce inhibits IRE1 phosphorylation and negatively modulate IRE1 mediated ER stress signaling. This exciting finding leads to our current hypothesis that this novel PP2C isoform is the endogenous IRE1 specific protein phosphatase and has an important role in regulating UPR in heart under pathological conditions. To rigorously test this hypothesis, we propose to accomplish the following three specific aims: Aim 1. we will determine the role of PP2Ce in regulating UPR in cardiomyocytes in culture. Aim 2, we will explore the molecular mechanisms of PP2Ce mediated regulation of IRE1 activity. Aim 3, we will determine the functional significance of PP2Ce mediated regulation in embryonic development in zebrafish. Aim4, we will use cardiac specific and inducible PP2Ce over-expressor and PP2Ce knockout mouse models to determine the impact of PP2Ce activity on cardiac function and ischemia reperfusion injury in intact animals. From these studies, we will establish the functional role and molecular mechanisms of a novel ER stress signaling component heart and shed new insights to the underlying mechanisms of heart failure.

DESCRIPTION (provided by applicant): Heart failure is a prevailing disease without effective treatment and represents a significant unmet medical need in the US. In response to pathological stresses, heart undergoes profound remodeling at molecular, cellular and organ levels. In previous studies, we and others have established that a stress-activated protein kinase, p38 plays a significant role in mediating pathological changes in heart under stress. We have demonstrated that constitutive activation of p38 in hearts leads to loss of contractility and pathological remodeling associated with pro- inflammatory cytokine induction. However, we have also observed that genetic inactivation of p38 leads to impaired survival and dysfunction under chronic stress or ageing. This paradox indicates complex roles for p38 mediated signaling in both deleterious and protective mechanisms in heart which has significant implications in the development of p38 targeted therapy for heart failure. To better understand the underlying mechanisms of p38 mediated signaling in heart, we have performed extensive studies at molecular, cellular and functional levels about p38 signaling complex and p38 mediated function. In particular, we have established that the auto-phosphorylation induced non-canonical p38 pathway is regulated by a novel interacting partner, Hsp90/Cdc37 complex. We have also discovered that p38 activity is critical to compensatory vascular remodeling in heart via paracrine cross-talk from cardiomyocytes to endothelial cells. Finally, we have demonstrated that a well established p38 downstream kinase MK2 has a selective contribution to p38 induced pathological changes in heart. These novel findings lead to our current hypothesis that diverse mechanisms in p38 activation and downstream targets contribute to specific roles of p38 signaling in both compensatory and pathological remodeling in heart. In the current proposal, we plan to advance our current knowledge of p38 mediated stress signaling by accomplishing the following specific aims: 1). Determine the molecular mechanism and the functional significance of non-canonical p38 kinase activation in heart. 2). Characterize the mechanisms underlying p38 mediated regulation of cardiomyocyte and endothelium cross-talk during pathological remodeling of heart. 3). Uncover the functional significance of downstream kinase MK2 in p38 mediated stress-response in heart. These studies will significantly advance our current knowledge in the disease mechanisms of heart failure and help to develop more effective therapy for the disease. PUBLIC HEALTH RELEVANCE: Stress kinase p38 is an important signaling pathway in stress-response. Our proposal will investigate the molecular mechanisms and functional role of a novel non-canonical activation pathway of p38 and its impact on crosstalk from myocyte to endothelial cells. These studies will improve our current understanding to the disease mechanisms of heart failure and provide potential insights for development of better treatment.

DESCRIPTION (provided by applicant): Oxidative stress has been increasingly recognized as a common feature among different forms of heart disease. Elevated reactive oxygen species (ROS) has been shown as a convergent signaling messenger leading to failing heart either of ischemic or non-ischemic origin. Despite significant progress in many areas of ROS related investigations, two fundamental issues remain unresolved and will constitute the center of this proposed investigation. The first question is who are the regulators of ROS in the diseased myocardium? The second question is what are the molecular targets of ROS and how do ROS-induced molecular modifications result in cardiac dysfunction? This application is inspired by our exciting data identifying PP2Cm as a novel regulator of ROS; and by the intriguing preliminary evidence that the cardiac proteasome complexes are a new class of molecular targets for ROS. Accordingly, the proposed investigation will address a novel aspect of ROS signaling: its modulation by PP2Cm, and it will embark on a largely unexplored area of research: the functional consequences of ROS elevation--its impact on the proteasome systems and their substrates. The application will determine the emerging role of PP2Cm in ROS biology; it will establish proteasome subunits as a new set of molecular targets for the elevated ROS; and it will systematically characterize perturbed protein degradation pathways in the normal and stressed myocardium. Furthermore, the application will identify potential therapeutic windows whereby disrupted protein quality control may be rescued. To accomplish our goals, two related models of cardiac stress--pressure overload by transverse-aortic constriction (TAC) and myocardial ischemic injury (I/R)-are employed. Three specific aims are proposed: Aim 1 will elucidate mechanisms underlying PP2Cm mediated protection of the heart; it will determine its role in governing ROS regulation and examine the impact of genetic perturbations of PP2Cm in TAC and I/R using the newly established PP2Cm genetic models in-house (the null/LacZ knock-in KO and the cardiac conditional inducible Tet-Off). Aim 2 will establish roles of the 20S and 26S proteasomes in the two stress models with respect to proteasome complex assembly, function, and degradation capacity; it will decipher molecular events underlying ROS damaged 20S and 26S proteasomes. It will apply a targeted proteomic approach to delineate the molecular modification of proteasome subunits; and it will define the functional significance of such modifications. Aim 3 will define functional consequences of ROS-injured 20S and 26S proteasomes in the two pathological models; it will characterize the substrate repertoire of 20S and 26S proteasomes in the normal and stressed myocardium. Our research plan is supported by a technology tool box combining established methods and innovative approaches assembled by the investigator team. It encompasses genetic models, proteasome biology, ROS biology, disease models, quantitative proteomics, and high-resolution imaging. Collectively, the proposed studies will conclusively characterize PP2Cm regulation of ROS in the stress myocardium; it will establish proteasome subunits as novel targets of ROS; and it will provide mechanistic insights into protein homeostasis in the two stress models.

DESCRIPTION (provided by applicant): The pathogenesis of heart failure is complex, involving heterogeneous genetic and environmental factors. Genome-wide association studies (GWAS) have had limited success for heart failure and the genetic factors contributing to human heart failure remain poorly understood. A major challenge in the field is to fully understand the heterogeneity of the disease in order to develop more effective and personalized therapies. In this proposal, we have developed a novel approach by using GWAS across a hybrid mouse diversity panel (HMDP) under a well defined pathological stressor to systematically identify genetic factors and molecular networks implicated in heart failure. We believe this novel approach has several major advantages. First, the HMDP consists of ~100 common inbred and recombinant inbred (RI) strains which have been either entirely sequenced or densely genotyped [over 140,000 single nucleotide polymorphisms (SNPs)]. Second, the insights learnt from mouse models of heart failure should provide relevant guidance for future mechanistic, epidemiological and genetic studies in human. Lastly, Chronic excessive adrenergic overdrive is a well recognized major contributor to human heart failure and understanding the genetic modulators to betaAR signaling would have a major impact. With precisely administered chronic treatment of isoproterenol, a non-selective betaAR agonist, we will be able to quantitatively inflict a pathological insult in a relatively high-throughput manner. Accordingly, we propose to 1). Identify genetic loci in mouse contributing to cardiac responses to chronic beta-adrenergic stimulation by quantitative analysis of cardiac function and remodeling in the HMDP mice in response to chronic isoproterenol stimulation, and association analysis with an efficient mixed model algorithm to identify regions of the genome and potential candidate genes linking to the different features of cardiac response to chronic beta-adrenergic stimulation. 2). Model pathways contributing to regulation of heart function and hypertrophy by global expression array analyses of hearts before and after isoproterenol treatment to map loci contributing to differences in gene expression that are associated with chronic beta-adrenergic response, and construction co-expression networks to identify subnetworks associated with chronic beta-adrenergic response. In short, the systems approach designed in this proposal will bring novel insights to genes and their interacting networks implicated in betaAR signaling and heart failure.

DESCRIPTION (provided by applicant): Heart failure is one of the most important diseases in the US and the world. Loss of contractility and blunted response to adrenergic stimulation are common pathophysiological features of a failing heart. Cardiac SR calcium cycling is a highly regulated process and its abnormalities play a major role in heart failure. Recently, our laboratory has identified a novel isoform of protein phosphatase 2C (PP2Ce) which has the following interesting features. PP2Ce is highly expressed in heart and the protein is targeted specifically on SR membrane of cardiomyocytes. PP2Ce has specific activity towards p-PLN without significant impact on p-RyR2. PP2Ce protein has a rapid turn- over rate and its expression is significantly induced by prolonged 2AR stimulation at post-transcriptional level. PP2Ce expression suppresses 2AR mediated induction in calcium transients and contractility, and promotes failure following ischemia/reperfusion injury. PP2Ce inactivation sustains 2AR induced contractility, protects against I/R injury and attenuates pressure-overload induced hypertrophy and heart failure. These findings lead to our exciting new hypothesis that PP2Ce is a novel phosphatase of PLN with a significant contribution to 2AR signaling and functional regulation in stressed hearts. In this proposal, we aim to uncover the regulatory mechanisms of PP2Ce expression and the functional significance of PP2Ce mediated signaling. Specifically, we plan to accomplish the following three aims: Specific aim 1: To investigate the molecular basis and cellular impact of PP2Ce-mediated PLN dephosphorylation. We will determine the interaction between PP2Ce and PLN, and the impact of PP2Ce expression/inactivation on SR calcium homeostasis. Specific aim 2: To investigate the regulatory mechanism of PP2Ce expression. We will dissect the contributing factors in PP2Ce protein expression, PLN targeting and 2AR mediated regulation of its turn-over. Specific aim 3: To determine the functional role of PP2Ce activity in intact heart. We will determine the functional impact of PP2Ce expression and inactivation in response to I/R injury and pressure-overload. In addition, we will determine functional significance of PLN in PP2Ce mediated cardiac protection and pathological remodeling.

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.

DESCRIPTION (provided by applicant): Transcriptome reprogramming is a key process of pathological remodeling in heart. In mammalian transcriptome, a significant portion of the genes produce more than one transcript species due to alternative RNA splicing. However, the contribution of alternative RNA splicing to total transcriptome complexity and its regulatory mechanism in heart failure is still poorly understood and understudied. Based on RNA sequencing and extensive validation studies, we have discovered that global pattern of alternative RNA splicing adapts a fetal-like profile in diseased hearts, including a highly conserved mutually exclusive splicing for all members of the transcriptional factor Mef2 family, Mef2a, Mef2c and Mef2d. We further find that Fox1 is a muscle enriched trans-acting RNA splicing factor that regulates this specific Mef2 splicing event in heart, producing splicing variants with distinct transcriptional activities and different functional impact in heart. Fox1 expression is diminished in mouse and human failing hearts. Inactivation of Fox1 in zebrafish causes developmental defects and cardiac dysfunction. Most remarkably, restoring Fox1 expression in mice significantly attenuates cardiac hypertrophy and dysfunction induced by pressure-overload. Therefore, alternative RNA splicing is a highly regulated process that significantly contributes to the transcriptome programming in heart. It has an important and previously underappreciated impact on cardiac development and pathogenesis. These exciting new findings lead to our current hypothesis that Fox1-MEF2 is a novel regulatory circuit in cardiac transcriptional network with a pivotal role in heart failure. In this proposal, we will expore this novel hypothesis at molecular and functional levels in multiple model systems in order to fully establish the underling mechanism and the functional importance of Fox-1 mediated RNA splicing regulation in heart failure. More specifically, we plan to accomplish in Aim 1: to determine the specific contribution of Fox-1 to transcriptome complexity in cardiomyocytes; in Aim 2: to investigate the molecular basis and functional impact of Mef2a splicing variants in heart; in Aim 3: to establish the functional impact of Fox-1-Mef2 circuit in cardiac hypertrophy and heart failure. These studies will provide exciting new insights to cardiac transcriptome regulation in normal development and diseases, and promising new targets for therapeutic development.

DESCRIPTION (provided by applicant): Congestive heart failure is a complex disease involving multiple genetic and environmental factors. Three years ago, the laboratories of Dr. Yibin Wang, a molecular biologist with expertise in heart failure, and Dr. Aldons Lusis, a geneticist working in the area of cardiovascular disease, joined forces to perform a genetic screen in a hybrid mouse diversity panel (HMDP) to identify genes contributing to common forms of heart failure. For the past two years, this work has been supported by a multi- PI R21. This support enabled us to complete the preliminary screen and identified over 30 genome-wide significant loci harboring genes contributing to different aspects of cardiac pathologies induced by chronic stimulation of the ?-adrenergic agonist isoproterenol (ISO), including hypertrophy, fibrosis, and cardiac dysfunction and remodeling. Moreover, gene expression profiles were obtained from all HMDP mouse hearts in control mice and following ISO treatment. These rich datasets containing genetic information detailed cardiac phenotype parameters and comprehensive cardiac transcriptome profiles from 107 inbred strains of mice will allow us to harness the power of genetics and systems approaches to identify novel molecular pathways contributing to the specific aspects of cardiac pathology during heart failure. Indeed, we observed a dramatic diversity of heart failure phenotypes among all HMDP strains following ISO stimulation and discovered a number of genetic loci and gene modules with significant association with cardiac hypertrophy and fibrosis. These data support the overall hypothesis that common genetic variants have a major contribution to the pathogenesis of heart failure. Uncovering the mechanistic basis of these newly discovered HF associated genes and their interactions via systems approach is the overarching goal of this proposal. Specifically, in Aim 1, we will extend our systems studies to discover genes and gene modules significantly associated with cardiac pathology induced by chronic angiotensin II treatment (AngII). We will identify unique and common genes involved in ?AR vs. ?AR-specific pathogenesis in heart. In Aim 2, we will investigate the molecular mechanisms underlying a candidate gene associated with heart failure, Miat, that encodes a long-non-coding (lnc)RNA with a previously unknown function in heart. In Aim 3, we will investigate the mechanism and functional role of Abcc6, a GWAS candidate gene, in stress induced cardiac fibrosis. These studies will reveal the underlying genetic contributions to specific features of heart failure, and the uncovered novel pathology associated genes and their interaction should provide new insights to the mechanism of the disease.

? DESCRIPTION (provided by applicant): For personalized genomic medicine to be a reality, two things must happen: First, we need to directly establish the role of genetic variation in disease incidence and progression, rather than studying genetic associations and molecular mechanisms in isolation from each other. Second, we must investigate the networks of biological molecules responsible for complex diseases like cardiovascular disease as emergent molecular phenotypes, while at the same time using targeted strategies to establish causal relationships. Together, these innovations can lead to an understanding of how genetic variability combines with environmental stimuli to influence disease susceptibility. The goal of this multi-PI grant is to advance the field toward genomic medicine for common forms of heart failure. It is well established that global changes in gene expression accompany the transition through cardiac hypertrophy and on to failure in animals and humans, causing cellular remodeling and deterioration of cardiac function. We reason that cues from the primary DNA sequence, modification of DNA (i.e. methylation) and chromatin-associated proteins (e.g. CTCF) and noncoding RNA (e.g. C5) combine to specify genomic structure and thereby gene expression. In this model, genomic conformation determines the range of phenotypic possibilities in an individual subjected to pathological stimuli by favoring some gene/protein expression profiles and disfavoring others. Our overall hypothesis is that epigenomic features, including DNA methylation and chromatin accessibility, set the baseline plasticity of chromatin structure and are influenced by genetics and environmental stimuli, such that some individuals are more susceptible than others to heart failure. We reason that global regulators of chromatin accessibility, including the novel epigenetic modifier C5 and the structural protein CTCF, play central roles in disease associated gene reprogramming. In the first aim, we will identify how transcription is regulated by the local chromatin landscape at genes, by dissecting the genetic contribution to DNA methylation (BS-seq) and chromatin accessibility (FAIRE- seq) in the normal and stressed heart. In the second aim, we will determine how intermediate chromatin domains are regulated in heart failure by exploring the role of a cardiac-specific lncRNA C5 to target deposition of heterochromatic histone modifications. In the third aim, we will identify the mechanisms of global chromatin conformation in cardiac health and disease by determining the involvement of the master genome architectural protein CTCF in cardiac phenotype. The long-term goals of these studies are to determine the mechanisms for how genetic variation controls differential disease susceptibility and to investigate epigenomic features as both biomarkers for cardiac pathology and causal components of cellular dysfunction.

Abstract Transcriptome reprogramming is central to cardiac hypertrophy and pathological remodeling. Recent advances in genomics have dramatically expanded the scope and function of the cardiac transcriptome to cover much beyond the coding genes to include many non-coding genes. In particular, a vast cohort of long non-coding RNAs (so called lncRNAs) have been identified in cardiac transcriptome, yet, much of their functions remain unexplored. In preliminary studies leading to this proposal, we discovered a novel cardiac-enriched lncRNA expressed in mouse, rat and human heart which is demonstrated to be essential to pressure overload-induced cardiac hypertrophy. Furthermore, we demonstrated that this lncRNA interacts specifically with the chromatin modifying complex PRC2 to modulate H3K27me2/3 levels on pathological genes in stressed heart, thus we named this lncRNA as Cardiac Hypertrophy Associated Epigenetic Regulator (Chaer). Most intriguingly, we found Chaer transiently interacts with PRC2 transient at the onset of hypertrophic stimulation in an mTOR dependent manner. This transient interaction appears to be important to the early onset but not the subsequent progression of cardiac hypertrophy and heart failure. Therefore, the Chaer-PRC2 interaction appears to be an early epigenetic check-point necessary for hypertrophic gene expression and remodeling in the heart. This discovery highlights two potentially very important roles for lncRNAs in transcription regulation: one as a molecular switch to link epigenetic modifiers with cellular stress signals, and another as a molecular chaperon to orchestrate target specificity in the context of specific tissues for ubiquitous epigenetic modifiers. To further establish this novel concept, we propose to vigorously investigate the mechanism of Chaer mediated cardiac gene regulation by achieving the following three specific aims: Aim 1. To uncover the molecular basis for hypertrophic signal regulated interaction between Chaer and PRC2. Aim 2. To investigate how Chaer interaction regulates Ezh2 function in response to hypertrophic stimulation. Aim 3. To establish the functional impact of Chaer-PRC2 interaction on cardiac epigenetic modulation and transcriptome reprogramming during cardiac hypertrophy. Accomplishing these aims will fill a major gap in our current knowledge of epigenetic regulation in cardiac hypertrophy and advance our mechanistic understanding of inducible, locus-specific chromatin modification in heart cells.

Abstract Metabolic remodeling is an integral part of pathogenic process of heart failure. From an unbiased transcriptome analysis focusing on known metabolic pathways, we unexpectedly found that branched chain amino acids (BCAA) catabolic pathway is one of the most significantly affected in mouse failure hearts. Subsequently, we revealed that BCAA catabolic defect and the resulted intra-cardiac accumulation of branched- chain keto acid (BCKA) are common metabolic features in human failing hearts. The detrimental impact of BCKA accumulation on cardiac function is associated with its direct effect on mitochondrial ROS induction and complex I specific inhibition. Most importantly, genetic inhibition of BCAA catabolic activity promoted pressure-overload induced heart failure while restoring BCAA catabolic activity and reducing BCKA accumulation significantly blunted the onset of heart failure. These exciting new findings established, for the first time, a direct and causal role of BCAA catabolic defect in heart failure, and provide proof of concept evidence to treat heart failure by targeting BCAA catabolic activity. These preliminary data lead to our novel hypothesis that stress-induced BCAA catabolic defect results in cardiac accumulation of BCKA which exerts detrimental effect on heart via impairment of mitochondria function and ROS induction (Figure 1). In this proposal, we will investigate the validity of our hypothesis via vigorous in vivo and in vitro examination, and establish the therapeutic potential of restoring BCAA catabolic activity for heart failure. Specifically, we will accomplish the following three specific aims: Aim 1. To determine cell-autonomous contribution of BCAA catabolic defect in cardiomyocyte to the pathogenesis of heart failure: Using novel mouse model, we will genetically impair BCAA catabolic activity specifically in adult cardiomyocytes and examine the direct impact on cardiac function and pathological remodeling under basal as well as in response to pressure-overload or chronic ISO stimulation. Aim 2. To unravel the cellular and molecular basis of BCKA induced cardiac dysfunction: We will determine both in vitro and in vivo the specific impact of BCKA accumulation on mitochondrial function, the connection between complex I inhibition and ROS induction, and impact of BCKA accumulation on myocyte viability and pathological remodeling. Aim 3 To validate the therapeutic potential of targeting BCKD Kinase for HF therapy. we will test the function impact of restoring BCAA catabolic activity by genetically or pharmacologically inhibiting BCKD kinase on the pathological progression of HF. Together, this project will uncover a novel and important aspect of pathological remodeling in heart failure, fill a significant gap of knowledge in our current understanding of cardiac pathogenesis, and help to identify novel therapeutic target for this major disease.