Jeffery D. Molkentin - US grants

PROJECT SUMMARY (See instructions): The overall emphasis of this extension R37 application is to understand the molecular pathways that control cardiac gene expression and the hypertrophic growth of the myocardium. The focus of this proposal is to analyze the GATA4, 5, and 6 transcription factor family as regulators of both embryonic heart development and during pathological and physiological growth of the adult heart. GATA factors are known to directly regulate the expression of most cardiac-expressed structural genes, thus facilitating the differentiation of , cardiomyocytes during early heart development. In the adult heart, GATA4 and GATA6 transcription factors are re-employed where they function as important regulators of the hypertrophic gene program in response to pathophysiologic stimulation. Indeed, the hypertrophic response of the adult heart involves re-expression of many fetal genes, suggesting that the developmental and disease gene programs share common regulatory events, potentially through GATA4/5/6. Our unifying hypothesis states that GATA4, 5, and 6 are required for both the establishment and maintenance of the cardiac differentiation-specific gene program as well as the growth response of the adult heart during stress stimulation. Over the past 4 years of this award we published several manuscripts that directly addressed each of the 3 original specific aims, including making cardiac-specific Gata4 null mice, Gata6 heart-specific null mice, and inducible transgenic mice that express either GATA4 or GATA6 in the heart. We also generated mice that had a knock-in mutation in Gata4 at serine 105, changing this amino acid to alanine to prevent its phosphorylation and transcriptional induction with hypertrophic stimulation. Our results were published in the very best journals and they established the role that both GATA4 and GATA6 play in the adult heart as necessary transcriptional regulators of the hypertrophic response. We will continue working on all three specific aims in the coming 5 year extension period, as well as add 2 new specific aims to identify additional mechanisms whereby GATA factors regulate cardiac hypertrophy, and to address redundancy between GATA4 and GATA6 in the heart.

DESCRIPTION (provided by applicant): The heart undergoes hypertrophic enlargement in response to both physiologic and pathophysiologic stimulation. Hypertrophy allows the myocardium to adapt to routine alterations in workload associated with developmental maturation, physiologic challenge, or injury. A finite number of intracellular signaling pathways have been implicated as regulators of the cardiac hypertrophic response including, mitogen-activated protein kinase (MAPK), protein kinase C (PKC), PI3K/Akt, and calcineurin-NFAT. Our previous studies have suggested a critical role for the calcium activated phosphatase calcineurin (PP2B) and its downstream transcriptional effector NFAT (nuclear factor of activated T-cells) in regulating the cardiac hypertrophic response. Calcineurin is an attractive candidate molecule for investigation as a hypertrophic transducer given its unique ability to dynamically respond to alterations in intracellular calcium handling. Indeed, data generated from both gain and loss of function approaches in genetically modified mouse models support the hypothesis that calcineurin functions as an important transducer of the cardiac hypertrophic response. Here we propose to investigate the downstream effectors of calcineurin in the heart and to investigate the role that calcineurin plays in regulating physiologic and/or adaptive growth responses of the myocardium. To accompolish this objective, the following specific aims and approaches are proposed: 1) To define calcineurin's role as a regulator of cardiac mass in response to physiologic stimuli. Specifically, CnBl-loxP targeted mice will undergo inducible Cre-mediated recombination in the heart to examine the hypothesis that calcineurin regulates the steady-state mass of the myocardium in response to physiologic stimuli. NFAT-dependent reporter mice will also be investigated to assess the hypothesis that calcineurin is a long-term regulator of hypertrophic growth. 2) To define the downstream effectors of calcineurin that mediate the hypertrophic growth of the myocardium. Both dominant negative NFAT and MEF2 inducible transgenic mice will be characterized to evaluate the hypothesis that these factors are necessary calcineurin transducers in the heart. 3) To characterize a novel class of NFAT interacting proteins (Nip-1 and Nip-2) in the heart. Yeast 2-hybrid screening has identified a novel class of cardiac-expressed NFAT inhibiting factors that will be further evaluated in vitro and in vivo.

The long term goal of this project is to understand intracellular reactive signaling pathways involved in cardiac hypertrophy. In response to various forms of heart disease the myocardium undergoes hypertrophic growth to compensate for a loss in cardiac output. While this response is initially beneficial, it eventually leads to pathology and heart failure. Despite years of investigator, the molecular pathways that result in cardiac hypertrophy remain largely unknown. The transcription GATA4 has been shown to play an important role in regulating the hypertrophic response at the transcriptional level. To investigate the molecular mechanisms whereby GATA4 regulates the hypertrophic responses, a yeast 2-hybrid screen was performed which identified the transcription factor NF-AT3 (nuclear factor of activated T-cells). Studies in T-cells have demonstrated that NF-AT factors are sequestered in the cytoplasm until activated by the phosphatase calcineurin in response to stress or stimuli that increase intracellular calcium. Our recent results demonstrate that the myocardium also utilizes this stress-response signaling pathway. Transgenic mice expressing a constitutively active form of calcineurin or a constitutively nuclear NF-AT3 protein in the heart develop substantial hypertrophy resulting in heart failure. Furthermore, inhibition of endogenous calcineurin activity with the drug cyclosporin A prevents angiotensin II and adrenergic-mediated hypertrophy of cultured neonatal cardiomyocytes. These data suggest a model whereby calcineurin regulates cardiac hypertrophy by effecting the transcription factor NF- AT3 and its ability to activated hypertrophic response genes. However, the physiologic relevance of this novel signaling pathway is presently uncertain. To this end, the goals of the proposed study are: 1) to define the mechanisms of calcineurin hypertrophic signaling in cultured neonatal cardiomyocytes. 2) To use transgenic models of intrinsic heart disease to determine the role of calcineurin signaling in genetically mediated myopathic responses. 3) To determine the role of calcineurin signaling in models of extrinsic reactive hypertrophy. 4) To determine if calcineurin is activated in human heart failure. Transgenic and pathophysiologic animal models as well as in vitro culture systems are proposed to test the hypothesis that calcineurin/NF-AT act as a parallel regulatory pathway for cardiac reactive responses.

DESCRIPTION (provided by applicant): In recent years, much investigation has centered on identifying and characterizing the intracellular signal transduction pathways that control cardiac hypertrophy and heart failure. Numerous intermediate signaling pathways have been implicated as important regulators of cardiac hypertrophy including mitogen-activated protein kinase (MAPK), calcineurin-NFAT, PKC, and many others. However, the role that MAPK signaling plays in mediating cardiac hypertrophy is currently an area of considerable debate. Currently, there is little consensus in the literature as to which of the 3 MAPK signaling branches (ERKs, p38 kinases, or JNKs) are important hypertrophic regulators, and almost nothing has been extended to the heart in vivo. Accordingly, we propose, in vivo, an in depth analysis of the ERK MAPK signaling pathway to characterize its role as a cardiac hypertrophic mediator and its role in cardiac apoptosis. Preliminary studies suggest that the ERK MAPK pathway, but not JNK or p38 MAPK can induce cardiac hypertrophy in vivo and promote protection from apoptotic stimuli. The ERK MAPK signaling pathway induces cardiac hypertrophy, in part, by directly acting on the zinc-finger-containing transcription factor GATA4. Given these data, we propose the hypothesis that an ERK-GATA4 signaling pathway is necessary for mediating cardiac hypertrophy in vitro and in vivo. To test this, the hypertrophic potential of MEK1 dominant negative mice and ERK1/2 knock-out mice will be characterized in vivo. The role that the ERK MAPK pathway plays in mediating cardioprotection (anti-apoptosis) will also be analyzed in response to ischemia/reperfusion. Lastly, the necessity of GATA4 as a downstream hypertrophic transducer of ERK MAPK signaling will be evaluated both in vitro and in vivo. These approaches will suggest the extent to which ERK MAPK and GATA4 are necessary in the development of cardiac hypertrophy in response to pathophysiologic stimuli.

[unreadable] DESCRIPTION (provided by applicant): Mitochondria comprise approximately 30% of the total intracellular volume of a mammalian cardiomyocyte. Not surprisingly, subtle alterations in mitochondrial function or membrane potential can have a dramatic influence on cardiomyocyte energy production and ultimately, the life or death individual cell. Indeed, cellular injury or stress stimulation directly elicit alterations in mitochondrial architecture, membrane potential, and oxidative capacity, which can be associated with ah irreversible loss of mitochondrial matrix contents and integral membrane protein constituents such as cytochrome C oxidase, followed soon thereafter by activation of intracellular proteases and DNA fragmentation enzymes associated with apoptosis and necrosis. An emerging paradigm places mitochondrial permeability transition pore (MPTP) formation as a central event precipitating cardiac myocyte apoptosis or necrosis following ischemic injury and during progressive heart failure. This project will test the hypothesis that MPTP is a primary mechanism responsible for driving myocardial cell death following ischemia- reperfusion injury or in response to long-standing cardiomyopathy. Both gain- and loss-of-function approaches will be implemented in the mouse as a means of dissecting the molecular determinants of MPTP and potential therapeutic opportunities. Specific aim 1 will define the mechanism of MPTP formation and its functional consequence in regulating cardiac myocyte apoptosis. Specific aim 2 will determine if inducible MPTP formation regulates cardiac myocyte apoptosis in vivo, while Specific aim 3 will target VDAC1/3 and cyclophilin D as a means of blocking MPTP formation in the heart. Even though inhibition of MPTP formation with pharmacologic agents often prevents cell death following catastrophic stimuli in diverse cell-types, the necessity of MPTP formation in mediating cardiomyocyte cell death following ischemic injury or long-standing cardiomyopathy has not been elucidated. Moreover, the identity and key functions of the putative MPTP components have not been subjected to genetic gain- or loss-of-function analysis in vivo, leaving the true identity of the complex unresolved. A greater understanding of the key constituents that comprise the MPTP, as well as further characterizing its functional dominance in the heart, will likely suggest novel approaches for treating human heart disease associated with cell death. [unreadable] [unreadable] [unreadable]

Given the relatively poor prognosis associated with end-stage heart failure, investigators and clinicians have long searched for therapies that alleviate or reverse the progressive loss of pump function that typifies a failing heart. The predominant strategy employed over the past two decades has been based on pharmacologic manipulation of cardiac contractility, aimed at agonizing or antagonizing beta-adrenergic receptor activity or the downstream accumulation of cAMP. Here we have identified a novel signaling pathway that utilizes PKCa as a feedback regulator to dampen cardiac contractility, presumably in response to periods of enhanced inotropic drive. Indeed, loss of PKCalpha through gene targeting revealed a hypercontractile phenotype, while PKCalpha overexpression in transgenic mice or acutely in isolated adult rat myocytes showed reduced contractility, supporting our first hypothesis that PKCalpha functions within a negative contractility feedback pathway in the heart. Augmentation in cardiac function observed in the absence of PKCalpha rescued a model of pressure overload-induced heart failure, supporting our second hypothesis that selective enhancement of cardiac contractility can benefit a failing heart. To examine these two hypotheses we will: 1) define the biochemical mechanism whereby PKCalpha regulates cardiac contractility, 2) investigate the ability of inducible gain- or loss-of-function for PKCalpha to alter the progression towards heart failure in two distinct mouse models of disease, 3) examine the ability of acute pharmacologic inhibition of PKCa to potentially benefit a large animal model of heart failure. Collectively, the proposed course of investigation will span a biochemical and mechanistic evaluation of a PKCalpha function in vitro through a physiologic assessment of PKCalpha in animal models of heart failure suggesting disease ramifications in humans. Thus, this component will address the central theme of the SCCOR proposal pertaining to signaling pathways and cardiac contractile responsiveness, through investigation of single gene-function correlations.

21+ years old; Address; Adult; Area; Autocrine Systems; Binding; Binding (Molecular Function); CSBP1; CSBP2; CSPB1; Calcineurin; Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Cardiac; Cardiac Diseases; Cardiac Disorders; Cardiac Myocytes; Cardiac Output; Cardiocyte; Cardiomyopathy, Dilated; Cell Communication and Signaling; Cell Nucleus; Cell Signaling; Cell-Extracellular Matrix; Cellular Expansion; Cellular Growth; Collaborations; Complementary DNA; Congestive Cardiomyopathy; Cytoplasm; DNA, Complementary; Death, Sudden; Dephosphorylation; Dilated Cardiomyopathy; EC 2.7; EC 2.7.2-; ECM; ERK 2; ERK MAP Kinases; EXIP; Endocrine; Event; Extracellular Matrix; Extracellular Signal Regulated Kinases; Extracellular Signal-Regulated Kinase 2; Extracellular Signal-Regulated Kinases; Extracellular Signal-Regulated MAP Kinases; Family; GATA-4 protein; GATA-binding protein 4; GATA4 protein; GATA4 transcription factor; Gene Expression; Gene Targeting; Generalized Growth; Generations; Genes; Growth; HTRPY; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Heart myocyte; Human; Human, Adult; Human, General; Hypertrophy; In Vitro; Individual; Injury; Intermediary Metabolism; Intracellular Communication and Signaling; JN Kinase; JNK; JNK Mitogen-Activated Protein Kinases; JNK1; JNK1 Kinase; JNK1 protein; JNK1A2; JNK21B1/2; Kinases; Literature; MAP Kinase 1; MAP Kinase 2; MAP Kinase 8; MAP Kinase 8 Gene; MAP kinase; MAPK; MAPK1; MAPK1 Mitogen-Activated Protein Kinase; MAPK14; MAPK14 gene; MAPK2 Mitogen-Activated Protein Kinase; MAPK8; MAPK8 Mitogen-Activated Protein Kinase; MAPK8 gene; METBL; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Membrane; Metabolic Processes; Metabolism; Mice; Mice, Transgenic; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 2; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Molecular; Molecular Interaction; Murine; Mus; Muscle Cells, Cardiac; Muscle Cells, Heart; Muscle, Cardiac; Muscle, Heart; Mxi2; Myocardium; Myocytes, Cardiac; Neonatal; Nucleus; P42MAPK; PKC; PP2B; PRKM1; PRKM14; PRKM15; PRKM8; PTK Receptors; Pathologic; Phosphatases; Phosphohydrolases; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Phosphomonoesterases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiologic; Physiological; Physiology; Play; Process; Programs (PT); Programs [Publication Type]; Protein Dephosphorylation; Protein Kinase C; Protein Phosphatase-2B; Protein Phosphorylation; Proteins; RNA Stability; RTK; Reagent; Receptor Protein-Tyrosine Kinases; Regulatory Protein; Reporting; Role; SAP Kinase-1; SAPK/JNK; SAPK1; SAPK1 Mitogen-Activated Protein Kinase; SAPK1/JNK; SAPK2A; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signal Transduction Systems; Signaling; Specificity; Stimulus; Stress; Stress-Activated Protein Kinase JNK1; Stress-Activated Protein Kinase gamma; Sudden Death; Surface; Targetings, Gene; Testing; Threonine/Tyrosine Protein Kinase; Tissue Growth; Transducers; Transgenic Mice; Transmembrane Receptor Protein Tyrosine Kinase; Transphosphorylases; Tyrosine Kinase Growth Factor Receptor; Tyrosine Kinase Linked Receptors; Tyrosine Kinase Receptors; Zinc Finger Domain; Zinc Finger Motifs; Zinc Fingers; adult human (21+); autocrine; biological adaptation to stress; biological signal transduction; c-jun Amino-Terminal Kinase; c-jun Kinase-1; c-jun N-Terminal Kinase; c-jun N-Terminal Kinase 1; cDNA; cardiac failure; cardiac hypertrophy; cardiac muscle; cardiomyocyte; cell growth; extracellular signal related kinase; gene product; genetic regulatory protein; heart disorder; heart muscle; heart output; in vivo; jun-NH2-Terminal Kinase; loss of function; membrane structure; novel; ontogeny; p38; p38 MAPK Gene; p38Alpha; p42 MAP Kinase; p42 MAPK; p42(Mapk) Kinase; p42(Mitogen-Activated Protein Kinase); paracrine; programs; reaction; crisis; receptor coupling; regulatory gene product; response; social role; stress response; stress-activated protein kinase 1; stress; reaction; therapeutic target; transcription factor

Address; Adult; Area; autocrine; Binding (Molecular Function); Calcineurin; Cardiac; Cardiac Myocytes; Cardiovascular Physiology; cell growth; Cell Nucleus; Collaborations; Complementary DNA; computerized data processing; Cytoplasm; Dilated Cardiomyopathy; Endocrine; Event; Family; GATA4 transcription factor; Gene Expression; Gene Targeting; Generations; Genes; Growth; Heart; Heart Diseases; Heart Hypertrophy; Hypertrophy; In Vitro; in vivo; Individual; Literature; MAP Kinase Gene; MAP2K1 gene; MAPK14 gene; MAPK3 gene; Mediating; Mediator of activation protein; Membrane; Mitogen-Activated Protein Kinases; Mus; Myocardium; N-terminal; Neonatal; novel; paracrine; Pathologic; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; programs; Protein Dephosphorylation; Protein Kinase C; Reagent; receptor coupling; Receptor Protein-Tyrosine Kinases; Reporting; response; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Stimulus; Stress; stress-activated protein kinase 1; Surface; Testing; transcription factor; Transducers; Transgenic Mice; Zinc Fingers

The muscular dystrophies are inherited disorders that largely affect striated muscle tissue resulting in progressive muscle weakness, wasting, and in many instances, premature death. Many characterized mutations in humans that cause muscular dystrophy (MD) result from alterations in structural attachment proteins that affix the underlying contractile proteins to the basal lamina, providing rigidity to the skeletal muscle cell membrane (sarcolemma). Loss of select attachment proteins in the dystrophin-glycoprotein complex (DGC) permits contraction-induced membrane tears and influx of calcium that is thought to cause cellular degeneration and necrosis of muscle fibers. During this necrotic process cytokines, chemokines and growth factors are released as part of the inflammatory and repair process, although induction of fibrosis and scarring are an unwanted side effect that worsens disease. One prominent cytokine is transforming growth factor-B ( TGFB ) that serves a master regulator ofthe fibrotic response and worsening of muscle pathology in MD. While fibroblasts are directly regulated by TGFB, other cytokines and growth factors from the myofiber are hypothesized to be necessary for fibroblast activation and a productive fibrotic response. Thus, here we will test the relatively novel hypothesis that myofibers themselves directly respond to TGFB in promoting the fibrotic response and tissue pathology in MD by generating secondary signals to resident fibroblasts. In two specific aims we will examine both the canonical (SMAD2/3) and non-canonical (TAK1-p38a-JNK) TGFB signaling pathways within myofibers to determine their role in transducing the fibrotic response outward to fibroblasts. Our approach will utilize conditional gene-targeted mice to disrupt Smad2/3 and p38a, as well as skeletal muscle-specific transgenic mice with inhibited JNK1/2 and TGFB receptor signaling. Our preliminary data show that p38a deletion in myofibers, or mice with loss of Jnk1, have reduced fibrosis and MD severity in the 6-sarcoglycan deficient background, suggesting a pathologic linkage with non-canonical TGFB signaling. Moreover, deletion of periostin, which is a TGFB -inducible extracellular matrix (ECM) protein that promotes effective TGFB signaling, similarly reduced MD severity in a mouse model of disease. These results support the overall focus of this project on TGFB signaling through the myofibers in mediating fibrosis and worsening of MD.

DESCRIPTION (provided by applicant): The ER/SR compartment in a cardiomyocyte is highly specialized for controlling calcium fluxing in excitation- contraction coupling (ECC), as well as for regulating protein synthesis and stress responsiveness to unfolded proteins. The traditional ER stress response involves sensing of calcium and unfolded or damaged proteins in the ER through 3 distinct pathways that initiate a cascade of signaling to alter protein synthesis and other features of cellular adaptation to stress. We recently identified thrombospondin 4 (TSP4) as a stress-inducible factor that resides for a period of time in the ER/SR before being secreted to the extracellular matrix (ECM), where it alters the ER stress response. The heart expresses TSP1, TSP2, and TSP4, each of which is dramatically up-regulated following injury or stress stimulation. Interestingly, TSP4 is only expressed in heart and skeletal muscle, and it appears to be of an entirely different functional subclass from TSP1 and TSP2. We have identified a novel function for TSP4 as a cardiac inducible protein that dramatically enhances the content and function of the ER/SR resulting in greater contractility, increased activity of the adaptive ER stress response, and protection from heart failure-inducing stimuli. Thus, we hypothesize that TSP4 is a novel adaptive stress-response factor that benefits ER/SR function to provide cardioprotection. In this project we will: 1) determine if TSP4 protects the heart from failure through adaptive ER stress response pathway engagement, 2) investigate the ER stress response factors that mediate TSP4-dependent cardio-protection, and 3) determine how and where TSP4 signals the adaptive ER stress response. We will use TSP4 transgenic and gene-targeted mice to investigate these 3 specific aims, as well as numerous transgenic models with altered ER stress signaling or protein aggregation-based cardiomyopathy. Extensive in vitro molecular approaches are also proposed to identify the mechanism whereby TSP4 coordinates the protective ER stress response and benefits the heart. Finally, numerous collaborations with the Kranias and Robbins lab's are proposed to determine how TSP4 affects calcium handling and the unfolded proteins response. PUBLIC HEALTH RELEVANCE: The ER stress response appears to be a universal feature of all cardiomyopathies. However, there are very little data that directly examine if the ER stress response is beneficial or detrimental to the heart. We have identified a novel regulator of the ER stress response, TSP4, which appears to only engage the protective ER stress response. Understanding how and why TSP4 does this is of great medical relevance.

The protein kinase C (PKC) family of Ca^* and/or lipid-activated serine-threonine kinases function downstream of most membrane-associated signal transduction pathways, where in the heart they are critically involved in cellular protection, hypertrophy, and regulation of contractility. PKCalpha is the predominant PKC isofomri expressed in the mammalian heart where we and others have shown it to be associated with human heart failure, cardiac ischemia, and other disease stimuli. Previous analysis of PKCalpha KO mice and transgenic overexpressors has shown that this kinase serves as a fundamental regulator of cardiac contractility and Ca^* handling in myocytes, which affects how the heart responded to insults. Specifically, toss of PKCalpha from the heart or its inhibition with a dominant negative mutant protected from heart failure. Moreover, use of drugs with that inhibit PKCalpha also preserved cardiac contractility in vivo and restored ventricular function in mouse and rat models of heart failure. These studies have suggested the hypothesis that inhibition of PKCalpha, such as with the drug ruboxistaurin, can be translated into humans as a novel therapy for heart failure. However, before this is possible we need to better understand the mechanism of action of PKCalpha, as well as conduct translational studies in a pig model of Ml-induced heart failure. Thus, specific Aim #1 will investigate the mechanisms in cells and transgenic mice whereby PKCalpha regulates cardiac contractility and propensity to heart failure after injury, while Specific Aim #2 will involve an elaborate translational approach in a pig Ml model of heart failure using ruboxistaurin treatment, as well as gene therapy with AAV6-dnPKCalpha. If our studies are successful, we believe that ruboxistaurin (or other PKCa inhibitors) would represent an attractive agent to apply to the heart failure clinical setting post-MI, especially given its apparent safety in late phase human clinical trials

The cardiac myocyte has long been the primary focus of most studies attempting to elucidate the signaling mechanisms underlying heart failure. More recently the involvement of nonmyocytes has emerged as potentially just as important as myocytes in contributing to and controlling cardiac remodeling and progressive pathogenesis in heart failure. Specifically, the cardiac fibroblast and its ability to convert to myofibroblasts in promoting the fibrotic response and ventricular remodeling appears to be a highly underappreciated disease process with significant ramifications. Fibroblasts are activated in the heart in response to damage or due to neuroendocrine signaling, such as through Transforming Growth Factor Beta (TGFp). Here we hypothesize that the fibroblast responds to TGFp and other cytokines through select signaling pathways in promoting the fibrotic response and maladaptive remodeling in heart failure. We will examine both canonical (Smad2/3) and non-canonical (TAK1/p38a) TGFp signaling within fibroblasts to determine how these cells and their activation mediate disease in heart failure. All previous in vivo analyses of TGFp signaling and cardiac fibrosis have focused on the myocytes given available genetic tools. However, we have recently engineered a novel fibroblast-specific knock-in mouse iinodel to permit tamoxifen-regulated Cre activity in vivo. We will use this mouse to study fibroblast-based signaling during the development of cardiac disease. Aim #1 will determine the necessary function of canonical TGFp signaling and Smad proteins in mediating cardiac fibrosis within the cardiac fibroblast itself. Aim #2 will exaniine the role that non-canonical TGFp signaling plays through TAKI and p38a MAPK in mediating cardiac fibrosis, andj once again, our focus will be on signaling within the cardiac fibroblast only. Aim #3 will examine a novel pathway that is calcium-TRPC6 activated and works in conjunction with TGFp signaling and other cytokines to pi^omote myofibroblast transdifferentiation in the heart and disease. These 3 specific aims will suggest for the first time the autonomous role for select signaling pathways from within the cardiac fibroblast in mediating myofibroblast transdifferentiation and fibrotic disease in the diseased heart. A number of potential therapeutic angles are suggested from the content of our project and emerging preliminary data. RELEVANCE (See instructions): Our work focuses on the cardiac fibroblast, a cell in the heart that is known to be important in the scarring processes and remodeling that occur after cardiac injury. We intend to study how this cell functions during these disease processes and attempt to modulate its actions so that we can impact favorably on cardiac disease and heart failure.

The cardiac myocyte has long been the primary focus of most studies attempting to elucidate the signaling mechanisms underlying heart failure. More recently the involvement of nonmyocytes has emerged as potentially just as important as myocytes in contributing to and controlling cardiac remodeling and progressive pathogenesis in heart failure. Specifically, the cardiac fibroblast and its ability to convert to myofibroblasts in promoting the fibrotic response and ventricular remodeling appears to be a highly underappreciated disease process with significant ramifications. Fibroblasts are activated in the heart in response to damage or due to neuroendocrine signaling, such as through Transforming Growth Factor Beta (TGFp). Here we hypothesize that the fibroblast responds to TGFp and other cytokines through select signaling pathways in promoting the fibrotic response and maladaptive remodeling in heart failure. We will examine both canonical (Smad2/3) and non-canonical (TAK1/p38a) TGFp signaling within fibroblasts to determine how these cells and their activation mediate disease in heart failure. All previous in vivo analyses of TGFp signaling and cardiac fibrosis have focused on the myocytes given available genetic tools. However, we have recently engineered a novel fibroblast-specific knock-in mouse iinodel to permit tamoxifen-regulated Cre activity in vivo. We will use this mouse to study fibroblast-based signaling during the development of cardiac disease. Aim #1 will determine the necessary function of canonical TGFp signaling and Smad proteins in mediating cardiac fibrosis within the cardiac fibroblast itself. Aim #2 will exaniine the role that non-canonical TGFp signaling plays through TAKI and p38a MAPK in mediating cardiac fibrosis, andj once again, our focus will be on signaling within the cardiac fibroblast only. Aim #3 will examine a novel pathway that is calcium-TRPC6 activated and works in conjunction with TGFp signaling and other cytokines to pi^omote myofibroblast transdifferentiation in the heart and disease. These 3 specific aims will suggest for the first time the autonomous role for select signaling pathways from within the cardiac fibroblast in mediating myofibroblast transdifferentiation and fibrotic disease in the diseased heart. A number of potential therapeutic angles are suggested from the content of our project and emerging preliminary data. RELEVANCE (See instructions): Our work focuses on the cardiac fibroblast, a cell in the heart that is known to be important in the scarring processes and remodeling that occur after cardiac injury. We intend to study how this cell functions during these disease processes and attempt to modulate its actions so that we can impact favorably on cardiac disease and heart failure.

ABSTRACT This is a first renewal application for a pre- and postdoctoral training program, the title and theme of which is ?Understanding Cardiovascular Disease Mechanisms?. The University of Cincinnati and Children?s Hospital has a renowned legacy spanning more than 4 decades of previous NIH T32 support for cardiovascular study, and this current T32 represents the only CV program in Cincinnati. Our collective 18 faculty in this renewal have placed 238 of their past trainees into academics over their careers, 153 of whom have run, or currently run independent research programs. In the past 4 years of this program, our 17 trainees published 43 papers and 5 received independent grant funding during their support period, while 5 graduates have matriculated to jobs in scientific careers. The overall scientific emphasis of our training program will continue to build from a basic platform of cardiovascular physiology, cell biology, biochemistry and pharmacology, but will also incorporate the latest approaches in biomedical research, as well as incorporating clinical and translational approaches. The cardiovascular environment at Cincinnati Children?s and the University of Cincinnati is considered one of the very best in the country. Our 18 TG faculty are all NIH funded (some 47 NIH funding components amongst them as PI status), 165 collaborative papers published together, and they are employing the very latest technologies and approaches with outstanding core support. The leadership consists of the co- PIs Drs. Evangelia Kranias and Jeffery Molkentin, both of whom have a long track record of working together (20 years), as well as having excellent mentorship credentials. The Executive Committee (2 members), Internal Advisory Committee (4 members) and External Advisory Committee (3 members) are highly engaged cardiovascular researchers who will continue to help ensure the quality of the training program. The renewal requests continuation of the funding of 3 pre- and 3 postdoctoral trainee positions. Predocs are selected by the Internal Advisory Committee from a vast and outstanding pool of candidates amongst 7 departmental graduate programs, while postdoctoral candidates are selected based on being accepted into a mentor?s laboratory and then passing the screening process by the Internal Advisory Committee and co-PIs. The mentoring program and evaluation process for the program are highly structured and oversight occurs on many levels. Trainees and mentors are evaluated every 6 months with IDPs processing. The proposed educational training curriculum is highly structured and state-of-the-art. Recruitment of minorities has been successful in the past with our faculty, and it will remain a top priority.

Abstract Ca2+ elevations in the heart can serve as a signal for augmented energy output from the mitochondria by directly increasing the activity of the electron transport chain and associated dehydrogenases. However, at the same time sustained elevations in Ca2+ that occurs acutely after myocardial infarction injury can cause cardiomyocyte necrotic and apoptotic death through opening of the mitochondrial permeability transition pore (PTP). The mitochondrial Ca2+ uniporter (MCU) complex imports Ca2+ across the inner membrane into the mitochondrial matrix where it can affect both energy production and PTP opening during acute ischemic injury. Hence the MCU complex and many of the more recently described genes that constitute it could be novel therapeutic targets for drug design with the goal of reducing cardiomyocyte death or altering cardiac metabolic performance. The genes that comprise the MCU were only recently identified in the past 4 years; hence the field is still in its infancy with respect to genetically correlating mitochondrial Ca2+ regulation with cardiac physiology and pathophysiology in vivo. Here we propose to use mice lacking many of these key molecular regulators of mitochondrial Ca2+ handling to decode and differentiate between physiological and pathological Ca2+ signals at baseline and with disease. Our overarching goal is to examine how mitochondrial Ca2+ influx and efflux regulates cardiac life, death and metabolism. However, no single cardiac laboratory in our field has the underlying expertise to both characterize the complex biophysics of mitochondrial Ca2+ handling and at the same time employ the necessary mouse genetics and cell biology to truly achieve the stated goals of this project. Hence, we have implemented a seamless collaborative dual-PI proposal that will be 50/50 effort between the Molkentin and Bers laboratory, to wed the very best in mouse molecular genetics and cardiac physiology with innovative assessment of mitochondrial and intracellular Ca2+ imagining and PTP activity, respectively. Our Aims will be: 1) to characterize the function of the newly identified MCU complex genes as well as other new genes underlying mitochondrial Ca2+ regulation the heart, 2) To assess MCU gene function in underlying cardiac physiology, metabolism and after ischemic injury, and 3) To assess PTP dynamics and the physiologic versus pathophysiologic states of the PTP in regulating mitochondrial Ca2+ and cell death. The 2 PIs have a strong track record of working together with multiple shared publications and joint grants. Hence, they represent an ideal melding of 2 rather divergent laboratory skill sets that are needed to truly understand mitochondrial Ca2+ regulation and its effect on cardiac physiology and disease responsiveness.

Abstract The role that stem or progenitor cells play in promoting cardiac regeneration has been the topic of rigorous scientific discussion over the past 15 years. A number of prominent laboratories have shown data whereby select sources of adult stem/progenitor cells can transdifferentiate and repopulate large areas of infarction or chemically injured myocardium with new cardiomyocytes that fully restore ventricular function. One stem/progenitor cell that has been highly touted as a true cardiac regenerative cell type is that expressing the cell surface marker for cKit (CD117). However, other laboratories have not observed the ability of select progenitors cells or cKit+ CPCs to generate new myocytes by transdifferentiation when injected, nor did they observe appreciable generation of new myocytes from endogenous stem cell sources. The entire field has become a contentious affair these past 5 years with essentially 2 diametrically opposed camps that continue to publish data supportive of their original observations. Hence, here we are proposing a novel approach whereby 2 laboratories from each camp will work together in a blinded manner with full exchange of all reagents and animal models to generate consensus data. This dual-PI application will also rely almost exclusively on mouse genetics and lineage tracing approaches so that more definitive data will arise. The three specific aims will address the overarching hypothesis that injection of exogenous progenitor cells into the heart results in cardioprotection through a paracrine mechanism of action. We will not address the transdifferentiation hypothesis as this seems to have been largely discredited. The paracrine hypothesis involves the generation of new endothelial cells from endogenesis cKit+ CPCs and the enhancement of ventricular perfusion in and around the area of myocardial infarction. It also may involve the augmentation of endogenous myocyte proliferation from existing myocytes, protection of myocytes from apoptosis in the border zone by paracrine factors and protective remodeling of the extracellular matrix, all of which will be investigated as part of the larger ?paracrine hypothesis? A highly structured experimental approach is proposed along with a system for blinded exchanges of biologic samples between the 2 laboratories. The goal is to generate a decisive data set based entirely on more rigorous standards and approaches, with the hope of bringing consensus to the cardiac stem cell field. The 2 PIs have a long track record of working together with multiple shared publications and joint grants.

Abstract The muscular dystrophies are inherited disorders that largely affect striated muscle tissue resulting in progressive muscle weakness, wasting, and in many instances, premature death. Many characterized mutations in humans that cause muscular dystrophy (MD) result from alterations in structural attachment proteins that affix the underlying contractile proteins to the basal lamina, providing rigidity to the skeletal muscle cell membrane (sarcolemma). Loss of select attachment proteins in the dystrophin-glycoprotein complex (DGC) permits contraction-induced membrane tears and influx of calcium that causes cellular degeneration and necrosis of muscle fibers. During this necrotic process cytokines, chemokines and growth factors are released as part of the inflammatory and repair process, although induction of fibrosis and scarring are an unwanted side effect that worsens disease. One prominent cytokine is transforming growth factor-? (TGF?) that serves a master regulator of the fibrotic response and worsening of muscle pathology in MD. While fibroblasts are directly regulated by TGF?, no one has yet to examine the function of the fibroblast in skeletal muscle directly in vivo, as a mediator or fibrosis and muscular dystrophy. Here we generated a unique genetic model in the mouse that will selectively modulate the activity of the cardiac and skeletal muscle fibroblast in vivo and in dystrophic mouse models of disease. Thus, here we will test the novel hypothesis that myofibroblasts play a selective role in mediating fibrosis and tissue remodeling in heart and skeletal muscle in response to cellular dropout from MD, while resident myofibers and cardiomyocytes in their respective tissues underlie physiologic ECM / collagen production and basal lamina production during development and as part of ongoing homeostasis. The application has 2 comprehensive specific aims: 1) To genetically parse the role of myofibroblasts in skeletal muscle and heart during MD in the mouse, 2) To examine how TGF?, SMAD2/3 and p38? signaling mediate disease in MD through the myofibroblast in vivo. The application will attempt to definitively address the function of the activated fibroblast (myofibroblast) in muscle during MD disease onset and progression. It will also attempt to elucidate the importance of TGF? signaling in mediating myofibroblast formation and disease activity in vivo, as both canonical and non- canonical pathways will be genetically dissected.

Abstract The cardiac myocyte has long been the primary focus of studies attempting to elucidate the regulatory aspects underlying cardiac development and disease. However, recently the involvement of nonmyocytes has emerged as potentially just as important as myocytes in contributing to and controlling cardiac remodeling during development and progressive pathogenesis in ischemia-induced heart failure. More specifically, the cardiac fibroblast and its ability to convert to myofibroblasts in promoting ECM production, ventricular remodeling and the fibrotic response have been underappreciated as critical regulator of cardiac biology. Here we propose a dual-PI application led by developmental and adult disease-based cardiac investigators to address key areas of fibroblast biology that span the early postnatal heart up through the failing and fibrotic adult heart. Previously, the field had not been able to carefully annotate the functional aspects of the fibroblast within the developing and diseased heart, in part because of a lack of appropriate genetic tools that specifically target this cell-type. More recently we and others have generated a few critical genetically modified mouse models that specifically target resident cardiac fibroblasts, as well as all activated fibroblasts and myofibroblasts in the heart. Thus, here we can now test the novel and overarching hypothesis that activated fibroblasts and myofibroblasts play selective roles in early neonatal ventricular maturation, regeneration and heart development, which is recapitulated in the adult heart in response acute and chronic ischemia-induced disease states. The dual-PI application has 3 specific aims: 1) To define sub-stages and functions of cardiac fibroblasts and myofibroblasts during postnatal development and in the adult heart following acute and chronic disease stimulation, 2) To identify crosstalk mechanisms between cardiac fibroblasts and myocytes in the developing and diseased heart, and 3) To define Tcf21- mediated contributions to fibroblast lineage expansion and commitment in the developing postnatal heart and in the adult heart after ischemic and acute injury. Collectively, these specific aims will uncover stages of fibroblast differentiation during development, regeneration and hypertrophy across both models of interstitial fibrosis and replacement fibrosis. Such an understanding will lay the foundation for future studies into specific therapeutic pathways to target in treating longstanding fibrotic heart disease states or to enhance the regenerative capacity of the heart.