Jeffrey Robbins - US grants

This proposal deals with the developmental regulation of the myosin heavy chain (MHC) gene family during embryogenesis and terminal muscle differentiation in the chick limb. The MHC genes are expressed in a developmental-stage-specific and tissue-specific manner. The overall aim of this project is to understand the structural modifications of the myosin genes which accompany, and may be responsible for their differential transcriptional activation. A MHC gene expressed specifically in the embryonic fast white muscle will be compared with a gene which is expressed only in the adult fast white fibers. Using DNAse I treatments of chromatin the nuclease hypersensitivity of the 5' -regions of the expressed and non-expressed genes during development in the different tissues will be measured in order to catalogue the hypersensitive sites, and correlate them with the transcriptional activation or inactivation of the genes. These data will then be used to map subdomains of sequence within the hypersensitive regions that are resistant to exonuclease III, in order to discern the binding of protein factors during the activation and deactivation of these genes. The degree of methylation will be measured, first on a gross level by using restriction enzymes that do, or do not recognize methylated sequences. This analysis will be followed by "genomic sequencing" which is able to detect the degree of methylation at all of the possible sites in the chosen sequences. These studies are directed toward the long term goal of understanding the molecular basis for the specific and regulated expression of the myosin gene family. By cataloging these basic structural parameters it becomes possible to anticipate the functional consequences and undertake a rational search for the factors which regulate the MHC genes' expression.

The structural underpinning of the essential physiological role of the heart resides in the synthesis and maintenance of those proteins that are responsible for the contractile mechanics, the polypeptides found in the sarcomere. The theme of this proposal is study both differentiation and development of the mammalian heart using gene targeting to produce defined mutations in four different genes, and to generate animal models in the mouse that will be used for molecular genetic, biochemical, cytological and physiological studies on fundamental mechanisms. In Project 1 the alpha-cardiac myosin heavy chain will be studied. Using gene targeting techniques, both ablation and site directed mutagenesis on structural and regulatory sequences will be carried out resulting in mice which carry the defined mutation in the correct chromosomal context. This Project will delineate the physiological importance of myosin isoform shifts in the myocardium, define transcriptional regulatory cassettes that are active in vivo, and will delineate structural sequences responsible for the unique ATPase activities of the different cardiac myosins. Project 2 uses the identical technological approach, but will analyze tropomyosin isoforms. This complements the analyses in Project 1, since the tropomyosin isoforms are generated at the post-transcriptional level, and interact with the myosins in the contractile apparatus. Similarly, Project 3 will analyze, again by gene targeting techniques, the structure/function relationships between the other major protein of the contractile apparatus, alpha-cardiac actin. Project 4 deals with a more basic facet of cardiomyogenesis, the basic fibroblast growth factor. While Projects 1, 2, and 3 focus on the proteins that are expressed during the terminal stages of cardiogenesis, basic fibroblast growth factor plays a role both in the differentiation of early mesoderm and in the later developmental stages. The factor's impact on the growth and development of both the myoblast as well as the other major cell type in the myocardium, the fibroblast, will be determined using gene targeting strategies. The entire group of projects is supported in their technological and analytical thrusts by 3 cores: the Embryonic Stem Cell Core, a Pathology Core and a Physiology Core which will use both whole hearts and isolated tissues to examine the cardiac mechanics of the resultant mutants. Taken together, these Projects should reveal specific and fundamental information on a mutation(s) effect(s) on cardiac differentiation, development and function.

This Component's long term objectives are twofold; 1) to understand normal and abnormal cardiac function in terms of the various contractile protein isoforms that are expressed; and 2) to explore the early developmental events that underlie normal and abnormal cardiogenesis. Towards the first goals, we will use transgenics to overexpress normal and mutated myosin light chains i the heart in a compartment specific manner such that athe protein complement of the heart is changed. Using cloned light chains and our cardiac specific promoters, a series of conditional transgenic animals will be made such that over expression and ectopic- isoform expression studies, as well as structure-activity studies can be done. Both the acute and chronic effects upon heart structure and function will be measured. These studies will use conditional expression transgenic models such that proteins encoded by the transgenes can be precisely modulated up/down during the animals' lifetime. The resultant mice will be analyzed at the molecular, biochemical, cellular and whole organ levels. In this manner, we will define whether or not a developing myopathy is due to a mutated protein and/or to stoichiometric perturbations in the levels of the contractile proteins that assemble to form the functional sarcomere. The second goal, to explore early cardiogenesis, involves the definition of the downstream targets of the morphogen, retinoic acid. We will use the a myosin heavy chain promoter to target a constitutively active retinoic acid receptor to the developing heart. The initial descriptive characterization will be followed by a concerted effort to identify the downstream genes that are activated, using a novel, dual screening approach. First, genomic fragments will be selected on the basis of t heir ability to bind the constitutively active retinoic acid receptor. These fragments will then be used to screen a normal genomic library. Clones isolated in this manner will then be screened with a probe population isolated via a differential PCR display obtained from developing hearts isolated from the mouse containing the constitutively active retinoic acid receptor and the nontransgenic littermates. In this manner, we hope to identify clones that are activated/deactivated by the morphogen's presence.

DESCRIPTION (provided by applicant): The objective of this application is to mechanistically dissect the cardiac-autonomous pathologies, which include cardiomyocyte hypertrophy and valve disease, that occur in Noonan syndrome. Our immediate goals are to carry out comprehensive studies using inducible, cardiac-specific expression of both the normal and mutated forms of the tyrosine phosphatase, Shp-2 in the different cardiac cell populations. These studies will be complemented by an inducible, cardiac-specific gene ablation of ptpn11 in order to discern protein function at different developmental times. The following SPECIFIC AIMS are directed towards this goal: SPECIFIC AIM I will explore the cardiomyocyte autonomous effects of Shp-2 expression in order to dissect the primary and secondary effects on hypertrophy and valve dysfunction. Both wild type (WT) and mutated protein will be expressed only in the cardiomyocyte population in standard transgenics and in transgenics under inducible control. The hypothesis is that expression of the Noonan mutation Shp-2 Gln79Arg, will result in cardiomyocyte hypertrophy. A second hypothesis is that cardiac pathogenesis is due to a gain of function: that is, high levels of wild type Shp-2 will have the same phenotype as animals with modest expression of Shp-2 Gln79Arg. SPECIFIC AIM 2 will carry out the complementary studies in the relevant non-cardiomyocyte populations to define the role that the Shp-2 mutation plays during cardiac cushion formation and development of the outflow tract. Both the WT and mutated protein will be expressed during development in the endothelial population only. SPECIFIC AIM 3 will explore loss of function of the normal protein by carrying out an inducible, cardiomyocyte-specific knock-out using the MerCreMer system developed in our Division. We hypothesize that the effects of Shp-2 loss of function will differ radically depending upon the developmental time and in this manner the role of Shp-2 in controlling normal cellular processes in the heart can be explored. SPECIFIC AIM 4 will explore the signaling pathways downstream of Shp-2 in cardiomyocytes. We hypothesize that Shp-2 signals through activation of the MAP kinase (MAPK) pathway in cardiomyocytes and that ablation of Shp-2 will blunt MAPK signaling while over-expression of WTShp- 2 or Shp-2 Gln79Arg will result in increased MAPK activity or inappropriate MAPK activity through the ERK branch in response to various stimuli.

Project 5's long term goal is to investigate both the basic biology underlying normal cardiac development and function and the physiological consequences of the myosin heavy chain (MyHC) isoform shifts that occur during the development of heart failure. Many of the signaling pathways activated during compensated hypertrophy and decompensated failure result in fetal gene program activation. Consistent with these data, it has recently been shown that substantial amounts of the alpha-myosin heavy chain gene transcript are present in the normal human heart, and that these levels are dramatically down regulated during heart failure. What is the functional significance of the different cardiac proteins that constitute the main component of the pump? Can modulation of the myosin heavy chain isoform content impact significantly upon the heart's ability to maintain normal cardiac output under normal or pathologic conditions? Project 5 addresses these questions by modulating the myosin isoform content of the mouse heart. SPECIFIC AIM 1 will, using cardiac-specific transgenic expression in the mouse, replace the normal alpha-MyHC isoform with the beta1 beta- MyHC protein. Analyses at the molecular, cellular, whole organ and whole animal levels over the lifetime of the transgenic cohorts will provide a comprehensive picture of the consequences of varying myosin isoform content. SPECIFIC AIM 2 will challenge these mice, using both a surgically-induced pressure overload model, and breeding them into a number of genetically defined, cardiac-compromised transgenic lines, in order to define how altered motor content impacts on the progression and severity of cardiac disease. SPECIFIC AIM 3 will test the relevance of the mouse models to the human condition by creating the exact isoform shift that occurs in man in a transgenic rabbit heart. The alpha-MyHC will be linked to the beta-MyHC promoter in order to effect a partial or complete replacement of the normal beta-MyHC in the rabbit heart. The effects at the molecular and cellular levels, as well as hypertrophy and failure. Finally, SPECIFIC AIM 4 will measure both the RNA and protein levels for the alpha- and beta- MyHC's in populations with defined heart disease. These data will establish the normal and abnormal levels of these molecules during various, well-defined stages of human cardiac disease. The levels of the V1 and V3 will be correlated with the mechanical properties of the fibers. Together, these experiments will, for the first time, unambiguously establish the structure-function relationships of the cardiac myosin isoforms in the intact animal and will help establish the consequences of altered isoform content on compensated hypertrophy and its progression to heart failure.

DESCRIPTION (provided by applicant): The Program Project Grant will integrate aspects of signal transduction that underlie normal and abnormal cardiovascular function. Gain- and loss-of-function approaches include cell culture, gene targeting and cardiac-specific transgenesis. Signaling pathways in normal cardiac development and function, the basic biology of cardiac signal transduction, as well as the actions on the final targets will be studied. The Program consists of 4 Subprojects and 3 Cores. Subproject 1: Phosphorylation and function of the contractile proteins focuses on the myofibrillar proteins, exploring how the contractile apparatus is tuned to match prevailing conditions. Using cardiac specific transgenesis, proteins in which the relevant sites are modified such that they cannot be phosphorylated, or act as if they were chronically phosphorylated, will replace the endogenous TnI or MyBP-C protein complements. Subproject 2: The calcineurin/NFAT pathway in heart development will explore the regulatory cascades that control differential gene expression and morphogenesis of the developing heart to determine if calcineurin signaling through the NFAT family of transcription factors drives programmatic changes in contractile protein gene expression during cardiac development. Suproject 3: The ERK-MAPK signaling branch in the heart will explore the ERK-MAPK pathway's role in inducing cardiac hypertrophy and promoting protection from apoptotic stimuli. The hypertrophic potential of MEK1 dominant negative mice and ERK1 knockout mice will be characterized. The role ERK-MAPK pathway's role in cardioprotection will be analyzed as will the transcriptional mechanism whereby MEK1-ERK1/2 signaling mediates cardiac hypertrophy. Subproject 4: Rab GTPase protein transport regulation in heart disease. The Rab protein family controls subcellular protein trafficking and the individual actions of myocardial Rab proteins will be explored in the heart using gain-of-function approaches in both cardiomyocytes and in transgenic animals. The Administrative Core (A) will serve as the organizational focus, The Histo-Pathology/Physiology Core (B) will provide an integrated central facility for the necessary histology and pathology, as well as for the physiological analyses. The Adenovirus Core (C) will prepare virus for subprojects 2, 3 and 4, and neonatal rat cardiomyocytes, which will be needed for subprojects 3 and 4.

DESCRIPTION (provided by applicant): The objective is to create animal models that are relevant to human cardiovascular disease. The immediate goals are to explore the functional consequences of up-regulation of the alpha-myosin heavy chain protein (alpha-MHC) in the rabbit heart, both under basal conditions as well as under conditions when normal cardiovascular function is challenged. Stable and elevated levels of alpha-MHC will be achieved by cardiac-specific transgenesis in the rabbit heart, whose myosin complement accurately reflects that of the human myocardium, testing the mechanistic implications of this isoform's presence for cardiovascular function. We hypothesize that altering alpha-MHC levels will be relatively benign under basal conditions and cardioprotective as the heart fails. In SPECIFIC AIM 1, we will define the phenotypes of rabbits with varying amounts of alpha-MHC being expressed in the ventricle. The effects of both low and moderate replacement will be determined at the motor, cellular, fiber and whole organ/animal levels under basal conditions. The different TG rabbits will establish the physiological importance of the different myosin isoforms under basal conditions in a "beta-MHC" heart and will test the hypothesis that replacement of the normal beta-MHC complement with either high or low levels of alpha-MHC is innocuous under normal unstressed conditions. SPECIFIC AIM 2 will test the effects of varying amounts of ventricular alpha-MHC on the ability of the rabbit heart to tolerate ischemia. We hypothesize that stable expression of low amounts of alpha-MHC will be beneficial for maintaining cardiovascular function under ischemic conditions. However, expression of alpha-MHC at significantly higher levels (40-50%) may alter cardiomyocyte biochemistry so dramatically as to negatively impact on the organ's ability to tolerate stress. SPECIFIC AIM 3 will test the effects of varying amounts of ventricular alpha-MHC on the ability of the rabbit heart to tolerate gradual increase in afterload, by inducing pressure-overload via trans-aortic coarctation soon after birth and allowing the animals to "grow into" the band during the adolescent and early adult stages. Again we hypothesize that in this model, modest replacement with alpha-MHC will be beneficial. SPECIFIC AIM 4 will test the effects of varying amounts of ventricular alpha-MHC on the ability of the rabbit heart to tolerate pacing induced heart failure. Our working hypothesis is that the alpha-MHC expressing TG animals will exhibit significantly less morbidity and mortality. Together with the models above, it will provide a comprehensive picture of the alpha-MHC's effects on the development of hypertrophy, dilation and failure.

The objective is to create animal models that are relevant to studying human cardiac contractility and heart failure. The immediate goals are to explore the functional consequences of up-regulation of the alpha-myosin heavy chain protein (alpha-MHC) in the rabbit heart, both under basal conditions as well as under conditions when normal cardiovascular function is challenged. Stable and elevated levels of alpha-MHC will be achieved by cardiac-specific transgenesis in the rabbit heart, whose myosin complement accurately reflects that of the human myocardium, testing the mechanistic implications of this isoform's presence for cardiovascular function. We hypothesize that altering alpha-MHC levels will be relatively benign under basal conditions and cardioprotective as the heart fails. In SPECIFIC AIM 1, we will define the phenotypes of rabbits with varying amounts of alpha-MHC being expressed in the ventricle. The effects of both low and moderate replacement will be determined at the motor, cellular, fiber and whole organ/animal levels under basal conditions. The different TG rabbits will establish the physiological importance of the different myosin isoforms under basal conditions in a "beta-MHC" heart and will test the hypothesis that replacement of the normal beta-MHC complement with either high or low levels of beta-MHC is innocuous under normal unstressed conditions. Specific Aim 2 will test the effects of varying amounts of ventricular beta-MHC on the ability of the rabbit heart to tolerate ischemia. We hypothesize that stable expression of low amounts of beta-MHC will be beneficial for maintaining cardiovascular function under ischemic conditions. However, expression of alpha-MHC at significantly higher levels (40-50%) may alter cardiomyocyte biochemistry so dramatically as to negatively impact on the organ's ability to tolerate stress. Specific Aim 3 will test the effects of varying amounts of ventricular alpha-MHC on the ability of the rabbit heart to tolerate gradual increase in aflerload, by inducing pressure-overload via trans-aortic coarctation soon after birth and allowing the animals to "grow into" the band during the adolescent and early adult stages. Again we hypothesize that in this model, modest replacement with alpha-MHC will be beneficial. Specific Aim 4 will test the effects of varying amounts of ventricular alpha-MHC on the ability of the rabbit heart to tolerate pacing induced heart failure. Our working hypothesis is that the alpha-MHC expressing TG animals will exhibit significantly less morbidity and mortality. Together with the models above, it will provide a comprehensive picture of the alpha-MHC's effects on the development of hypertrophy, dilation and failure.

DESCRIPTION (provided by applicant): The long term objective of this application is to understand the roles that pre-amyloid oligomers (PAO) may play in human heart failure. We noted that cardiomyocytes from diseased hearts contain a protein that reacts to antibodies able to detect a toxic protein(s), PAO, which is normally associated with the amyloid-based neurodegenerative diseases. Subsequently, we found that PAO is present in cardiomyocytes derived from a limited number of human heart failure patients of different etiologies, implying that it may be an important mediator of cardiovascular disease. We think that the 1B crystallin mutant mouse, which also accumulates PAO in the cardiomyocytes, is a uniquely useful and relevant system that models a heretofore understudied phenomenon, accumulation of PAO in the cardiomyocytes of both adult and pediatric heart failure patients. The goals, therefore, are to understand the pathogenic pathway that results in PAO accumulation, determine its toxicity in cardiomyocytes and define potential therapeutic targets or modalities for the resultant dilated cardiomyopathy and heart failure that occurs as a result of CryABR120G expression. Aim 1 will test the hypothesis that cardiomyocyte accumulation of PAO is widespread in the human heart failure population, across age groups and disease types. Aim 2 will use inducible, cardiomyocyte-specific CryABR120G (a mutant protein causative for human muscle disease), expression to test the hypothesis that PAO-mediated heart failure can be reversed. Aim 3 will express anti-apoptotic factors in CryABR120G cardiomyopathic hearts to determine if prevention of programmed cell death can, in the face of continuous CryABR120G expression, prevent heart failure or even reverse existing disease. We hypothesize that despite the occurrence of cardiomyocyte apoptosis in CryABR120G hearts, programmed cell death is peripheral and collateral to the primary etiology that transits the hearts toward failure in this model. These studies have the potential of establishing broad linkages between the neurodegenerative and cardiovascular diseases and identifying new targets for interfering with processes that occur in a broad range of cardiovascular disease. We have found that the heart contains a protein that is normally associated with neurodegenerative diseases. This toxic protein is only found in diseased hearts and our experiments will examine exactly when and where the protein accumulates and how it causes cardiovascular disease.

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DESCRIPTION (provided by applicant): The overall emphasis of this renewal application is to understand the molecular pathways that control cardiac hypertrophy and homeostasis. Specifically we propose to continue our investigation of calcineurin-NFAT signaling in the heart as a regulatory of pathologic hypertrophy, but also baseline function. We will continue to investigate the signaling relationships that underlie calcineurin's hypertrophic functionality in the heart, with an emphasis on characterizing novel interacting partners with known regulatory implications. Thus we hypothesize that calcineurin-NFAT signaling are critical mediators of cardiac disease responsiveness through highly integrated complexes with other signaling effectors. Indeed, our preliminary data shows a large number of novel signaling effectors that interact with calcineurin in programming the hypertrophic response, and these will be characterized here. We will also investigate a number of novel hypotheses related to non-hypertrophic functions of calcineurin, as well as continue our analysis of hypertrophic regulatory mechanisms. Finally, this renewal application will also attempt to address the source or microdomain of Ca2+ that activates calcineurin signaling in the heart through an in depth and mechanistic assessment of several channels and Ca2+ regulatory proteins. Thus, we hypothesize that select microdomains associated with specific Ca2+ channels can directly communicate with calcineurin outside of contractile Ca2+, thus regulating the hypertrophic response. To examine these various hypotheses we propose 2 distinct but functionally inter-related Specific Aims. We have observed that mice lacking all calcineurin from the heart die for unknown reasons and so very poor ventricular performance. Specific Aim #1 will evaluate the function of RCAN (formerly known as MCIP) proteins in modulating calcineurin-NFAT signaling, as well as in promoting calcineurin interaction with novel targets in the heart through higher-order complexes. For example, we have determined that RCAN1 directly interacts with TAB2 in generating a novel signaling circuit that allows TAK1 and calcineurin to co-regulate one another in programming the hypertrophy response. Characterization of such novel interactions might suggest other critical functions of calcineurin in the heart, possibly explaining why it is required for viability. Specific aim #2 will examine the source of Ca2+ that communicates with calcineurin in facilitating its activity in the heart, distinct from total contractile Ca2+. Specifically, we will examine the ability of L-type Ca2+ channels, T-type channels, TRPC channels, and the IP3 receptor to directly communicate with calcineurin by providing a highly local Ca2+ signal outside of excitation-contraction coupling. Thus, the current application will attempt to address the most salient issues and remaining frontiers associated with calcineurin-NFAT signaling in the heart.

Our goal is to define the role(s) played by the fibroblast during fibrosis, cardiac remodeling and the development of heart failure in two mouse models of human disease. Fibrosis is increasingly recognized as an important contributor to progressive heart disease and failure but there is a striking deficit in our understanding of fibroblast-based signaling and its role in these processes. We hypothesize that TGFp signaling processes that are fibroblast-based play a critical role the fibrotic response in sarcomere- based and non-sarcomere-based disease. We will precisely ablate discrete signaling pathways in the fibroblast during cardiac disease development. Two models will be used. Model 1 is a sarcomere protein- based model in which an N-terminal fragment of cardiac myosin heavy chain C (cMyBP-C) can be inducibly expressed specifically in the cardiomyocyte. This fragment uniquely presents in the failing or diseased human heart. Expression of this polypeptide results in cardiac hypertrophy, fibrosis and transition to failure. Model 2 utilizes a mutant aB crystallin (CryAB'^^^¿¿) that is causative for human skeletal and cardiac disease, but is a non-sarcomeric protein-based model of cardiac failure. This model is also characterized by hypertrophy, extensive fibrosis and heart failure. Aim 1 will test the hypothesis that canonical TGF(3 signaling plays a critical role in fibrosis during hypertrophy and failure in the cMyBP-C truncation model. The necessity and sufficiency of different pathways active in the fibroblast will be defined by breeding the transgene into a novel inducible kniockout set of mice in which we are able to ablate either canonical or non-canonical TGF(3 signaling. The mutant cMyBP-C allele will be bred into a mouse line with activated fibroblast-specific expression of inducible Cre. Offspring containing both the transgene and inducible Cre will then be bred into lines with either a smad2/3-loxP or tgfprl-loxP allele. Aim 2 will test and define the role of TGFp signaling in our CryAB^^^¿¿ model in a similar set of experiments. These aims will define the role of fibroblast-based signaling. Aim 3 willdetermine, in both the CryAB and cMyBP-C models, the importance of non-TGFp cytokine signaling during cardiac disease. The experiments outlined in Aims 1 and 2 will be repeated, but on a genetic background in which we are able to modulate non-canonical TGFp signaling in the fibroblast. We hypothesize that non-canonical TGFp signaling plays a key role in fibrosis during sarcomere-protein based cardiac disease. To'test that, we carry out the experiments described above, but in Tak1- and p38a-loxP targeted mice. Those lines will be crossed to the inducible fibroblast-specific Cre alleles. RELEVANCE (See instructions): Fibrosis often occurs during the development of heart disease. In fact, fibrosis remains a hallmark of hypertrophic cardiomyopathy and is a substrate for arrhythmogenic events, pump dysfunction and, eventually, heart failure. We propose to study how a major cell type in the heart, the fibroblast, contributes to these disease processes. Understanding the roles these cells play will open up novel therapeutic possibilities for impacting favorably on cardiac disease.