Molecular Signaling in Hypertrophic Cardiomyopathy

Harvard Medical School, Boston MA

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Abstract

DESCRIPTION (provided by applicant): Sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (HCM), sporadic HCM, pediatric HCM and HCM of the elderly and occur in approximately 1 million people in the US. The molecular mechanisms by which these mutations produce the clinical features of LVH remain largely unknown. We have produced mouse models that carry selective human mutations, characterized the development of histopathology, assessed candidate molecules for triggering hypertrophic signaling, and performed comprehensive (SAGE) transcriptional profiling early and late in pathologic remodeling of ventricular myocardium. Phenotypic characterization of genetically identical HCM mice demonstrated that responses to sarcomere protein gene mutations are complex, activating different molecular pathways in different myocytes within the same heart. These different cellular pathways must be activated by different environmental factors. The central hypotheses of this application is that different myocyte populations will be distinguished by different expression profile signatures and that definition of these RNA signatures will help to identify key molecules that are involved in directing each facet of the hypertrophic response. Our previous efforts to identify transcriptional signatures of HCM have involved using existing techniques to assess RNA expression in the entire left ventricle of HCM mice. Our initial efforts to identify RNA profile signatures were confounded by three technical problems: 1) Existing transcriptional profiling technologies did not allow assessment of RNAs that are expressed at low levels;2) Cardiac tissue was treated as a homogenous cell population;3) The response to sarcomere protein gene mutations varies considerably even between genetically identical mice. Here we propose two approaches to overcome the technical difficulties encountered in characterizing the hypertrophic response. First, we will isolate specific cell populations in which a particular molecular marker of a hypertrophic response has been activated. For example, we will use a marker gene in which the (3-myosin heavy chain (MHC) gene promoter drives a fluorescent yellow protein to isolate cells in which this molecular hypertrophy marker is activated. Second, we have recently developed a modified RNA profiling method, we have termed PMAGE (polony multiplex analysis of gene expression), which provides about 100 fold more sensitivity than existing techniques. We propose to define the role of proteins whose expression is altered in different myocyte subsets. Specifically we propose to: 1) Isolate mouse myocyte populations with shared molecular responses to HCM mutations. 2) Employ a highly sensitive RNA profiling technique PMAGE to define RNA profiles in mouse myocyte populations. 3) Assess roles of signaling proteins in hypertrophic pathways triggered by sarcomere gene mutations. 4) Assess RNA profiles and screen candidate genes for mutations in human HCM samples.