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. ? ? ?
| Patel, Parth N; Gorham, Joshua M; Ito, Kaoru et al. (2018) In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing. Curr Protoc Hum Genet 97: |
| Sharma, Arun; Toepfer, Christopher N; Schmid, Manuel et al. (2018) Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes. Curr Protoc Hum Genet 96:21.12.1-21.12.12 |
| Sharma, Arun; Toepfer, Christopher N; Ward, Tarsha et al. (2018) CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells. Curr Protoc Hum Genet 96:21.11.1-21.11.20 |
| Garfinkel, Amanda C; Seidman, Jonathan G; Seidman, Christine E (2018) Genetic Pathogenesis of Hypertrophic and Dilated Cardiomyopathy. Heart Fail Clin 14:139-146 |
| Sharma, Arun; Mücke, Michael; Seidman, Christine E (2018) Human Induced Pluripotent Stem Cell Production and Expansion from Blood using a Non-Integrating Viral Reprogramming Vector. Curr Protoc Mol Biol 122:e58 |
| Hueneke, Rocco; Adenwala, Adam; Mellor, Rebecca L et al. (2017) Early remodeling of repolarizing K+ currents in the ?MHC403/+ mouse model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 103:93-101 |
| Saddic, Louis A; Sigurdsson, Martin I; Chang, Tzuu-Wang et al. (2017) The Long Noncoding RNA Landscape of the Ischemic Human Left Ventricle. Circ Cardiovasc Genet 10: |
| Ito, Kaoru; Patel, Parth N; Gorham, Joshua M et al. (2017) Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing. Proc Natl Acad Sci U S A 114:7689-7694 |
| Alamo, Lorenzo; Ware, James S; Pinto, Antonio et al. (2017) Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes. Elife 6: |
| Burke, Michael A; Chang, Stephen; Wakimoto, Hiroko et al. (2016) Molecular profiling of dilated cardiomyopathy that progresses to heart failure. JCI Insight 1: |
Showing the most recent 10 out of 60 publications
