This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.This project seeks to define the molecular mechanisms controlling the growth and proliferation of heart muscle cells or cardiac myocytes in the developing embryo. Dysregulation of heart muscle proliferation in developing segments or chamber precursors of the heart leads to major forms of congenital heart disease in humans, including chamber hypoplasia and septal and valve defects causing chronic illness and early death.The research described in this proposal will investigate the interaction of two regulators of embryonic cardiac proliferation, the zinc finger domain-containing transcription factor Gata4, and its associated co-factor Smarcd3. Independent mutation or loss of function of either gene results in poor growth or hypoplasia of the right ventricle of heart due to decreased proliferation of myocyte precursors. This leads to the hypothesis that critical genes regulating right ventricle growth are subject to dual regulation by Gata4 and Smarcd3 through direct binding of Smarcd3 to Gata4, and recruitment of Smarcd3's associated SWI/SNF chromatin remodeling complex to genes binding Gata4 at their promoter regions. Experiments described in this proposal will first examine the molecular determinants of Gata4-Smarcd3 binding, both to investigate the potential for regulation by growth factor signaling of this interaction, and as a guide to designing dominant interfering isoforms of either Gata4 or Smarcd3. Overexpression of these isoforms by lentiviral transduction in the P19CL6 model cardiac myocyte system will specifically disrupt activation of Gata4-Smarcd3 joint target genes. Comparison of resulting expression profiles to those from control P19CL6 cells and cells subjected to RNA interference knockdown of Gata4 and Smarcd3 will identify a focused population of candidate genes for testing by chromatin immunoprecipitation for direct activation by Gata4 and Smarcd3. Assay of resulting candidate genes by overexpression and conditional knockdown in-vitro and in-vivo will provide the first steps in determining a molecular roadmap for right ventricular proliferation, and insight as to how the embryo regulates the size, shape and structure of its heart. Such insight will also be of great utility to future therapeutic efforts seeking to identify, culture and expand cardiac stem or progenitor cells for use in post-injury grafting and cardiac repair.
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