NEGATIVE CELL CYCLE REGULATION IN CARDIAC MUSCLE CELLS

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DESCRIPTION (adapted from the applicant's abstract): The long-term goal of this project is to understand the molecular mechanisms that regulate the permanent withdrawal of ventricular cardiomyocytes from the cell cycle during terminal differentiation. The applicant has isolated a gene from juvenile rat ventricles by cDNA subtraction with fetal ventricular cDNA. Northern blot analysis shows expression of the gene in neonatal, juvenile, and adult ventricles but not in fetal ventricles. In neonates, its expression is higher in ventricles than other organs. The deduced amino acid sequence shares about 50% similarity to Growth Arrest-Specific gene 1 (gas1) which suppresses mitosis. Hence, the isolated gene is named c-gas1 for cardiac gas1. Transient expression of c-gas1 in fetal ventricular myocytes results in a significant reduction in DNA synthesis. The applicant hypothesizes that c-gas1 is a developmentally regulated gene which suppresses ventricular myocyte proliferation during and after terminal differentiation. To test this hypothesis, the following specific aims are proposed: 1) To determine the cellular localization of c-gas1 protein in ventricular myocytes by immunofluorescence staining and confocal microscopy. 2) To determine whether abolishing the expression of c-gas1 by antisense oligonucleotides will permit terminally differentiated ventricular myocytes to enter the cell cycle. 3) To identify the cellular factors in terminally differentiated ventricular myocytes that directly interact with c-gas1 protein by the yeast double-hybrid system. 4) to examine the effect of overexpressing c-gas1 in fetal ventricular myocytes and the effect of inactivation of both alleles of the c-gas1 gene on myocardial growth and development.