Growth of cardiac myocytes is traditionally divided into two categories; namely the increase in cell numbers which is observed during embryonic development (hyperplastic growth), and the increase in cell mass which is observed during neonatal life (hypertrophic growth). The gene products which regulate these different types of myocardiocyte growth remain largely uncharacterized. The experiments in this proposal are designed to identify and clone gene which regulate proliferation in myocardiocytes. the experiments are based on a transgenic model system in which expression of the SV40 large T antigen oncogene was targeted to the cardiac atria with the Atrial Natriuretic Factor promoter. The transgenic mice develop unilateral right atrial hyperplasia; the hyperplastic atria are comprised of differentiated, dividing myocytes. This model demonstrates that adult myocardiocytes can re-enter the cell cycle in response to viral oncogene expression. Further analysis of the transgenic mice has revealed several properties which provide experimental inroads to identify and clone other genes which control observations. Specifically, we will: 1) Identify and clone the gene which promotes constitutive myocardiocyte hyperplasia in the transgenic model. A genetic variant has been identified which is able to complement T antigen and induce constitutive hyperplasia in atrial myocytes. A combination of genetic and molecular approaches is proposed to identify and clone this gene. 2) characterize the growth requirements of myocardiocytes in culture. We have generated a putative cardiac cell line derived from the transgenic tissue. These myocardiocytes divide rapidly in culture, retain a highly differentiated cardiocyte phenotype, can be repeatedly passaged, and exhibit spontaneous electrical and contractile activities. Clone genes which, when altered, produce a hyperplastic response in transgenic myocardiocytes. Atrial hyperplasia in the transgenic model can follow a pattern which is dependent upon stocastic genetic alterations (mutations). We propose to use a variation of standard oncogene transfection studies to clone the genes which facilitate a hyperplastic response. 4) Identify the array of genes which are expressed specifically in proliferating myocardiocytes. We will perform comprehensive screens to identify gene products who's expression is specifically induced (or repressed) during myocardiocyte proliferation. 5) Further develop second transgenic model system in which T antigen is targeted to cardiac myocytes with the alpha-Myosin Heavy Chain promoter. Initial experiments indicated that ventricular myocytes can also exhibit a hyperplastic response to T antigen. We will characterize ventricular hyperplasia in this new model, and further we will apply the approaches described above to this new system. These experiments will help resolve a fundamental question in cardiology; namely what genes regulate myocardiocyte growth. Once these genes are identified, studies aimed at assessing stimuli (hormonal or chemical) which mediate their expression may ultimately provide a therapeutic basis for myocardiocyte regeneration in vivo.
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