Cardiac development, and cardiac hypertrophy involved specific switches in contractile protein gene expression. Little is known about the mechanisms of such gene switches, partly because cardiac cell lines are not available and primary cell culture models have been difficult to adapt to a molecular analysis of gene regulation. Our efforts under this project have been directed toward the goal of establishing such models both for the purpose of analyzing growth- and hypertrophy-associated gene switching, as well as for analysis of gene regulatory elements via transfection of cloned cardiac specific genes. Using these model systems, we have shown that stimulation of the alpha 1-adrenergic receptor induces cardiac myocyte hypertrophy with a concomitant switch in the expression of the mRNA encoding sarcomeric actins and myosin heavy chains. Using run-on transcription assays, we show that the actin gene switch is under transcriptional control. These results establish a linkage between stimulation of a specific cell surface receptor and a gene- specific alteration in transcription in context of myocardial cell growth and hypertrophy. In order to study the molecular mechanisms governing transcription of cardiac specific genes we have also established a transfection model allows detailed analysis of the cis regulatory regions governing cardiac genes. Using the cloned gene encoding cardiac troponin T (cTNT), we have identified the regions required for cell specific expression of this gene. Multiple upstream regions control expression of this gene and some of these regions are specific for expression in cardiac cells, while others appear to govern skeletal muscle specific expression. The mechanism(s) by which these regulatory regions control cTNT transcription are not yet known but at least some of these regions appear to contain cell specific promoter elements while others are enhancer elements. In the current application we propose to use site-directed mutagenesis, chimeric promoters and DNA footprinting to make a derailed analysis of the cis elements governing cardiac specific expressing of the cTNT gene. Those studies will allow precise identification of the nucleotide sequences which control expression o the cTNT gene to be made and will permit preliminary isolation of the proteins involved in that regulation. To extend those studies, we propose to develop a functional assay in which the interaction of cis and trans regulatory components can be studied in vitro. The long range goals of this project are to clone the genes encoding these regulatory factor(s) to permit detailed analysis of their function at the molecular level.
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