The heart responds to various hemodynamic and humoral stimuli by changes in myocyte size and function. Left ventricular hypertrophy such as arises from hypertension or valvular disease is a sensitive predictor for morbidity and mortality due to myocardial dysfunction. In the present application we propose to test the hypothesis that myocyte contractile activity per se regulates the transcription of the (alpha)-myosin heavy chain (alpha-MHC) gene. We have identified a cis-acting element at -47 bp from the transcription initiation site, designated hemodynamic response element (HME), that is both necessary and sufficient to confer contractile responsiveness in spontaneously contracting cardiomyocytes in culture. The HME contains an E-box motif whose binding protein (designated HRP) is antigenically related to USF (upstream stimulatory factor). We propose to clone HRP from a neonatal rat cardiomyocyte expression library by its ability to bind specifically to the HME sequence. Expression studies of the cloned HRP is cultured cardiac myocytes will confirm its role in contractile-mediated (alpha-)-MHC transcription. In vitro studies will determine its dimerization, DNA binding and transcription activation properties, and whether phosphorylation of HRP occurs in response to the mechanogenic stimulus. The role PKC(Z) in the signal transduction pathway and its potential localization to the nucleus will be determined. We propose to study the combinatorial effects of thyroid hormone (TH) and contractile activity on transcriptional regulation of the (alpha)-MHC gene to understand the rapid induction of this gene at birth when both TH and cardia hemodynamics are changing. The interaction of these activators with general transcription factors (TATA box binding protein and TAFs) will be studied to determine the molecular basis of workload- and hormone-regulated (alpha)-MHC transcription. To verify the transcriptional role of HRP in vivo we propose to overexpress HRP in the hemodynamically unloaded heart to directly assess its effect on (alpha)-MHC promoter activity and to determine whether PKC(Z) phosphorylation of HRP is required for its transcriptional activation. These studies will advance our understanding of the molecular pathways of workload regulated cardiac growth and function.
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