The application is a continuation of studies on the mechanism involved in the cAMP-induced regulation of alpha MHC gene transcription. Recent work in the investigator's laboratory has demonstrated that in primary cultures of fetal rat heart myocytes the elevation of intra-cellular cAMP results in up-regulation of alpha MHC and down-regulation of beta MHC mRNA expression. Using several deletion constructs in transient transfection assay, the investigator has identified a 31 base-pair fragment, which contains a hybrid motif with overlapping sequence of E-box and MCAT box (EM element) which confers both muscle specific and cAMP-inducible expression of the gene. The protein that recognizes this element appears to be indistinguishable from the previously characterized TEF-1 transcription factor. The applicant's goal, therefore, is (1) to characterize the protein which interacts with the cAMP-inducible element (EM) in the rat alpha MHC gene promoter by way of isolating and characterizing the cDNA encoding the cardiac specific TEF-1 isoform binding to the EM element, (2) clone and characterize the cDNA of a putative partner protein involved in the interaction, (3) study the cAMP-dependent phosphorylation of factors binding to the EM element (4) define the role of phosphorylation in protein factors which bind to DNA and recognize other proteins in the protein-protein interaction, and (5) verify the in vitro studies by in vivo experiments by constructing transgenic mice and by targeted ablation of selected loci by isolating cardiac specific isogenes from 129 S.V. mouse. It is understood that the proposed experiments will further elucidate the role of signaling pathways in the coupling of the hemodynamic load to the compensatory growth of the heart.
