Regulation of cardiac alpha-myosin heavy chain gene transcription by a contractile-responsive E-box binding protein

The hemodynamic workload imposed on the heart modulates the expression of the cardiac-specific alpha-myosin heavy chain (MHC) gene. A hemodynamic responsive element (HME) has been mapped to an E box motif (CACGTG) located at position -47 of the promoter. The present studies showed that the HME is sufficient to confer contractile responsiveness to a heterologous promoter, the simian virus thymidine kinase gene, when expressed in cultured neonatal rat ventricular myocytes. Proximity of the HME to the TATA box of the alpha-MHC promoter appear necessary for high levels of basal transcription and for the four-fold induction in response to the contractile stimulus. An HME binding protein, approximately 43 kDa, was isolated from a neonatal rat ventricular myocyte cDNA library with sequence homology to the human upstream stimulatory factor-1 (hUSF1). Electrophoretic mobility shift assay showed that the in vitro translation product of the rat USF1 cDNA bound to the alpha-MHC HME motif and was recognized by an antibody to hUSF1. Overexpression of recombinant rat USF1 in spontaneously contracting cultured cardiomyocytes significantly increased activity of a cotransfected alpha-MHC promoter/luciferase reporter plasmid containing the HME motif plus core promoter elements (-40/+32), suggesting a role of rat USF1 in the contractile-mediated activation of the gene.