Identification of a contractile-responsive element in the cardiac alpha-myosin heavy chain gene.

The mechanisms by which the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene responds to contractile activity was studied in cultured cardiomyocytes and in vivo. Deletion analysis of the alpha-MHC promoter transiently transfected into neonatal rat cardiomyocytes localized the contractile-responsive element within -80 to -40 base pairs of the transcriptional start site. Mutational analysis of an E-box motif at position -47 showed that it was necessary for the contractile response both in cultured cardiomyocytes and in the intact heart. Competition gel mobility shift experiments indicated that the protein-DNA complex formed within the -39 to -59 base pair region could be competed by the E-box element at -309 of the alpha-MHC gene and that base substitutions within an E-box motif at -47 eliminated the protein-DNA complex. To identify the contractile-responsive nuclear protein, antibodies specific for E12/E47, an E-box binding basic-helix-loop-helix (bHLH) protein, and antibodies recognizing upstream stimulatory factor (USF), a widely expressed bHLH-leucine zipper transcription factor, were studied for their ability to inhibit cardiomyocyte nuclear protein binding to the E-box motif at -47. Anti-USF antibody abolished formation of the protein-DNA complex, thus identifying the protein as antigenically related to USF and demonstrating that bHLH-leucine zipper proteins are involved in the contractile-induced expression of the cardiac alpha-MHC gene.