Abstract
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.
Highlights
The cardiac myocyte responds directly to mechanical stimuli such as load, stretch, or contractility by changes in cell mass and by alterations in specific gene expression resulting in changes in contractile function [1,2,3,4,5]
We have shown that the contractile activity per se of cultured neonatal rat cardiac myocytes modulates the expression of the myosin heavy chain (MHC) genes [1, 5, 19]
Identification of a Contractile-responsive Region by Deletion Analysis of the ␣-MHC Promoter—To identify the region of the ␣-MHC gene that is both necessary and sufficient for contractile responsiveness, the activities of deletion mutants of the 5Ј-flanking sequences were determined by transient transfection of neonatal cardiac myocytes
Summary
Isolation and Culture of Neonatal Rat Cardiac Myocytes—Ventricular myocytes were isolated from hearts of 2-day-old rat pups by collagenase digestion as described previously [5, 19]. For the preparation of nuclear extracts, cardiomyocytes were plated onto collagen-coated 75-cm tissue culture flasks and maintained in DMEM/F-12 medium containing ITS plus T3 for 48 h prior to harvest. Plasmids containing 5Јflanking regions of the ␣-MHC gene were co-transfected with a constitutively active RSV (Rous sarcoma virus long terminal repeat) -galac-. Cells in each 35-mm well were exposed for 6 h to DMEM/F-12:PC-1 (2:1) medium containing Lipofectin reagent (Life Technologies, Inc.), 2.5 g of ␣-MHC promoter/luciferase reporter plasmid, and 0.25 g of pRSVZ. A second series of promoter constructs was generated that terminated at the downstream position ϩ32 and extended upstream to positions Ϫ388, Ϫ163, Ϫ80, and Ϫ40 These sequences were generated using polymerase chain reaction to amplify the regions from the plasmid pSVOMCAT. The supernatant resulting from centrifugation at 9500 ϫ g was used for both chloramphenicol acetyltransferase (CAT) and luciferase (LUC) analyses as described previously [8, 22]. ␣-MHC promoter activity is expressed as luminescence units normalized to percent CAT conversion (LUC/CAT) in the same volume of homogenate
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