Functional overload increases beta -MHC promoter activity in rodent fast muscle via the proximal MCAT (beta e3) site

Department of Physiology and Biophysics, University of California, Irvine, California 92697 Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, -MHC isoform. Different length rat -MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of 3,500, 914, 408, and 215 bp promoters increased in response to 1 wk of OL. The smallest 171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the 171 and 408 bp region were performed. The 408 bp promoters containing mutations of the e1, distal muscle CAT (MCAT; e2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT ( e3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the e3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the e3 site functions as a putative OL-responsive element in the rat -MHC gene promoter. gel mobility shift assay; plantaris muscle; direct gene transfer; dual luciferase; -myosin heavy chain