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

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, [beta]-MHC isoform. Different length rat [beta]-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 [beta]e1, distal muscle CAT (MCAT; [beta]e2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT ([beta]e3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the [beta]e3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the [beta]e3 site functions as a putative OL-responsive element in the rat [beta]-MHC gene promoter. gel mobility shift assay; plantaris muscle; direct gene transfer; dual luciferase; [beta]-myosin heavy chain