Mechanical overload leads to enlargement of adult striated muscle and distinct changes in both myosin phenotype and contractile properties. Remaining critical questions in adult-stage muscle biology involve determining the DNA regulatory element(s) and nuclear protein factor(s) which transcriptionally induce beta myosin heavy chain expression in mechanical overloaded plantaris muscle and to test their possible role in fiber specific expression. We propose experiments for two levels of inquiry. The fine level involves: 1) identifying interactions between nuclear factor(s) and DNA elements(s) shown to be necessary for in vivo induction of beta myosin heavy chain by mechanical overload, using mobility shift, DNAse I footprinting and methylation interference assays, and 2) isolating cDNA(s) encoding nuclear factor(s) involved in beta myosin heavy chain induction by mechanical overload using expression cloning. Northern analysis will determine developmental, tissue and fiber specific expression patterns of these nuclear factor(s). The gross level involves the generation and analysis of transgenic mice harboring: 1) transgenes carrying PCR site-directed mutations of DNA element(s) involved in beta myosin heavy chain induction by mechanical overload. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase specific activity following mechanical overload and in muscles containing various fiber types, and 2) transgenes overexpressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. Northern analysis will assess the impact of overexpression of nuclear factors on striated muscle phenotype. The proposed work will identify and test the in vivo function of nuclear factor(s) and beta myosin heavy chain promoter element(s) involved in beta myosin heavy chain fiber specific expression and induction by mechanical overload. In vivo overexpression of nuclear factor(s) will identify their potential roles in regulation of other gene(s), muscle enlargement and development. These experiments are expected to identify potential protein/DNA targets for therapies aimed at providing countermeasures to derangements in muscle phenotypes and growth patterns attributable to muscle diseases.
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