This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. Mechanical overload (MOV) 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) required for fiber-type specific expression (FSE) and MOV induced changes in muscle gene expression. This proposal?s major focus is to identify the DNA element(s) and nuclear factor(s) which transcriptionally induce beta myosin heavy chain (bMHC) expression in MOV plantaris muscle and to test their possible role in FSE. We propose experiments for two levels of inquiry. The fine level involves: 1) identifying interactions between nuclear factor(s) and DNA element(s) shown to be necessary for in vivo induction of bMHC by MOV, using mobility shift, DNAse I footprinting and methylation interference assays, and 2) isolating cDNA(s) encoding nuclear factor(s) involved in bMHC induction by MOV using expression cloning. Northern analysis will determine developmental, tissue and FSE patterns of these nuclear factor(s). The gross level involves the genera-tion and analysis of transgenic mice harboring: 1) transgenes carrying PCR site-directed mutations of DNA element(s) involved in bMHC induction by MOV. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase (CAT) specific activity following MOV and in muscles containing various fiber types, and 2) transgenes over-expressing 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 viv function of nuclear factor(s) and bMHC promoter element(s) involved in bMHC FS and induction by MOV. In vivo overexpression of nuclear factor(s) will identif 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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