ALTERED MECHANICAL LOADS AND SKELETAL MUSCLE PHENOTYPE

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One major outstanding question in muscle biology involves determining the molecular mechanisms by which alterations in mechanical load lead to changes in muscle fiber phenotype. A quintessential marker of myofiber phenotype and function is the myosin heavy chain (MyHC) since it is a major determinate of maximum unloaded velocity of shortening (Vmax) of skeletal muscle fibers. By developing a mouse model of decreased mechanical loading [hindlimb nonweight bearing (NWB)] to investigate transcriptional regulation of the betaMyHC gene we have previously identified in vivo the first NWB responsive promoter region (-450 to -294). We also showed that this NWB region contains a negative element (-332 to -311; termed dbetaNRE-S) that binds two different proteins identified ONLY in NWB-soleus nuclear extracts. Thus, the major focus of this proposal is to identify the nuclear factor(s) that transcriptional repress betaMyHC gene expression under NWB conditions, and to test their possible role in fiber-specific expression (FSE). Proposed experiments at the fine level involve: 1) isolating cDNAs encoding nuclear transcription factor(s) that bind the dbetaNRE-S using expression cloning. Northern analysis will determine developmental, tissue and FSE pattern of the nuclear factor(s). The gross level involves the generation and analyses of transgenic mice harboring: 1) a transgene carrying PCR site- directed mutation of the dbetaNRE-S element to determine its authenticity as an NWB-E. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase (CAT) specific activity in NWB-soleus muscle extract, and 2) transgenes overexpressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. RNA and protein analyses will assess the impact of cDNA overexpression on whole striated muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. The proposed experiments will identify and test the in vivo function of nuclear factor(s) and betaMyHC cis-element(s) involved in NWB induced transcriptional repression of betaMyHC gene expression and possibly its FSE. In vivo overexpression of nuclear factor(s) will identify their potential roles in regulation of other muscle gene(s), muscle atrophy and development. These experiments are expected to identify potential DNA/protein targets for therapies aimed at providing counter-measures against the slow-to-fast muscle phenotype and debilitating loss of postural function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.