In response to functional overload, existing adult skeletal muscle fibers enlarge (hypertrophy) and express a different subset of contractile protein isoforms. One of the most profound phenotypic changes is a large increase in the proportion of slow myosin heavy chain (Beta-MHC), most recently observed in rodent fast twitch muscle. Evidence to date suggests that this induction is controlled by the rate of transcription. Hypertrophy and isomyosin transitions, appear to represent important physiologic adaptations used by post-mitotic adult skeletal muscle cells to accommodate increased workloads. Although the Beta-MHC gene has been characterized structurally, little is known about the transcriptional regulation of this gene, particularly in response to an overload stimulus. The major focus is to identify and test whether DNA sequences required for high level constitutive (basal) and muscle specific expression in adult skeletal muscle are necessary and sufficient to confer induction of the human Beta-MHC gene by overload. We propose experiments involving two levels of characterization. The first or gross level will involve analysis of : 1) the kinetics of endogenous Beta-MHC MRNA induction in overloaded adult mouse muscle, 2) an in vitro analysis of DNA sequences located 5' to the start site of human Beta-MHC gene transcription by deletion mapping and gene transfer into cells in culture, 3) the generation of transgenic mice, and 4) an in vivo analysis of human Beta-MHC DNA sequences using transgenic mice. The latter experiment will: 1) confirm DNA sequence elements identified in cell culture experiments and possibly locate additional DNA sequence elements, 2) determine if muscle specific element (s) or distinct DNA sequence element (s) are required for Beta-MHC induction by overload, and 3) compare DNA sequence elements which are involved in Beta-MHC induction by overload to those which mediate high level constitutive expression of this gene in slow-twitch muscle. The second and fine level of analysis investigates interactions between nuclear protein factor(s) and DNA sequences elements shown by the gross analysis to be relevant for in vivo promoter function using mobility shift, DNAse I footprinting and methylation interference assays. The proposed studies will be some of the first to address mechanisms of gene regulation which underlie the phenotypic changes associated with skeletal muscle hypertrophy in adult transgenic mice. Knowledge gained from these studies will serve to expand our understanding of selected muscle disease states involving altered growth and isomyosin phenotypic conversions.
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