Development of Slow Skeletal Muscle Fibers

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DESCRIPTION (provided by applicant): Diversity among skeletal muscle fiber types is controlled by innervation-dependent and muscle fiber type specific cell signaling and downstream transcriptional regulation. A cell signaling pathway has been revealed that regulates slow myosin heavy chain 2 (MyHC2) gene expression in innervated fast and slow secondary avian muscle fibers. This signaling cascade, initiated by innervation-induced depolarization, is mediated by the inositol 1,4, 5 triphosphate receptor 1 (IP3R1). The goal of the proposed research is to elucidate the mechanism by which IP3R1 activity is regulated in a fiber type specific manner and to elucidate the mechanism by which controlled IP3R1 activity regulates expression of the slow MyHC2 gene and thereby muscle fiber type. The first aim of the proposal is to determine the muscle fiber type specific protein components of the IP3R1-associated protein complexes in fast versus slow muscle fibers. The identity of IP3R1-associated proteins will be established by mass spectrometry and western blot analysis. The second aim of the proposal is to determine the functional effects of altered IP3R1-associated protein-protein interactions and phosphorylation. IP3R1-mediated calcium release measurements will be made in fast and slow muscle fibers transfected with wild type and mutated IP3R1 expression constructs. In addition, IP3R1- mediated calcium release measurements will be made in fast and slow muscle fibers with altered IP3R1-associated cell signaling protein activities. The third aim of the proposal is to determine the effects of altered IP3R1 function on the transcriptional regulation of the slow MyHC2 gene and establishment of diverse muscle fiber types. The effects of altered IP3R1 activities in fast versus slow muscle fiber types on slow MyHC2 promoter activity and occupancy of transcriptional activating sites within the slow MyHC2 promoter will be determined. The result of these aims will be a synthesis of the structural data obtained from aim 1 and the functional data derived from aim 2 with the resulting regulation of slow MyHC2 gene expression and muscle fiber type identity. Altered calcium release mechanisms are associated with many disease states involving apoptosis and in skeletal muscle, muscular dystrophy. The significance of the proposed studies of regulation of IP3R1 activity has immediate relevance to understanding normal and disease-associated cellular function in skeletal muscle and other cell types.