Muscle contraction requires the orderly assembly and functional interaction of numerous proteins in the sarcomere. The most abundant sarcomeric protein is the motor protein, myosin, which in mammals is represented by at least eight closely related isoforms. Expression of these genes is precisely regulated in space and in time, but their functional relationships remain largely unknown.
In Aim I of the proposed experiments, transgenic approaches will be used to define the role of these genes in muscle development and function. Mice null for the expression of the two major skeletal myosin heavy chains (MyHC), fast IIb and fast IId have been obtained. Common phenotypes include decreased body mass and limb weakness. Distinct phenotypes include kyphosis, histopathology and physiological defects. Interestingly, both null strains exhibit compensation by other MyHC genes, but the compensating gene is different between the two strains. The basis for these phenotypes will be explored and the molecular basis for, and the timing of compensation will be determined. Because compensation has occurred, and yet the mice have strong phenotypes, the PI will test whether the MyHCIIa gene can functionally substitute for the MyHCIId gene by """"""""knocking"""""""" the IIa coding region into the IId locus. The sequence of the coding regions for all six human skeletal MyHC genes has been completed and these will be used in Aim II to determine the biochemical properties of the skeletal isoforms, including the roles of the two highly variable loops in the motor domain. To accomplish this goal, the motor domains will be expressed in baculovirus and their enzymatic and motile properties will be characterized. In addition to myosin's motor activity, it is a structural protein, self-assembling into the thick filament. The PI will continue to define the determinants of thick filament assembly using biochemical, phage display and cell culture approaches. Finally, in Aim III, the molecular and cellular biology of an unusual contractile cell type, the myofibroblast, which has features of both muscle and nonmuscle cells, will be explored. In vivo, these cells participate in tissue injury and in wound healing. The PI has shown that these cells express an extensive array of sarcomeric proteins (including six skeletal MyHC genes) and two myogenic regulatory factors, and yet they are not terminally or morphologically differentiated. The number, distribution and gene expression profiles of these cells will be explored in vivo in normal and pathological settings. Using cultured myofibroblast model systems, the role and organization of sarcomeric proteins in myofibroblast contractility will be determined.
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