The ultimate goal of these investigations is improved diagnosis and treatment of heritable myopathies through elucidation of underlying biochemical and genetic mechanisms. Understanding of these disorders requires correlation of phenotypic and genetic information. Phenotypic analyses of human myopathies in tissue culture have been limited by the restricted size of diagnostic muscle biopsies and the modest proliferative capacity of cultured human muscle cells. This laboratory has undertaken to overcome these obstacles by establishing permanent myogenic cell lines from biopsy material. The primary focus will be the mitochondrial encephalomyopathies, disorders of the electron transport chain, some of which may result from mutations within the 16,569 base pairs of the mitochondrial genome. Collaborative studies will be undertaken, also, of Duchenne muscular dystrophy, DMD, whose gene has been localized within several hundred thousand base pairs on the X-chromosome. Preliminary results from this laboratory have demonstrated the feasibility of obtaining human myogenic cell lines, established by reversible transformation with the early region of SV40. In other studies defects of oxidative metabolism have been demonstrated in differentiating muscle cultures from patients with mitochondrial encephalomyopathies. Other groups have demonstrated a muscle-specific transcript encoded within the putative DMD region of the X-chromosome. This proposal seeks, first, to improve establishment and maintenance of human myogenic cell lines through modifications of the SV40 large T antigen procedure and by transfection with other oncogenes, some of which may extend proliferation of human myoblasts without full transformation; second, to identify precisely the defective component of the electron transport chain and the location of its encoding gene, through studies of oxidative metabolism and mitochondrial protein synthesis in cell lines from patients with mitochondrial encephalomyopathies, and in cybrids (produced from nuclei and cytoplasm of control and patient cell lines, respectively); third, to study expression of putative DMD gene sequences in Lyonized normal and DMD myogenic cell lines established from DMD heterozygotes also heterozygous for the X- linked marker enzyme, G6PD. It is anticipated that permanent myogenic cell lines will also prove useful for phenotypic analyses of other heritable myopathies, expression of which may require myogenic differentiation.
