We intend to study developmentally regulated isoenzyme transitions of phosphorylate, phosphofructokinase, adenylate deaminase and phosphoglycerate mutase in uninnervated and innervated human muscle cultures from normal and genetically deficient muscle using immunocytochemical, histochemical and electrophoretic techniques. These studies will lead to a better understanding of factors regulating isoenzyme transitions and the metabolic consequences, when such transitions fail to occur due to genetic defects. We also propose to develop permanent human myoblast cell lines from normal and genetically-deficient muscle by transforming myoblasts derived from primary muscle with a temperature-sensitive mutant SV40 virus. Myoblast transformants, unlike cultured cells from primary tissues do not become senescent, are free of fibroblast contamination, and can be induced to differentiate by temperature shift. These cell lines will provide unlimited supplies of large samples for studying normal and genetically deficient human muscle at early states of differentiation, and will permit a more detailed analysis of human muscle development at the molecular level.
