Alterations of enzymatic activities during muscle differentiation in vitro

The activity of creatine phosphokinase, myokinase, and glycogen phosphorylase in muscle cells grown under controlled culture conditions was investigated. Cell fusion and formation of multinucleated muscle fibers were found to be closely associated with a severalfold increase in enzyme activity. The correlation is manifested both in primary cultures and during the differentiation of myogenic cell lines which were maintained for several months under conditions of continuous multiplication. Cytochemical staining for glycogen phosphorylase showed that the increased activity is localized within the multinucleated fibers. Experiments in which the duration of cell fusion was controlled by the concentration of Ca²⁺ in the nutritional medium suggested that the increase in enzyme activity is largely dependent on the continuation of the fusion process. Application of actinomycin D to cultures during the stage of rapid increase in enzymatic activity did not prevent activity from increasing for several hours, whereas application of inhibitors of protein synthesis did inhibit the increase in activity. In the presence of cyclohexamide, the process of cell fusion was almost totally inhibited while in the presence of actinomycin D fusion continued for several hours. These experiments suggest the possibility that the messenger RNA molecules which specify the synthesis of proteins essential for cell fusion and increased enzymatic activity are formed at a developmental stage preceding cell fusion.