Manipulation of myogenesis in vitro: reversible inhibition by DMSO

Abstract

A system has been developed for the detailed analysis of the transition from proliferative myoblast to differentiated muscle cell. Dimethylsulfoxide (DMSO) prevents the terminal differentiation of L8 myoblasts in vitro, and its effect is reversible. DMSO (2%) inhibits the fusion of myoblasts to form multinucleate myotubes, the normal increases in activity of creatine phosphokinase (CPK) and acetylcholinesterase, and the synthesis of alpha-actin and acetylcholine receptor protein. Upon removal of DMSO from the medium, a lag precedes the onset of differentiation. The potential to inhibit muscle differentiation reversibly is not specific to DMSO, but is shared by a number of compounds, including dimethylformamide, hexamethylbisacetamide and butyric acid, all potent inducers of gene expression in Friend erythroleukemia cells. L8 cells routinely cease DNA synthesis and initiate fusion and muscle protein synthesis once they are confluent. In the presence of DMSO, however, nearly all cells continue DNA synthesis, even several days after reaching confluence. Protein synthetic patterns of DMSO-inhibited cells are almost indistinguishable from those of untreated myoblasts and distinct from differentiated myotubes. It appears that cells exposed to DMSO are locked indefinitely in a proliferative myoblast stage of development and are unable to enter the Go phase of the cell cycle necessary for initiation of differentiation. DMSO coordinately inhibits all the differentiative parameters measured. In contrast, cytochalasin B uncouples normally linked differentiative events so that fusion is inhibited while muscle-specific protein synthesis proceeds. DMSO has similar effects on both cytochalasin B-treated and fusing control cultures, suggesting that its primary effect is exerted not at the level of fusion but earlier in the differentiative time-table. Once fusion and the synthesis of muscle-specific proteins are well under way, the addition of DMSO is ineffective and differentiation continues in its presence. The potential to manipulate muscle gene expression in vitro makes this system particularly useful for the detailed analysis of the processes involved in the transition to the differentiated state and for determining the linkage of developmental events.