Metalloendoprotease inhibitors that block fusion also prevent biochemical differentiation in L 6 myoblasts ( creatine kinase / commitment )

The effect of metalloendoprotease inhibitors on the biochemical differentiation of the rat skeletal muscle line, L6, was investigated. Confluent unfused L6 cells exposed briefly to 1,10-phenanthroline, a chelator of divalent metal cations, or continuously to dipeptide amide metalloendoprotease substrates that are blocked at the NH2-terminals, Ncarbobenzyloxyserylleucyl amide and N-carbobenzyloxyglycylleucyl amide, did not fuse or express creatine kinase, myosin heavy chain, or a-actin. These effects were reversible and dose-dependent. Exposure to N-carbobenzyloxylglycylglycyl amide, which is not a metalloendoprotease inhibitor, had no effect. As the differentiation in a culture progressed, 1,10phenanthroline became less effective in blocking the accumulation of creatine kinase and myosin heavy chain. Exposure of partially fused cultures to N-carbobenzyloxyseryfleucyl amide prevented any further accumulation of muscle-specific proteins. In confluent cultures where cell division was blocked before the onset of differentiation, N-carbobenzyloxyserylleucyl amide still prevented fusion and the induction of creatine kinase. This indicates that these inhibitors do not act by interfering with the cell cycle. Experiments that measured DNA synthesis rates, plating efficiencies, and the effects ofsequential dipeptide and dimethyl sulfoxide treatments indicate that L6 myoblasts do not irreversibly withdraw from the cell cycle when exposed to N-carbobenzyloxyserylleucyl amide. These results are consistent with the role of a metalloendoprotease in initiating the terminal differentiation of cultured muscle cells. The rat skeletal muscle cell line, L6 (1), undergoes a developmental program in culture similar to muscle cells in vivo. Sparse myoblasts proliferate until confluency, after which they begin withdrawing from the cell cycle (Go state) and subsequently lose their proliferative capacity (commitment) (2, 3). These committed myoblasts then fuse their plasma membranes to form myotubes and biochemically differentiate by synthesizing large amounts of muscle-specific proteins, such as proteins for the contractile apparatus, creatine kinase, and the acetylcholine receptor. These processes result in a terminally differentiated muscle fiber and are collectively referred to as myogenesis. Couch and Strittmatter (4) showed that the metalloendoprotease (MEPr) inhibitors, 1,10-phenanthroline and certain N-carbobenzyloxy dipeptide amides, prevent the Ca2l-dependent fusion of primary rat myoblasts. They identified a MEPr activity in the cytosol of L6 myoblasts that is inhibited by the same compounds that block myoblast fusion (5). These inhibitors also prevent Ca2+-dependent fusion in vesicle secretory systems (6), suggesting that MEPrs may have a general role in catalyzing biological membrane fusion. Our laboratory has recently identified dramatic changes in major plasma membrane glycoproteins during myogenesis of L6 myoblasts. These changes were blocked by the MEPr inhibitors that block fusion (7). We now report that these same MEPr inhibitors also prevent the increased synthesis of muscle-specific proteins during myogenesis. We hypothesize that a MEPr is involved in initiating the terminal differentiation of L6 myoblasts.