Calpain and calpastatin in myoblast differentiation and fusion: effects of inhibitors.
Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca(2+)-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-beta and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of mu-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.
Barnoy
S
S
Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel.
Glaser
T
T
Kosower
N S
NS
D000818:Animals; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002459:Cell Fusion / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D009132:Muscles / Q000166:cytology* / Q000187:drug effects; D051381:Rats
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain