Calpains and Myogenesis

There is now a considerable literature suggesting that the calpain/calpastatin system may be involved in myogenesis (1 –7 ). Primary myoblasts in culture at first proliferate, but then usually cease to divide, in response to complex signals. The cells then migrate, align, and fuse to form the multinucleated syncytium or myotube, a process that clearly involves major remodeling of the cell membrane (1 ). Many factors such as growth factors, hormones, calpain antibodies, and inhibitors may be added to the culture system to investigate the mechanisms of cell fusion. The emphasis in this chapter is, however, on the use of antisense oligonucleotides based on calpain or calpastatin cDNA sequences to investigate the role of this protease system in the cell fusion process (1 ,3 ). At the present time, this appears to represent the only reported use of antisense methods in the calpain field. We describe here protocols for working with myoblast cultures that permit investigation of the roles of calpain and calpastatin in myogenesis, in carefully controlled and highly reproducible conditions. The use of reverse transcriptase-polymerase chain reaction (RT/PCR) methods to quantify mRNA levels of members of the calpain system in these myoblast cultures has been described elsewhere (1 ).