Abstract
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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Balcerzak D., Cottin P., Poussard S., Cucuron A., Brustis J. J., and Ducastaing A. (1998) Calpastatin-modulation of m-calpain activity is required for myoblast fusion. Eur. J. Cell Biol. 75, 247–253.
Cottin P., Brustis J. J., Poussard S., Elamrani N., Broncard S., and Ducastaing A. (1994) Ca2+-dependent protease (calpains) and muscle cell differentiation. Biochim. Biophys. Acta 1223, 170–178.
Balcerzak D., Poussard S., Brustis J. J., Elamrani N., Soriano M., Cottin P., and Ducastaing A. (1995) An antisense oligodeoxynucleotide to m-calpain mRNA inhibits myoblast fusion. J. Cell. Set 108, 2077–2082.
Dourdin N., Brustis J. J., Balcerzak D., Elamrani N., Poussard S., Cottin P., and Ducastaing A. (1997) Myoblast fusion requires fibronectin degradation by exteriorized m-calpain. Exp. Cell Res. 235, 385–394.
Kwak K. B., Chung S. S., Kim O. M., Kang M. S., Ha D. B., and Chung C. (1993) Increase in the level of m-calpain correlates with the elevated cleavage of filamin during myogenic differentiation of embryonic muscle cells. Biochim. Biophys. Acta 1175, 243–249.
Kumar A., Shafiq S., Wagdaonkar R., and Stracher A. (1992) The effects of protease inhibitors, leupeptin and E64d, on differentiation of C2C12 myoblasts in tissue culture. CellMol. Biol. 38, 477–483.
Schollmeyer J. E. (1986) Role of Ca2+ and the Ca2+-activated protease in myoblast fusion. Exp. Cell Res. 162, 411–422.
Debus E., Weber K., and Osborn M. (1983) Monoclonal antibodies to desmin, the muscle intermediate filament protein. EMBO J. 12, 2305–2312.
Alterio J., Courtois X., Bechet D., and Martelly I. (1990) Acidic and fibroblast growth factor mRNA are expressed by skeletal muscle satellite cells. Biochem. Biophys. Res. Commun. 166, 1205–1212.
Deluca C. I., Davies P. L., Samis J. A., and Elce J. S. (1993) Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit. Biochim. Biophys. Acta 1216, 81–93.
Deluca C. I., Samis J. A., Graham E. J., Back D. W., and Elce J. S. (1991) Partial cDNA and genomic clones for the large subunit of rat calpains I and II. FASEB J. 5, A 830.
Sorimachi H., Imajoh-Ohmi S., Emori Y., Kawasaki H., Ohno S., Minami Y., and Suzuki K. (1989) Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m-and mu-types: specific expression of the mRNA in skeletal muscle. J. Biol. Chem. 264, 20,106–20,111.
Ishida S., Emori Y., and Suzuki K (1991) Rat calpastatin has diverged primary sequence from other mammalian calpastatins but retains functionally important sequences. Biochim. Biophys. Acta 1088, 436–438.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc.
About this protocol
Cite this protocol
Cottin, P. et al. (2000). Calpains and Myogenesis. In: Elce, J.S. (eds) Calpain Methods and Protocols. Methods in Molecular Biology™, vol 144. Humana Press. https://doi.org/10.1385/1-59259-050-0:173
Download citation
DOI: https://doi.org/10.1385/1-59259-050-0:173
Publisher Name: Humana Press
Print ISBN: 978-0-89603-632-1
Online ISBN: 978-1-59259-050-6
eBook Packages: Springer Protocols