Microinjection of Calpastatin Inhibits Fusion in Myoblasts

• Published in: Experimental cell research 1999-02, Vol.247 (1), p.293-303
• Main Authors: Temm-Grove, Constance J., Wert, David, Thompson, V.F., Allen, R.E., Goll, Darrel E.
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Calcium-Binding Proteins - metabolism; Calcium-Binding Proteins - pharmacology; calpain; calpastatin; Cell Differentiation - drug effects; Cell Division - drug effects; Cell Fusion - drug effects; Cells, Cultured; Desmin - analysis; Mice; microinjection; Microinjections; Muscle Fibers, Skeletal - cytology; Muscle Fibers, Skeletal - metabolism; Muscle Fibers, Skeletal - physiology; myoblast fusion; Rats; satellite cells; Stem Cells - cytology; Stem Cells - metabolism; Stem Cells - physiology; Vinculin - analysis
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1998.4362

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12myoblasts contained detectable amounts of μ-calpain before fusion. The levels of calpastatin in C2C12myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.