Calcium regulation of skeletal myogenesis. II. Extracellular and cell surface effects

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Suppression of fusion is obtained by lowering medium [Ca2+] and re-initiated by raising medium [Ca2+]. Previously, we showed that such changes in medium [Ca2+] produced concomitant changes in myoblast [Ca2+] and that a critical cellular concentration of calcium must be present in myoblasts for fusion to occur. In this study, we report on further investigations on the relationship between myoblast [Ca2+] and fusion and also present data which suggest that an outer cell surface pool of calcium is involved in the fusion process. Cellular [Ca2+] must reach greater than 0.8 pmoles/cell and the medium [Ca2+] must be greater than 0.2-0.4 mM for myoblast fusion to occur. These conditions do not have a trigger effect on the entire myoblast population; instead, myoblast fusion was a dose-dependent linear response. If medium [Ca2+] was not maintained at 0.9 mM then cellular [Ca2+] decreased below a critical 0.8 pmoles/cell nucleus and fusion ceased. The cell surface pool of calcium was detected with the calcium antagonist lanthanum. A defined culture medium with 0.9 mM Ca2+ was used to maintain cell viability, and to prevent precipitation of medium components and changes in medium pH with La3+ (0.1 mM). La3+ did not enter the myoblasts as detected by electron microscopy, did not inhibit Ca2+ movement into the cells and the cellular [Ca2+] was sufficient to promote myoblast fusion. Under these conditions, myoblast fusion was inhibited. Morphologically, the fusion-suppressed myoblasts resembled those suppressed by lowered medium [Ca2+]. After removal of the fusion-block by washing, the myotubes that formed were equivalent to those present in unaltered cultures. These results suggest that a La3+ displaceable Ca2+ pool exists at the surface of myoblasts which is involved in myoblast fusion. Thus, it appears that myoblast fusion is dependent on the continuous presence of cell surface calcium and an adequate intracellular Ca2+. An influx of calcium alone is not sufficient to promote myotube formation.