Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the 45Ca-exchange method to determine free cellular Ca++, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca++). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca++] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca++ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca++] results in a release of the fusion-block and an increase in cellular Ca++ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca++] must exist. The latter suggests further that cell surface Ca++ was also critical.