Spontaneous and repetitive calcium transients in C2C12 mouse myotubes during in vitro myogenesis

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+](i)) in the mouse muscle cell line C2C12 during in vitro myogenesis. Three different patterns of changes in [Ca2+](i) were observed: (i) [Ca2+](i) oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+](i) spikes) and (iii) [Ca2+](i) spikes detectable in the entire myotube (global [Ca2+](i) spikes), [Ca2+](i) oscillations and localized [Ca2+](i) spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2C12 cells. Global [Ca2+](i) spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+](i) oscillations and localized [Ca2+](i) spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+](i) spikes, membrane currents and fluorescence signals were measured simultaneously, These experiments revealed that global [Ca2+](i) spikes were correlated with an inward current, Moreover, while the depletion of the Ca2+ stores blocked [Ca2+](i) oscillations and localized [Ca2+](i) spikes, it only reduced the amplitude of global [Ca2+](i) spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+](i) changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+](i) spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.