CHARACTERIZATION OF SPONTANEOUS AND ACTION POTENTIAL-INDUCED CALCIUM TRANSIENTS IN DEVELOPING MYOTUBES INVITRO

We have investigated the onset and maturation of action potential- and calcium-induced calcium release from the sarcoplasmic reticulum durin g the differentiation of excitation-contraction coupling in skeletal m uscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo-3 revealed the dynamics, t ime course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contr actions in well-differentiated myotubes are accompanied by brief (200- 500 ms) increases in the concentration of free cytoplasmic calcium. Th ese transients are modulated by sub-threshold concentrations of caffei ne, resulting in a plateau of elevated calcium. Two novel types of cal cium transients were observed in non-contracting myotubes. 1) Fast loc alized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. 2) Upon addition of caffeine, propagating calcium waves a re generated (35-70 mum/s velocity), which are accompanied by contract ures. Aside from caffeine sensitivity, calcium waves and contraction-r elated sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, the se transients may represent calcium-induced calcium release in quiesce nt and active myotubes, respectively. Following one calcium-induced ca lcium release event, myotubes become refractory to new calcium-induced transients; however, action potential-induced transients and FLTs are not blocked. This suggests that these transients occur by distinct re lease mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation.