Isolated myofibers from rat skeletal muscle in suspension: cellular morphology and calcium homeostasis.

Myofiber suspensions with 80% viability (morphological intactness) were prepared from rat m. flexor digitorum brevis by a 6-hour collagenase treatment. The viable fibers had intact sarcolemmas and generally showed continuous basal laminae. The intracellular compartments exhibited normal muscular morphology. The suspended myofibers showed good tolerance against extracellular calcium (up to 30 mM), but were susceptible to 15 mM EGTA. Calcium ionophore A23187 induced contraction, with loss of viability in both calcium-containing and nominally calcium-free Krebs-Ringer medium. EGTA prevented the ionophore effect, but the muscle cells collapsed as soon as calcium was replenished. A calcium paradox, as observed with perfused heart, could not be induced in isolated skeletal myocytes. This suspended cell system should provide a useful tool for investigating relationships between metabolic parameters and muscle physiology. The extracellular environment can be manipulated easily, and cellular responses of individual skeletal muscle fibers can be measured and visualized.