CALCIUM TRANSIENTS IN INTACT RAT SKELETAL-MUSCLE FIBERS IN AGAROSE-GEL

Intact single fibers enzymatically dissociated from rat flexor digitor um brevis muscle were suspended in 0.5% low-melting-temperature agaros e gel to minimize fiber movement during action potentials or trains of action potentials. Resting Ca2+ concentration ([Ca2+]) and changes in [Ca2+] were monitored using the fluorescent calcium indicator fura 2. The time course and waveform of [Ca2+] transients during an action po tential or trains of action potentials in fibers in agarose were calcu lated using kinetic parameters previously determined to correct for th e calcium-fura 2 kinetic delay. Half times of the calculated calcium t ransients for single action potentials were 30-fold briefer than the o riginal fura 2 signals. To confirm the time course and waveform of the calculated calcium transients, changes in [Ca2+] were monitored using the more rapidly equilibrating calcium indicator mag-fura 2. [Ca2+] t ransients for fibers containing fura 2 had very similar time courses a nd waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. Th e advantages of the agarose gel suspension are discussed.