Calcium transients in intact rat skeletal muscle fibers in agarose gel

Intact single fibers enzymatically dissociated from rat flexor digitorum brevis muscle were suspended in 0.5% low-melting-temperature agarose 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 potential or trains of action potentials in fibers in agarose were calculated using kinetic parameters previously determined to correct for the calcium-fura 2 kinetic delay. Half times of the calculated calcium transients for single action potentials were 30-fold briefer than the original 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+] transients for fibers containing fura 2 had very similar time courses and waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. The advantages of the agarose gel suspension are discussed.