The effect of cromakalim on intracellular [Ca2+] in isolated rat skeletal muscle during fatigue and metabolic blockade

The effects of the ATP-sensitive K+ channel (K-ATP channel) opener cromakalim on excitation-contraction (E-C) coupling were studied in skeletal muscle during fatiguing and non-fatiguing activity. Intracellular calcium concentration ([Ca2+](i)) was monitored using the fluorescent indicator fura-2 in isolated single skeletal muscle fibres enzymatically dissociated from rat flexor digitorum brevis. A protocol of tetanic stimulation (50 Hz for 300 ms) with progressively shorter durations between tetani was used to induce E-C coupling failure in these cells. Cromakalim (100-800 mu M) had little effect on peak [Ca2+](i) during twitch and non-fatiguing tetanic stimulation. However, with 0.4 s between tetani, 100 mu M cromakalim decreased peak tetanic [Ca2+](i) from 1.47 +/- 0.11 mu M to 835 +/- 55 nM, but did not affect resting [Ca2+](i) (control, 220 +/- 40 nM; with cromakalim, 171 +/- 33 nM). Cyanide (2 mM) decreased tetanic [Ca2+](i) and increased resting [Ca2+](i) during the stimulus protocol; with 0.4 s between tetani, peak [Ca2+](i) was 820 +/- 50 nM and resting [Ca2+](i) was 443 +/- 32 nM. The ability of cromakalim to inhibit E-C coupling was enhanced by the presence of cyanide. Complete blockade of metabolism by cyanide and iodoactetate (0.1 mM) caused a marked rise in resting [Ca2+](i) and inhibition of the tetanic rise of [Ca2+](i). With cromakalim (100 mu M) present, E-C coupling failed during metabolic blockade but without a significant increase in resting [Ca2+](i). These results are consistent with a role for the K-ATP channel in the failure of Ca2+ release during fatigue.