The activity-induced reduction of myofibrillar Ca2+ sensitivity in mouse skeletal muscle is reversed by dithiothreitol

The aim of this study was to further characterize the reduction of myofibrillar Ca2+ sensitivity in mouse muscle which has been observed after fatigue at 37 degrees C. Muscle bundles and single fibres were isolated from mouse flexor digitorum brevis muscle and studied at 37 degrees C. The single fibres were injected with the Ca2+ indicator indo-1. Muscle fatigue was produced by 0.4 s tetani repeated at 4 s intervals until force had fallen to less than 50% of initial. Excitation-contraction coupling was assessed by measuring the cytosolic calcium concentration ([Ca2+](i)) during tetani, and the maximum Ca2+-activated force and the myofibrillar Ca2+ sensitivity were estimated from a series of tetani at different stimulation frequencies. Two main results were found. (i) The reduction of Ca2+ sensitivity only occurred when the muscle was intensely stimulated leading to fatigue. When the muscle was rested for 10 min at 37 degrees C there was no significant change in Ca2+ sensitivity. (ii) If the membrane-permeant thiol-specific reducing agent dithiothreitol (0.5 mM) was applied to the muscle for 2 min following the fatigue protocol, the reduction in Ca2+ sensitivity was reversed. Dithiothreitol had no effect on Ca2+ sensitivity in unfatigued preparations. There was no effect of fatigue or dithiothreitol on tetanic [Ca2+](i) or on the maximum Ca2+-activated force. These results suggest that intense activity of skeletal muscle at 37 degrees C causes the production of reactive oxygen species which oxidize a target protein. We propose that critical sulphydryl groups on the target protein(s) are converted to disulphide bonds and this reaction reduces Ca2+ sensitivity.