Mitochondrial and myoplasmic Ca2+ in single fibres from mouse limb muscles during repeated tetanic contractions

Previous studies on single fast-twitch fibres from mouse toe muscles have shown marked fatigue-induced changes in the free myoplasmic Ca2+ (Ca2+i), while mitochondrial Ca2+ remained unchanged. We have now investigated whether muscle fibres from the legs of mice respond in a similar way. Intact, single fibres were dissected from the soleus and extensor digitorum longus (EDL) muscles of adult mice. To measure Ca2+i, indo-1 was injected into the isolated fibres. Mitochondrial Ca2+ was measured using Rhod-2 and confocal laser microscopy. Fatigue was induced by up to 1000 tetanic contractions (70 Hz) given at 2 s intervals. In soleus fibres, there was no significant decrease in tetanic Ca2+i at the end of the fatiguing stimulation, whereas tetanic force was significantly reduced by about 30 %. In 10 out of 14 soleus fibres loaded with Rhod-2 and subjected to fatigue, mitochondrial Ca2+ increased to a maximum after about 50 tetani; this increase was fully reversed within 20 min after the end of stimulation. The force-frequency curve of the non-responding soleus fibres was shifted to higher frequencies compared to that of the responding fibres. In addition, eight out of nine Rhod-2-loaded EDL fibres showed similar changes in mitochondrial Ca2+ during and after a period of fatiguing stimulation. The stimulation-induced increase in mitochondrial Ca2+ was reduced when mitochondria were depolarised by application of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, whereas it was increased by application of an inhibitor of the mitochondrial Na+/Ca2+ exchange (CGP-37157). In conclusion, isolated slow-twitch muscle fibres show only modest changes in tetanic force and Ca2+i during repeated contractions. The increase in mitochondrial Ca2+ does not appear to be essential for activation of mitochondrial ATP production, nor does it cause muscle damage.