In frog skeletal muscle fibres subjected to repeated intermittent tetanic contractions, force production decreases due to reduced tetanic Ca²⁺ transients. Relaxation of force also slows due to slowed Ca²⁺ uptake into the sarcoplasmic reticulum (SR). Repeated tetanic stimulation also causes mitochondrial Ca²⁺ to increase, which may be detrimental to muscle performance. We were interested to determine if muscle fibres from a slow-twitch mammalian muscle showed similar changes during a bout of repetitive activity.

Adult NMRI mice were killed by cervical dislocation. Intact single fibres were mechanically dissected from the soleus muscles. Clips were attached to the tendons. One clip was attached to a force transducer and the other to an adjustable holder. The fibre length was set to the length giving maximum tetanic force. A total of 21 fibres was used in these experiments. In seven fibres, free myoplasmic Ca²⁺ ([Ca²⁺]_i) was measured using the fluorescent indicator, Indo-1. In the remaining 13 fibres, the free Ca²⁺ in the mitochondrial matrix was monitored using changes in the Rhod-2 fluorescent signal (expressed in arbitrary units, a.u.). Fibres were stimulated repeatedly with 70 Hz, 500 ms tetani at 2 s intervals for 1000 tetani or until force fell to 40 % of its initial value. All values shown are means ± S.E.M. Significance was tested with Student’s t test.

Tetanic [Ca²⁺]_i rose from 1.74 ± 0.26 mM in the first tetanus to 3.10 ± 0.87 mM (n = 7) in the tenth tetanus, while force fell to 93.5 ± 2.3 % (n = 7) of the starting value. Thereafter, tetanic [Ca²⁺]_i fell slightly and in the last tetanus of the series was 1.91 ± 0.50 mM (n = 27) and not significantly higher than control. Tetanic force also decreased slightly and was significantly reduced from control (P < 0.05) to 74 ± 6 % (n = 6) in the final tetanus. There was no evidence of any slowing of Ca²⁺ uptake into the SR as basal [Ca²⁺]_i was 71.2 ± 14.6 nM (n = 7) and 89.2 ± 15.8 nM (n = 7) prior to the first and last tetanus of the series, respectively.

In the 14 fibres loaded with Rhod-2 and stimulated as described above, 10 fibres showed a marked increase in the Rhod-2 signal (indicating that mitochondrial Ca²⁺ had increased) while the remaining four showed no change. In those fibres in which mitochondrial Ca²⁺ increased, a peak Rhod-2 signal of 9.1 ± 1.7 a.u. (n = 9) occurred within 50 tetani. Thereafter, the Rhod-2 signal fell slowly to about half of this peak value by the time stimulation was stopped. Interestingly, there was a significant inverse correlation between the amplitude of the Rhod-2 signal at the end of stimulation and the extent of force depression. Thus mitochondrial Ca²⁺ accumulation does not impair force production.

All procedures accord with current National guidelines.