1. The role of the myoplasmic free Mg2+ concentration ([Mg2+]i) in fatigue was studied in intact single fibres isolated from mouse skeletal muscle. Fatigue was produced by repeated tetanic stimulation. The fluorescent Mg2+ indicator furaptra was pressure injected into fibres. In vivo calibrations were performed to convert fluorescence signals into [Mg2+]i. 2. [Mg2+]i at rest was 0.78 +/- 0.05 mM (mean +/- S.E.M., n = 14). An increase of the extracellular [Mg2+] from 0.5 to 20 mM resulted in a small elevation of [Mg2+]i (86-mu-m in 5 min). Removal of extracellular Na+ did not affect [Mg2+]i. An intracellular alkanization of about 0.6 pH units gave a [Mg2+]i reduction of 65-mu-M. 3. During fatiguing stimulation [Mg2+]i initially remained almost constant and it then suddenly started to rise towards the end of the stimulation period. The onset of the [Mg2+]i rise was always followed by a rapid tension decline. In fatigue [Mg2+], was approximately twice as high as at rest. 4. Fibres were injected with MgCl2 to study if the rise in [Mg2+]i could explain the tension decline in fatigue. An elevation of [Mg2+]i was accompanied by a tension reduction but the [Mg2+]i for a given tension was generally much higher in rested fibres injected with MgCl2 than in fatigued fibres. Thus the rise in [Mg2+]i as such cannot explain the tension reduction in fatigue. 5. Injection of MgCl2 was also used to assess the intracellular Mg2+ buffering. The mean Mg2+ buffer power (i.e. the ratio of the change in [Mg2+]i to the amount of Mg2+ added) was 0.62. 6. ATP is the quantitatively most important binding site for Mg2+ at rest and ATP breakdown is then a likely source of the [Mg2+]i increase in fatigue. The role of ATP breakdown in the increase of [Mg2+]i was studied with metabolic inhibition: fibres were exposed to iodoacetic acid to inhibit glycolysis and cyanide to inhibit oxidative phosphorylation. The pattern during metabolic inhibition was similar to that observed during fatigue. After remaining almost constant during a lengthy period, [Mg2+]i rose rapidly and this rise preceded a period of rapid tension decline. The fibres thereafter went into rigor and [Mg2+]i stabilized at an elevated level; the mean [Mg2+]i increase in rigor was 1.30 mm. 7. We have used modelling to determine the likely change in the intracellular ATP concentration ([ATP]i) for the observed changes in [Mg2+]i. Our model could with reasonable accuracy predict (i) the intracellular Mg2+ buffer power, (ii) the change in [Mg2+]i due to an alkalinization, and (iii) the [Mg2+]i increase in rigor. 8. The modelled [ATP]i for the average [Mg2+]i in fatigue was 1.74 mM, which represents an [ATP]i reduction to about 30 % of the original. The time course of the [Mg2+]i recovery after fatiguing stimulation suggests that a large fraction of the ATP which broke down during fatigue was deaminated to IMP (inosine monophosphate). 9. In conclusion, fatigue was associated with a marked increase of [Mg2+]i. This increase was, however, too small to explain the tension reduction in fatigue. A likely scenario is instead that the [Mg2+]i increase reflects a breakdown of ATP and that this breakdown contributes to the tension decline in fatigue.
