Vacuole formation in fatigued single muscle fibres from frog and mouse

Force recovery from fatigue in skeletal muscle may be very slow. Gross morphological changes with vacuole formation in muscle cells during the recovery period have been reported and it has been suggested that this is the cause of the delayed force recovery. To study this we have used confocal microscopy of isolated, living muscle fibres from Xenopus and mouse to visualise transverse tubules (t-tubules) and mitochondria and to relate possible fatigue-induced morphological changes in these to force depression. T-tubules were stained with either RH414 or sulforhodamine B and mitochondrial staining was with either rhodamine 123 or DiOC6(3). Fatigue was produced by repeated, short tetanic contractions. Xenopus fibres displayed a marked vacuolation which started to develop about 2 min after fatiguing stimulation, reached a maximum after about 30 min, and then receded in about 2 h. Vacuoles were never seen during fatiguing stimulation. The vacuoles developed from localised swellings of t-tubules and were mostly located in rows of mitochondria. Mitochondrial staining, however, showed no obvious alterations of mitochondrial structure. There was no clear correlation between the presence of vacuoles and force depression; for instance, some fibres showed massive vacuole formation at a time when force had recovered almost fully. Vacuole formation was not reduced by cyclosporin A, which inhibits opening of the non-specific pore in the mitochondrial inner membrane. In mouse fibres there was no vacuole formation or obvious changes in mitochondrial structure after fatigue, but still these fibres showed a marked force depression at low stimulation frequencies ('low-frequency fatigue'). Vacuoles could be produced in mouse fibres by glycerol treatment and these vacuoles were not associated with any force decline. In conclusion, vacuoles originating from the t-tubular system develop after fatigue in Xenopus but not in mouse fibres. These vacuoles are not the cause of the delayed force recovery after fatigue.