Vacuole formation in fatigued single muscle fibres from frog and mouse

Force recovery from fatigue in skeletal muscle may be very slow. Gross morp hological changes with vacuole formation in muscle cells during the recover y period have been reported and it has been suggested that this is the caus e of the delayed force recovery. To study this we have used confocal micros copy of isolated, living muscle fibres from Xenopus and mouse to visualise transverse tubules (t-tubules) and mitochondria and to relate possible fati gue-induced morphological changes in these to force depression. T-tubules w ere stained with either RH414 or sulforhodamine B and mitochondrial stainin g was with either rhodamine 123 or DiOC(6)(3). Fatigue was produced by repe ated, short tetanic contractions. Xenopus fibres displayed a marked vacuola tion which started to develop about 2 min after fatiguing stimulation, reac hed a maximum after about 30 min, and then receded in about 2 h. Vacuoles w ere never seen during fatiguing stimulation. The vacuoles developed from lo calised swellings of t-tubules and were mostly located in rows of mitochond ria. Mitochondrial staining, however, showed no obvious alterations of mito chondrial structure. There was no clear correlation between the presence of vacuoles and force depression; for instance, some fibres showed massive va cuole formation at a time when force had recovered almost fully. Vacuole fo rmation was not reduced by cyclosporin A, which inhibits opening of the non -specific pore in the mitochondrial inner membrane. In mouse fibres there w as no vacuole formation or obvious changes in mitochondrial structure after fatigue, but still these fibres showed a marked force depression at low st imulation frequencies ('low-frequency fatigue'). Vacuoles could be produced in mouse fibres by glycerol treatment and these vacuoles were not associat ed with any force decline. In conclusion, vacuoles originating from the t-t ubular system develop after fatigue in Xenopus but not in mouse fibres. The se vacuoles are not the cause of the delayed force recovery after fatigue.