Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force-length relation and a steepening of the force-frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T-tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T-tubules were clearly delineated. Sulforhodamine B diffused out of the T-tubules with a half-time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the T-tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half-time of 6.3 ± 2.4 min and diffused out of the T-tubules with a half-time of 39 ± 4 s. Vacuole production was eliminated by application of 1 m ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T-tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na + and water exceeds the capacity of the T-tubules and causes vacuole production. The damage to the T-tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.

The effect of eccentric contractions on intracellular Na(+) concentration ([Na(+)](i)) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na(+)](i) was measured with either Na(+)-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na(+)](i) (7.2 +/- 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 +/- 3% of control and increased [Na(+)](i) to 16.3 +/- 1.6 mM (n = 6; P < 0.001). The rise of [Na(+)](i) occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na(+)](i) showed a spatially uniform distribution both at rest and after eccentric contractions. Gd(3+) (20 microM) had no effect on resting [Na(+)](i) or control tetanic force but prevented the rise of [Na(+)](i) and reduced the force deficit after eccentric damage. These data suggest that Na(+) entry after eccentric contractions may occur principally through stretch-sensitive channels.

The effect of eccentric contraction on force generation and intracellular pH (pH(i)) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pH(i) was measured with a pH-sensitive probe, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pH(i) regulation was studied by inducing an acute acid load with the removal of 20-40 mM ammonium chloride, and the rate of pH(i) recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pH(i) recovery by the buffering power. The resting pH(i) after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na(+)/H(+) exchanger is the dominant mechanism for the recovery of pH(i), this suggests that the impairment in the ability of the muscle to regulate pH(i) after eccentric contractions is caused by decreased activity of the Na(+)/H(+) exchanger.

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