Excitation‐induced Ca²⁺ influx and muscle damage in the rat: loss of membrane integrity and impaired force recovery

Abstract

Prolonged or unaccustomed exercise leads to loss of contractility and muscle cell damage. The possible role of an increased uptake of Ca 2+ in this was explored by examining how graded fatiguing stimulation, leading to a graded uptake of Ca 2+ , results in progressive loss of force, impairment of force recovery, and loss of cellular integrity. The latter is indicated by increased [14 C]sucrose space and lactic acid dehydrogenase (LDH) release. Isolated rat extensor digitorum longus (EDL) muscles were allowed to contract isometrically using a fatiguing protocol with intermittent stimulation at 40 Hz. Force declined rapidly, reaching 11% of the initial level after 10 min and stayed low for up to 60 min. During the initial phase (2 min) of stimulation 45 Ca uptake showed a 10-fold increase, followed by a 4-to 5-fold increase during the remaining period of stimulation. As the duration of stimulation increased, the muscles subsequently regained gradually less of their initial force. Following 30 or 60 min of stimulation, resting 45 Ca uptake, [ 14 C]sucrose space, and LDH release were increased 4-to 7-fold, 1.4-to 1.7-fold and 3-to 9-fold, respectively (P < 0.001). The contents of Ca 2+ and Na + were also increased (P < 0.01), a further indication of loss of cellular integrity. When fatigued at low [Ca 2+ ] o (0.65 mM), force recovery was on average twofold higher than that of muscles fatigued at high [Ca 2+ ] o (2.54 mM). Muscles showing the best force recovery also had a 41% lower total cellular Ca 2+ content (P < 0.01). In conclusion, fatiguing stimulation leads to a progressive functional impairment and loss of plasma membrane integrity which seem to be related to an excitation-induced uptake of Ca 2+ . Mechanical strain on the muscle fibres does not seem a likely mechanism since very little force was developed beyond 10 min of stimulation. Intense exercise, such as extended periods of running, strength training, sprinting or eccentric exercise, disrupts the normal ultrastructure of skeletal muscle (Waterman-Storer, 1991;Belcastro, 1993;Belcastro et al. 1998). Impaired force recovery seen after intense exercise can be due to structural damage such as myofibrillar and cytoskeletal disruptions often leading to Z-disc streaming (Hoppeler, 1986; WatermanStorer, 1991;Appell et al. 1992;Gibala et al. 1995), as well as disturbances to mitochondria and the SR-T-tubular system (Hoppeler, 1986;Gibala et al. 1995). Loss of sarcolemmal integrity has also repeatedly been reported after prolonged or unaccustomed exercise (e.g. Stupka et al. 2001;Clarkson & Hubal, 2002), contributing to the functional impairment.Several mechanisms have been proposed to explain the functional impairment and it is unlikely to be caused by one single factor. The causes of functional impairment could be either mechanical damage due to eccentric contractions (Armstrong, 1986(Armstrong, , 1990, loss of excitability (Nielsen & Overgaard, 1996;Clausen, 1996;Overgaard et al. 1999;Carlsen & Villarin, 2002), or Ca 2+ -induced damage (Gissel & Cl...

Skeletal Muscle Fatigue: Cellular Mechanisms

“…The combined effect of influx and efflux is that net Ca 2ϩ flux is normally quite small. Fast-twitch rat muscle stimulated in vitro at 40 Hz continuously or intermittently for a total of 30 s shows net uptake of 50 -100 mol Ca 2ϩ /kg wet wt (183,313), equivalent to ϳ5-10% of the Ca 2ϩ already present in the SR endogenously and representing Ͻ5% of the available SR Ca 2ϩ capacity. With continuous stimulation at 1 Hz for 2-4 h, fast-twitch rat muscles increase their Ca 2ϩ content to ϳ2.5 times the endogenous level, whereas slow-twitch muscle shows little increase (183), consistent with the endogenous Ca 2ϩ and maximum SR capacity observed in such muscle types (176,440).…”

Excitation-induced cell damage and β2-adrenoceptor agonist stimulated force recovery in rat skeletal muscle

“…After 30 -60 min of fatiguing stimulation, the cell membranes are leaky, as evidenced by an increased resting uptake of 45 Ca, resting [ 14 C]sucrose space, and release of LDH (22). The present results indicate that despite the damaged membranes, stimulation of the Na ϩ -K ϩ pump by hormones, a ␤ 2 -agonist and their second messenger cAMP, improves recovery of tetanic force.…”

“…The muscles were then allowed to rest or were fatigued for 5, 10, 30, or 60 min. Force development was recorded during the fatiguing stimulation and, as previously described (22), declined by 88% within 10 min (data not shown). Subsequently, force recovery was tested at 90 Hz (0.5-s pulse trains) at intervals up to 240 min after cessation of stimulation.…”

“…EDL muscles were exposed to fatiguing stimulation using a protocol previously shown to elicit muscle cell damage and long-lasting loss of force (22). This standard fatiguing stimulation protocol was applied in all experiments.…”

“…This was associated with a considerable reduction in tetanic force lasting for at least 240 min (22). In another study on rat muscles, we found that sarcolemmal leaks induced by electroporation and documented by increases in [ 14 C]sucrose space and LDH release were associated with a graded and partly reversible loss of force (11).…”