Glutathione depression alters cellular mechanisms of skeletal muscle fatigue in early stage of recovery and prolongs force depression in late stage of recovery.

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body wt/day) treatment for 5 days, which decreased GSH content to ∼10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N = 17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR.

Preconditioning contractions prevent the delayed onset of myofibrillar dysfunction after damaging eccentric contractions.

KEY POINTS: We examined the mechanisms underlying the positive effect of preconditioning contractions (PCs) on the recovery of muscle force after damaging eccentric contractions (ECCs). The mechanisms underlying the immediate force decrease after damaging ECCs differ from those causing depressed force with a few days' delay, where reactive oxygen species (ROS) produced by invading immune cells play an important causative role. PCs counteracted the delayed onset force depression and this could be explained by prevention of immune cell invasion, which resulted in decreased myeloperoxidase-mediated ROS production, hence avoiding cell membrane disruption, calpain activation and degenerative changes in myosin and actin molecules. ABSTRACT: Preconditioning contractions (PCs) have been shown to result in markedly improved contractile function during the recovery periods after muscle damage from eccentric contractions (ECCs). Here, we examined the mechanisms underlying the beneficial effect of PCs with a special focus on the myofibrillar function. Rat medial gastrocnemius muscles were exposed to 100 repeated damaging ECCs in situ and excised immediately (recovery 0, REC0) or after 4 days (REC4). PCs with 10 repeated non-damaging ECCs were applied 2 days before the damaging ECCs. PCs improved in situ maximal isometric torque at REC4. Skinned muscle fibres were used to directly assess changes in myofibrillar function. PCs prevented the damaging ECC-induced depression in maximum Ca2+ -activated force at REC4. PCs also prevented the following damaging ECC-induced effects at REC4: (i) the reduction in myosin heavy chain and actin content; (ii) calpain activation; (iii) changes in redox homeostasis manifested as increased expression levels of malondialdehyde-protein adducts, NADPH oxidase 2, superoxide dismutase 2 and catalase, and activation of myeloperoxidase (MPO); (iv) infiltration of immune cells and loss of cell membrane integrity. Additionally, at REC0, PCs enhanced the expression levels of heat shock protein (HSP) 70, HSP25, and αB-crystallin in the myofibrils and prevented the increased mRNA levels of granulocyte-macrophage colony-stimulating factor and interleukin-6. In conclusion, PCs prevent the delayed force depression after damaging ECCs by an HSP-dependent inhibition of degenerative changes in myosin and actin molecules caused by myeloperoxidase-induced membrane lysis and subsequent calpain activation, which were triggered by an inflammatory reaction with immune cells invading damaged muscles.

Muscle dysfunction associated with adjuvant-induced arthritis is prevented by antioxidant treatment.

BACKGROUND: In addition to the primary symptoms arising from inflamed joints, muscle weakness is prominent and frequent in patients with rheumatoid arthritis (RA). Here, we investigated the mechanisms of arthritis-induced muscle dysfunction in rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced in the knees of rats by injection of complete Freund's adjuvant and was allowed to develop for 21 days. Muscle contractile function was assessed in isolated extensor digitorum longus (EDL) muscles. To assess mechanisms underlying contractile dysfunction, we measured redox modifications, redox enzymes and inflammatory mediators, and activity of actomyosin ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase. RESULTS: EDL muscles from AIA rats showed decreased tetanic force per cross-sectional area and slowed twitch contraction and relaxation. These contractile dysfunctions in AIA muscles were accompanied by marked decreases in actomyosin ATPase and SR Ca(2+)-ATPase activities. Actin aggregates were observed in AIA muscles, and these contained high levels of 3-nitrotyrosine and malondialdehyde-protein adducts. AIA muscles showed increased protein expression of NADPH oxidase 2/gp91(phox), neuronal nitric oxide synthase, tumor necrosis factor α (TNF-α), and high-mobility group box 1 (HMGB1). Treatment of AIA rats with EUK-134 (3 mg/kg/day), a superoxide dismutase/catalase mimetic, prevented both the decrease in tetanic force and the formation of actin aggregates in EDL muscles without having any beneficial effect on the arthritis development. CONCLUSIONS: Antioxidant treatment prevented the development of oxidant-induced actin aggregates and contractile dysfunction in the skeletal muscle of AIA rats. This implies that antioxidant treatment can be used to effectively counteract muscle weakness in inflammatory conditions.

Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle.

The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release.

The effects of eccentric contraction on myofibrillar proteins in rat skeletal muscle.

The present study investigated the effects of eccentric muscle contractions (ECC) on the content of myofibrillar proteins (my-proteins) and the catalytic activity of myofibrillar ATPase (my-ATPase) in skeletal muscles. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC or isometric contractions (ISC) and used for measures of force output and for biochemical analyses, respectively. Whereas in ISC-treated muscles, full restoration of tetanic force was attained after 2 days of recovery, force developed by ECC-treated muscles remained depressed (P < 0.05) after 6 days. The total my-protein content and the relative content of myosin heavy chain (MHC) in total my-proteins were unaltered during 4 days of recovery after ECC, but fell (P < 0.05) to 55.9 and 63.4% after 6 days of recovery, respectively. my-ATPase activity expressed on a my-protein weight basis was unaltered immediately after ECC. However, it decreased (P < 0.05) to 75.3, 45.3, and 49.3% after 2, 4 and 6 days of recovery, respectively. Total maximal calpain activity measured at 5 mM Ca(2+) was significantly augmented (P < 0.05) after 2 days of recovery, reaching a level of threefold higher after 6 days. These alterations were specific for ECC and not observed for ISC. These results suggest that depressions in my-ATPase activity contribute to ECC-induced decreases in force and power which can take a number of days to recover.

Chicken breast attenuates high-intensity-exercise-induced decrease in rat sarcoplasmic reticulum Ca2+ handling.

This study was conducted to determine whether dietary chicken-breast extract (CBEX), a rich source of histidine-containing dipeptides, could modify exercise-induced changes in sarcoplasmic reticulum (SR) function. After 5 weeks of dietary CBEX, SR Ca2+-handling ability was examined in the vastus lateralis muscles of rats subjected to high-intensity running for 2.5 min. Dietary CBEX caused an approximately 15% and 45% increase (p<.01) in muscle carnosine and anserine concentrations, respectively. In resting muscles, depressions in SR Ca2+-ATPase activity were evoked by dietary CBEX without concomitant changes in SR Ca2+ uptake and release rates. The data confirm that high-intensity exercise depresses SR Ca2+ handling. In spite of the same run time, SR Ca2+ handling was reduced to a lesser degree in muscles of CBEX-containing-chow-fed rats than in standard-chow-fed rats (p<.05). These results suggest that dietary CBEX might attenuate deteriorations in SR Ca2+-handling ability that occur with high-intensity exercise.

Time course of changes in in vitro sarcoplasmic reticulum Ca2+-handling and Na+-K+-ATPase activity during repetitive contractions.

Time-dependent changes in sarcoplasmic reticulum (SR) Ca2+-handling and Na+-K+-ATPase activity, as assessed in vitro, were investigated in the superficial (GS) and deep regions (GD) of rat gastrocnemius muscles undergoing short-term (up to 30 min) electrical stimulation. There was a rapid and progressive loss of force output during the first 5 min of stimulation. For GS, significant depressions (P < 0.05) in SR Ca2+-uptake rate and Ca2+-ATPase activity were observed during only the first 1 min. No further reductions occurred with stimulation time. SR Ca2+-release rate was significantly (P < 0.05) decreased at 3 min. For GD, significant reductions (P < 0.05) in Ca2+-uptake rate, Ca2+-release rate and Ca2+-ATPase activity were manifested after 3, 5, and 5 min, respectively. A decay in Na+-K+-ATPase activity was found only in 1-min stimulated GD and 30-min stimulated GS. After 30 min, the depressed functions reverted to resting levels in GD but not in GS. The alterations in any variables examined were not parallel with changes in force output. These results suggest that, at least under the conditions used in this study, in vivo disruptions in cation regulation mediated by vigorous contractile activity would be attributable primarily to events other than structural alterations to the respective proteins.

Alterations in in vitro function and protein oxidation of rat sarcoplasmic reticulum Ca2+-ATPase during recovery from high-intensity exercise.

The hypothesis tested in this study was that the extent to which sarcoplasmic reticulum (SR) Ca(2+)-ATPase is oxidized would correlate with a decline in its activity. For this purpose, changes in the SR Ca(2+)-sequestering ability and the contents of carbonyl and sulfhydryl groups during recovery after exercise were examined in the superficial portions of vastus lateralis muscles from rats subjected to 5 min running at an intensity corresponding to maximal oxygen uptake (50 m min(-1), 10% gradient). A single bout of exercise elicited a 22.4% reduction (P < 0.05) in SR Ca(2+)-ATPase activity. The decreased activity progressively reverted to normal levels during recovery after exercise, reaching normal levels after 60 min of recovery. This change was paralleled by a depressed SR Ca(2+)-uptake rate, and the proportional alteration in these two variables resulted in no change in the ratio of Ca(2+)-uptake rate to Ca(2+)-ATPase activity. The contents of SR Ca(2+)-ATPase protein and sulfhydryl groups in microsomes were unchanged after exercise and during recovery periods. In contrast, the content of carbonyl groups in SR Ca(2+)-ATPase behaved in an opposite manner to that of SR Ca(2+)-ATPase activity. An approximately 80% augmentation (P < 0.05) in the carbonyl group content occurred immediately after exercise. The elevated carbonyl content decreased towards normal levels during 60 min of recovery. These results are strongly suggestive that oxidation of SR Ca(2+)-ATPase is responsible, at least in part, for a decay in the SR Ca(2+)-pumping function produced by high-intensity exercise and imply that oxidized proteins may be repaired during recovery from exercise.

Effects of high-intensity training and acute exercise on in vitro function of rat sarcoplasmic reticulum.

To evaluate the effects of high-intensity training and/or a single bout of exercise on in vitro function of the sarcoplasmic reticulum (SR), the rats were subjected to 8 weeks of interval running program (final training: 2.5-min running x 4 sets per day, 50 m/min at 10% incline). Following training, SR function, i.e., Ca2+-ATPase activity and Ca2+-uptake and release rates, was examined in homogenates of the superficial region of the vastus lateralis muscle from rats subjected to a single bout of treadmill running (50 m/min at 10% incline) for 2.5 min or to exhaustion. Training brought about a 12.4% increase (P < 0.05) in SR Ca2+-uptake rate in rested muscles. This change was not accompanied by alterations in Ca2+-ATPase activity, Ca2+-release rate, Ca2+ dependence of enzyme and protein contents of Ca2+-ATPase and ryanodine receptor. A single bout of high-intensity exercise to exhaustion evoked significant reductions (P < 0.05) in SR function, irrespective of whether or not the animals were trained. For 2.5-min run and exhausted rats, no differences existed between SR functions of untrained and trained muscles. These data suggest that high-intensity training may be capable of enhancing SR Ca2+-sequestering ability, and may not protect against decreasing SR function with high-intensity exercise.

Oxidation of myosin heavy chain and reduction in force production in hyperthyroid rat soleus.

We tested the hypothesis that a force reduction in hyperthyroid rat soleus muscle would be associated with oxidative modification in myosin heavy chain (MHC). Daily injection of thyroid hormone [3,5,3'-triiodo-L-thyronine (T3)] for 21 days depressed isometric forces of whole soleus muscle across a range of stimulus frequencies (P < 0.01). In fiber bundles, hyperthyroidism also led to pronounced reductions (P < 0.01) in both K+ - and 4-chloro-m-cresol-induced contracture forces. The degrees of the reductions were similar between these two contractures that were induced by distinct reagents. Treatment with T3 elicited a significant decrease ( approximately 14%; P < 0.05) in the relative content of MHC contained in myofibrillar proteins. The content of carbonyl groups in myofibrillar protein extracts was elevated (P < 0.05) by approximately 50% in T3-treated muscles. Immunoblot analyses on T3-treated muscles showed a greater increase (106%; P < 0.05) of the carbonyl content in MHC than in myofibrillar protein extracts. These data suggest that in hyperthyroidism the decrease in force production of skeletal muscles may stem primarily from failure in myofibrillar protein function resulting from oxidative modification of MHC.

Oxidation of sarcoplasmic reticulum Ca(2+)-ATPase induced by high-intensity exercise.

Ca(2+)-ATPase and Ca(2+)-pumping activities by the sarcoplasmic reticulum (SR) and the amounts of sulphydryl and carbonyl groups contained in the SR protein were examined in the superficial portion of the gastrocnemius and vastus lateralis muscles of the rat after high-intensity treadmill runs to exhaustion (average time to exhaustion: 363 s). Exercise at the estimated maximal O(2) uptake rate led to 16% and 34% reductions in SR Ca(2+)-ATPase activity ( P<0.01) and Ca(2+) uptake rate ( P<0.01), respectively. The carbonyl group content in SR Ca(2+)-ATPase, assessed by immunoblotting analysis, was increased by 127% after exercise ( P<0.05), while the sulphydryl group content in the purified SR fraction was unchanged. Consistent with the unchanged sulphydryl group content, treatment of homogenates with dithiothreitol, the disulphide reducing reagent, failed to restore the decreased catalytic activity of SR Ca(2+)-ATPase in exercised muscles. These findings show clearly that high-intensity, exhaustive exercise causes oxidation of SR Ca(2+)-ATPase protein and suggest that oxidation of amino acids, other than cysteine, in the SR Ca(2+)-ATPase may be responsible, at least in part, for exercise-induced inactivation of this enzyme.

Effect of endurance training and acute exercise on sarcoplasmic reticulum function in rat fast- and slow-twitch skeletal muscles.

Following 10 weeks of endurance training and in age-matched sedentary rats, sarcoplasmic reticulum (SR) Ca(2+)-uptake, Ca(2+)-release, and Ca(2+)-stimulated adenosinetriphosphatase (ATPase) activity were examined in homogenates of the plantaris and soleus muscles from rats subjected to moderate-intensity treadmill running to exhaustion. In order to examine the effects of acute exercise and/or training on SR Ca(2+)-handling capacity, comparisons between exhausted and non-exercised rats and between trained and untrained rats were performed. Our data confirm that Ca(2+)-sequestration by the SR from fast-twitch muscles is depressed after training. Immediately after exhaustive running, decreases in SR function occurred in both muscles, but were more pronounced in the soleus. In the plantaris, reductions in SR Ca(2+)-uptake rate and Ca(2+)-ATPase activity were observed in untrained rats only, while in the soleus they were adversely affected irrespective of training status. Although the average run time to exhaustion varied markedly between untrained and trained animals (untrained: 253.0 min; trained: 559.4 min), no differences existed with regard to the magnitude of decreases in SR function in the soleus after exercise. The mean rate of decline in SR Ca(2+)-handling capacity during acute exercise, as estimated from the run time and the extent of the decline, was more than twofold higher in untrained than in trained soleus. From the present study, it is unclear whether there exists a causal relationship between muscular fatigue and SR function because the run time to exhaustion was not significantly correlated with any of parameters indicative of SR Ca(2+)-handling capacity, but suggested that endurance training may be capable of delaying a progression of the deterioration in SR function that occurs during exercise.