Effects of single ingestion of arginine on mTORC1 activation in rat fast- and slow-twitch muscles / 体力科学

This study aimed to examine whether a single ingestion of arginine activates the mammalian target of rapamycin complex 1 (mTORC1) in rat fast- and slow-twitch skeletal muscles. In the first experiment, the rats were orally administered arginine (3 or 10 mmol/kg body weight) in water. The plantaris, gastrocnemius, and soleus muscles were excised 1 h after the administration. Immunoblot analysis showed that the administration with a higher dose (10 mmol/kg) resulted in increased phosphorylation of ribosomal S6 kinase (S6K) and ribosomal protein S6 only in the soleus muscles. The amounts of cellular arginine sensor for mTORC1 subunit 1 (CASTOR1) expressed were similar in these three muscles. In the second experiment, the plantaris and soleus muscles were excised 1 h after the administration of 10 mmol/kg of arginine. The binding of CASTOR1 to the GATOR2 complex was not detected in either muscle in co-immunoprecipitation and immunoblot analyses, irrespective of arginine administration. In the third experiment, a role of nitric oxide (NO) was elucidated. Treatment with an inhibitor of NO synthase blocked the arginine-induced increase in S6K phosphorylation. These results indicate that a single ingestion of arginine is capable of activating mTORC1 only in slow-twitch muscles and suggest that the activation may be mediated via NO, but not via the CASTOR1-GATOR2 complex pathway.

Characteristics and mechanisms of low-frequency muscle fatigue: alterations in skeletal muscle / 体力科学

Repeated contractions of skeletal muscle cause fatigue, as manifested by a reduced ability to produce force and slowed contraction. During studies of muscle fatigue, a phenomenon known as low-frequency fatigue (LFF) was observed in human skeletal muscles. It is characterized by a greater loss of force in response to low- versus high-frequency muscle stimulation and a long period of time for full recovery. This force deficit is most likely to be owing to disturbances in sarcoplasmic reticulum (SR) Ca²⁺ release and/or reductions in myofibrillar Ca²⁺ sensitivity. Studies on metabolites have implied that inorganic phosphate and Mg²⁺ might have some role in reduced SR Ca²⁺ release that occurs immediately after fatiguing contraction. In addition, recent experiments have shown that impaired myofibril function may relate to increased nitric oxide and hydroxyl radical production, whereas deterioration of SR function may be attributable to increased superoxide production, elevation of cytoplasmic Ca²⁺ concentration and/or decreased muscle glycogen. Finally, we will discuss possible proteins which are affected and contribute to the development of LFF.

EFFECTS OF ECCENTRIC CONTRACTIONS ON IN VITRO Na⁺-K⁺-ATPase ACTIVITY AND SARCOPLASMIC RETICULUM Ca²⁺-SEQUESTERING IN RAT SKELETAL MUSCLE / 体力科学

The purpose of this study was to examine the effects of eccentric contractions (Ecc) on cation (i.e., K⁺, Na⁺ and Ca²⁺) regulation in skeletal muscle. The left anterior crural muscles of male Wistar rats were subjected <i>in vivo</i> to either Ecc or isometric contrations (Iso) for 200 cycles. The extensor digitorum longus and tibialis anterior muscles were removed immediately after and 2, 4 and 6 days following contractions and used for measures of force output and biochemical analyses, respectively. Ecc led to a 75% decrease in maximal tetanic force. Decreased force output did not revert to pre-exercise levels during 6 days of recovery. Sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity was reduced by 52 and 60% 4 and 6 days after Ecc, respectively. The reduction in catalytic activity after 6 days was accompanied by a 63% decrease in SR Ca²⁺-ATPase protein and an approximately 3.5-fold increase in calpain activity. Na⁺-K⁺-ATPase acticity was decreased by 23% immediately after Ecc and restored during 2 days of recovery. These alterations were specific for Ecc and not observed for Iso. These results suggest that disturbances in cation regulation may account, at least partly, for Ecc-induced decreases in force and power which can take a number of days to recover and that the decrease in SR Ca²⁺-ATPase activity would result from the degradation of the enzyme.

EFFECT OF HIGH-INTENSITY TRAINING AND ACUTE EXERCISE ON Ca²⁺-SEQUESTERING FUNCTION OF SARCOPLASMIC RETICULUM : ROLE OF OXIDATIVE MODIFICATION / 体力科学

To investigate the influences of high-intensity training and/or a single bout of exercise on <i>in vitro</i> Ca²⁺-sequestering function of the sarcoplasmic reticulum (SR), the rats were subjected to 8 weeks of an interval running program (final training : 2.5-min running×4 sets per day, 50 m/min at 10% incline). Following training, both trained and untrained rats were run at a 10% incline, 50 m/min for 2.5 min or to exhaustion. SR Ca²⁺-ATPase activity, SR Ca²⁺-uptake rate and carbonyl group contents comprised in SR Ca²⁺-ATPase activity were examined in the superficial portions of the gastrocnemius and vastus lateralis muscles. For rested muscles, a 12.7% elevation in the SR Ca²⁺-uptake rate was induced by training. Training led to improved running performance (avg time to exhaustion : untrained-191.1 vs trained-270.9 sec ; <i>P</i><0.01). Regardless of training status, a single bout of exercise caused progressive reductions in SR Ca²⁺-ATPase activity and SR Ca²⁺-uptake rate. Increases in carbonyl content only occurred after exhaustive exercise (<i>P</i><0.05). At both point of 2.5-min and exhaustion, no differences existed in SR Ca²⁺-sequestering capacity and carbonyl content between untrained and trained muscles. These findings confirm the previous findings that oxidative modifications may account, at least partly, for exercise-induced deterioration in SR Ca²⁺-sequestering function ; and raise the possibility that in the final phase of acute exercise, high-intensity training could delay the progression of protein oxidation of SR Ca²⁺-ATPase.

RELATIONSHIP BETWEEN OXIDATION OF MYOFIBRILLAR PROTEINS AND CONTRACTILE PROPERTIES IN SOLEUS MUSCLES FROM HYPERTHYROID RAT / 体力科学

We tested the hypothesis that a force reduction in soleus muscles from hyperthyroid rats would be associated with oxidative modification of myofibrillar proteins. Daily injection of thyroid hormone [3, 5, 3’-triiodo-_L-thyronine (T₃)] for 21 days depressed isometric forces in whole soleus muscle across a range of stimulus frequencies (1, 10, 20, 40, 75 and 100 Hz) (<i>P</i><0.05). In fiber bundles, hyperthyroidism also led to pronounced reductions (<i>P</i><0.05) in both K⁺- and 4-chloro-<i>m</i>-cresol-induced contracture forces. The degrees of the reductions were similar between these two contractures. These reductions in force production were accompanied by a remarkable increment (103% ; <i>P</i><0.05) in carbonyl groups comprised in myofibrillar proteins. In additional experiments, we have also tested the efficacy of carvedilol, a non-selective β₁-β₂-blocker that possesses anti-oxidative properties. Treatment with carvedilol prevented T₃-induced oxidation of myofibrillar proteins. However, carvedilol did not improve the hyperthyroid-induced reductions in force production. These data suggest that oxidative modification of myofibrillar proteins may not account for the reductions in force production of hyperthyroid rat soleus muscle.

CHANGES IN SARCOPLASMIC RETICULUM Ca²⁺-SEQUESTERING CAPACITY DURING RECOVERY FOLLOWING HIGH-INTENSITY EXERCISE / 体力科学

The purpose of this study was to investigate changes in sarcoplasmic reticulum (SR) Ca²⁺-sequestering capacity in rat fast-twitch plantaris (PL) and slow-twitch soleus (SOL) muscles during recovery after high-intensity exercise. The rats were subjected to treadmill runs to exhaustion at the intensity (10% incline at 50 m/min) estimated to require 100% of maximal O₂ consumption. The muscles were excised immediately after exercise, and 15, 30 and 60 min after exercise. Acute high-intensity exercise evoked a 27 % and 38 % depression (<i>P</i><0.05) in SR Ca²⁺-uptake rate in the PL and SOL, respectively. In the PL, uptake rate remained lower (<i>P</i><0.05) at 30 min of recovery but recovered 60 min after exercise. These alterations were paralleled by those of SR Ca²⁺-ATPase activity. On the other hand, SR Ca²⁺-uptake rate in the SOL recovered 15 min after exercise. Unlike the PL, discordant time-course changes between SR Ca²⁺-ATPase activity and uptake occurred in the SOL during recovery. SR Ca²⁺-ATPase activities were unaltered with exercise and elevated (<i>P</i><0.05) by 25, 30 and 30% at 15, 30 and 60 min of recovery, respectively. These results demonstrate that SR Ca²⁺-sequestering ability is restored faster in slow-twitch than in fast-twitch muscle during recovery periods following a single bout of high-intensity exercise and suggest that the rapid restoration of SR Ca²⁺-sequestering ability in slow-twitch muscle could contribute to inhibition of disturbances in contractile and structural properties that are known to occur with raised myoplasmic Ca²⁺ concentrations.

EFFECTS OF THYROID HORMONE ON SARCOPLASMIC RETICULUM Ca2+ UPTAKE AND CONTRACTILE PROPERTIES IN RAT SOLEUS MUSCLE / 体力科学

The effect of hyperthyroidism on the contractile properties and Ca<SUP>2+</SUP> sequestering abilities by the sarcoplasmic reticulum (SR) in the soleus muscles was examined in rats treated with thyroid hormone (3, 5, 3'-triiodo-L-thyronine, T<SUB>3</SUB>) (300μg/kg body weight) for 3, 7 and 21 days. At the end of a given treatment period, the right or left soleus muscle was mounted isometrically at 30°C, and stimulated directly using supramaximal current intensity. A twitch contraction was elicited by a 1 msec square-wave pulse and a tetanic contraction by 20 Hz stimulation for 600 msec. To evaluate fatigue resistance, muscles were stimulated at 40 Hz for 350 msec with tetani repeated at an interval of 2 sec during a 4-min period. Another soleus muscle was used, for analysis of SR Ca<SUP>2+</SUP> -uptake rate and SR Ca<SUP>2+</SUP> -ATPase activity. Pronounced increases in SR Ca<SUP>2+</SUP> -uptake rate and ATPase activity were observed after T<SUB>3</SUB> treatment periods longer than 6 days. These alterations were accompanied by decreases in twitch and tetanic tension, half-relaxation time, and fatigue resistance. The T<SUB>3</SUB>-treated muscles stimulated at 20 Hz relaxed during the interval between successive stimuli, indicating that the mechanical fusion of tetanic contractions was incomplete. SR Ca<SUP>2+</SUP> uptake rate was significantly correlated both to tetanic tension and to fatigue resistance. These data suggest that there may be a causal relationship between changes in SR Ca<SUP>2+</SUP> uptake and the loss of muscular strength in the hyperthyroid soleus.

EFFECT OF DITHIOTHREITOL ON Ca<SUP>2+</SUP>-ATPase ACTIVITY OF SARCOPLASMIC RETICULUM IN RAT SKELETAL MUSCLE AFTER HIGH-INTENSITY EXERCISE / 体力科学

Although the precise mechanisms underlying the dysfunction of sarcoplasmic reticulum (SR) that occurs during skeletal muscle fatigue remain obscure, it has been hypothesized that it may be attributable to oxidation of critical sulfhydryl groups residing in SR Ca<SUP>2+</SUP>-ATPase protein by endogenously produced reactive oxygen species. In order to test this hypothesis, SR Ca<SUP>2+</SUP>-ATPase activities in the absence or presence of the disulfide reducing agent, dithiothreitol (DTT), were examined in muscle homogenates of the soleus muscles (SQL) and the superficial portions of the vastus lateralis muscles (VS) from the rat subjected to exhaustive running at 50 m/min on a 10% grade. Immediately after exercise, the catalytic activity of SR Ca<SUP>2+</SUP>-ATPase was significantly depressed in VS, but not in SQL. The loss of SR Ca<SUP>2+</SUP>-ATPase activity observed in VS was fully recovered after treatment with DTT (1 mM) . These recovery effects of a potent disulfide reducing agent suggest that important proteins of SR Ca<SUP>2+</SUP>-ATPase may be oxidized during high-intensity exercise and that the onset of muscular fatigue may be delayed by the improved function of the cellular antioxidant

Alterations in function of sarcoplasmic reticulum after acute high-intensity exercise / 体力科学

This study examined the impact of acute high-intensity exercise on the rate of Ca<SUP>2+</SUP>uptake and release and Ca<SUP>2+</SUP>-stimulated adenosine triphosphatase (ATPase) activity of the sarcoplasmic reticulum (SR) in the soleus muscle (SOL) and the superficial region of the vastus lateralis muscle (VS) of rats. The animals were run on a 10% grade at 50 m/min of a motorized treadmill until fatigued (The average time to exhaustion was 306 sec.) . At exhaustion, glycogen concentrations were 65% and 85% less in the SOL and VS, respectively. The rate of Ca<SUP>2+</SUP>release induced by 4-chloro-m-cresol was un-changed in fatigued SOL and VS. The rate of Ca<SUP>2+</SUP>uptake stimulated by adenosine triphosphate (ATP) was significantly lower following exercise in VS but not in SOL. This lower rate observed in VS was paralleled by decreased catalytic activity of SR Ca<SUP>2+</SUP>-ATPase. The rate of Ca2+ uptake measured using adenosine diphosphate and phosphocreatine, as substrate was lower than that of ATP in fatigued VS. These findings suggest that, in fast-twitch muscles, high-intensity exercise not only reduces SR Ca<SUP>2+</SUP>-ATPase activity but also elicits a decrease in creatine kinase activity, probably resulting from nitric oxide that is produced during exercise.

Effects of exhaustive exercise on sarcoplasmic reticulum ATPase. Comparison of short- and long-term exercise / 体力科学

Effects of short-term, high-intensity and long-term, moderate-intensity exercise on biochemically assessed sarcoplasmic reticulum (SR) ATPase protein were analyzed in muscle homogenates of the rat after treadmill runs to exhaustion (avg, time to exhaustion 2 min 48 sec and 1 h 29 min, respectively) . The exercise-induced changes in SR Ca<SUP>2+</SUP> -ATPase activity were muscle type-specific. After short-term exercise, a decrease in the activity occurred in the soleus muscle and the superficial region of the vastus lateralis muscle whereas long-term exercise depressed the rate of ATP hydrolysis in the soleus muscle and the deep region of the vastus lateralis muscle. The concentration of fluorescein isothiocyanate, a competitor at the ATP-binding site, for 50% inhibition of SR Ca<SUP>2+</SUP> -ATPase activity fluctuated only in the soleus muscle subjected to short-term exercise ; it was increased by 31%. This change occurring in the soleus muscle would elevate SR Ca<SUP>2+</SUP> -ATPase activity at a given concentration of ATP. The results presented here suggest that acute short-term exercise to exhaustion may exert a remarkably inhibitory factor on SR Ca<SUP>2+</SUP> -ATPase protein of slow-twitch muscle, which can overcome the positive effect probably arising from the phosphorylation of the phospholamban.

Effects of hindlimb suspension on patterns of myosin isoforms in rat soleus muscle / 体力科学

Using several electrophoretic techniques, this study examined the effects of 3 weeks hindlimb suspension on the patterns of isomyosins, myosin heavy chain (HC) isoforms and myosin light chain (LC) isoforms in the soleus muscle of the rat. The suspended soleus showed a shift in the HC isoform distribution with a marked increase in fast HC isoforms and a commensurate decrease in HCI. In addition, the change in the fast HC isoforms consisted of the expression of HCIId and HC IIb absent in the normal soleus. In contrast to HC isoforms, suspension did not lead to appreciable changes in LC isoform distribution. Analyses of electrophoresis under nondenaturing conditions demonstrated that the normal soleus expressing HCI and HCIIa isoforms contained two isomyooins. Although, of the two isomyosins observed in the normal soleus, the faster migrating band most likely represented the HCIIa-based one (FMas), its mobility was not identical with that of the HCIIa-based isomyosin (FMaf) found in fast-twitch muscles, migrating in the order FMaf>FMas. FMas was designated as intermediate isomyosin (IM) . Some of the suspended soleus contained slow isomyosin (SM) and IM whereas the others comprised FM 3 and/or FM 2 as well as SM and IM. In spite of the expression of HCIIb and HCIId in the suspended soleus, FM 3 and FM 2 observed in these muscles exhibited distinct mobilities from either HCIId-based or HCIIb-based isomyosins comprised in fast-twitch muscles. These results suggest that some of newly expressed HCIId and/or HCIIb isoforms in the suspended soleus are associated with not only fast but also slow LC isoforms and function as a constitutive element of the myosin molecule.

Two-dimensional electrophoretic analysis of myoproteins in developing rat skeletal muscle / 体力科学

Changes in myoproteins during development of rat skeletal muscle were investigated using two-dimensional gel electrophoresis. In M, soleus (SOL) which in adult, is composed predominantly of slow twitch fibers, fast type myosin light chains (fLC) were the major species and slow type light chains (sLC) were the minor species at birth. During development, the replacement rate of fLC to sLC sequentially occured so that LC patterns at 21 days postpartum were similar to adult where fLC were difficult to visualize. In contrast, M. extensor digitorum longus (EDL) always contained dominant fLC although sLC were found only for 5-9 days. LC 3 f became detectable at 5 days and gradually increased. In α-tropomyosin there were isozymes of fast and slow type based on difference in molecular weight, but not in β-tropo-myosin. Changes in isozymes of α-tropomyosin approximately corresponded with that in isozymes (fast and slow type) of LC in both EDL and SOL. During adult stage following birth, in EDL creatine kinase underwent a three-fold increase in molecular ratio to actin, whereas in SOL there was little change though increase took place transiently. These results suggest that with develoment many myoproteins change more dramatically in slow muscle than in fast muscle, and that transitions in LC isozymes and changes in distribution of histochemically typed muscle fibers may follow different time courses.