Comparative study on muscle function in two different streptozotocin-induced diabetic models.

AIMS: Streptozotocin (STZ) is widely used to study diabetic complications. Owing to the nonspecific cytotoxicity of high-dose STZ, alternative models using moderate-dose or a combination of low-dose STZ and a high-fat diet have been established. This study aimed to investigate the effects of these models on muscle function. METHODS: The muscle function of two STZ models using moderate-dose STZ (100 mg/kg, twice) and a combination of low-dose STZ and high-fat diet (50 mg/kg for 5 consecutive days + 45% high-fat diet) were examined using in vivo electrical stimulation. Biochemical and gene expression analysis were conducted on the skeletal muscles of the models immediately after the stimulation. RESULTS: The contractile force did not differ significantly between the models compared to respective controls. However, the moderate-dose STZ model showed more severe fatigue and blunted exercise-induced glycogen degradation possibly thorough a downregulation of oxidative phosphorylation- and vasculature development-related genes expression. CONCLUSIONS: Moderate-dose STZ model is suitable for fatigability assessment in diabetes and careful understanding on the molecular signatures of each model is necessary to guide the selection of suitable models to study diabetic myopathy.

GDE5/Gpcpd1 activity determines phosphatidylcholine composition in skeletal muscle and regulates contractile force in mice.

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.

Arginine ingestion inhibits phagocyte invasion in eccentrically contracted rat fast-twitch muscle.

Eccentric contraction (ECC) has been shown to induce leukocyte invasion into skeletal muscle, resulting in muscle inflammation. This study aimed to investigate whether prior ingestion of L-arginine (ARG), a nitric oxide precursor, inhibits ECC-induced macrophage invasion. Male Wistar rats received ARG in water for 7 days, beginning 3 days prior to ECC. ECCs were induced in the anterior crural muscles for 200 cycles. Three days later, the tibialis anterior and extensor digitorum longus muscles were excised for biochemical analysis and force measurement, respectively. ARG ingestion increased nitrite and nitrate levels in plasma and muscle, inhibiting force depression and reducing CD68 content in muscles subjected to ECC. ARG ingestion also ameliorated an ECC-induced increase in protein nitration, although neither ARG ingestion nor ECC induction affected protein carbonyl levels. The present results suggest that ingestion of ARG or ARG-rich foods may alleviate inflammation by attenuating phagocyte invasion in eccentrically contracted skeletal muscles.

Effects of eccentric contraction on force enhancement in rat fast-twitch muscle.

The aim of this study was to elucidate the effects of eccentric contraction (ECC) on force enhancement in rat fast-twitch skeletal muscle. Gastrocnemius (GAS) muscles were subjected to 200 ECCs in situ by electrical stimulation. Immediately before and after the stimulation, isometric torque produced by ankle flexion was measured at an ankle angle of 90°. After the second torque measurement, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analysis. ECC did not induce changes in the amount of degraded titin. After ECC, isometric torques in the GAS muscles were markedly reduced, especially at low stimulation frequency. ECC increased passive torque in whole muscle and passive force in skinned fibers. Passive force enhancement and the ratio of passive force to the maximal Ca2+ -activated force, but not residual force enhancement, were augmented in the skinned fibers subjected to ECC. An ECC-induced increase in titin-based stiffness may contribute to the increased PFE. These results suggest that skeletal muscle is endowed with a force potentiation system that can attenuate ECC-induced force reductions.

Effects of Leucine Ingestion and Contraction on the Sestrin/GATOR2 Pathway and mTORC1 Activation in Rat Fast-Twitch muscle.

BACKGROUND: Leucine activates the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in mammalian skeletal muscle. Recent studies have shown that Sestrin, a leucine sensor, might play a role in this process. However, it remains unknown whether Sestrin dissociates from GATOR2 in a dose- and time-dependent manner and whether an acute bout of muscle contraction augments this dissociation. OBJECTIVE: This study aimed to examine the effects of leucine ingestion and muscle contraction on the interaction between Sestrin1/2 and GATOR2 and on mTORC1 activation. METHODS: Male Wistar rats were randomly assigned to control (C), leucine 3 (L3), or leucine 10 (L10) groups. Intact gastrocnemius muscles were subjected to 30 repetitive unilateral contractions. The L3 and L10 groups were then orally administered 3 and 10 mmol/kg body weight of L-leucine 2 h after the end of the contractions, respectively. Blood and muscle samples were collected 30, 60, or 120 min after the administration. RESULTS: The blood and muscle leucine concentrations increased in a dose-dependent manner. The ratio of phosphorylated ribosomal protein S6 kinase (S6K) to total S6K (which indicates mTORC1 signaling activation) was markedly increased by muscle contraction and increased in a dose-dependent manner only in rested muscle. Leucine ingestion but not muscle contraction increased Sestrin1 dissociation from GATOR2 and Sestrin2 association with GATOR2. A negative relationship was observed between the blood and muscle leucine concentrations and the Sestrin1 association with GATOR2. CONCLUSIONS: The results suggest that Sestrin1, but not Sestrin2, regulates leucine-related mTORC1 activation via its dissociation from GATOR2 and that acute exercise-induced mTORC1 activation involves pathways other than the leucine-related Sestrin1/GATOR2 pathway.

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.

Eccentric muscle contraction potentiates titin stiffness-related contractile properties in rat fast-twitch muscles.

This study was conducted to examine the effects of an acute bout of eccentric muscle contraction (ECC) on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ to undergo 200 repeated ECCs. Immediately after the cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. Small heat shock protein αB-crystallin in the muscle fraction enriched for myofibrillar proteins was increased by ECC. ECC resulted in an increase in the titin-based passive force. Protein kinase A-treatment decreased the passive force only in ECC-subjected but not in rested fibers. ECC decreased the maximum Ca2+-activated force at a sarcomere length (SL) of 2.4 µm and had no effect on myofibrillar-Ca2+ sensitivity at 2.6-µm SL. In both rested and ECC-subjected fibers, these two variables were higher at 3.0-µm SL than at 2.4- or 2.6-µm SL. The differences in the two variables between the short and long SLs were greater in ECC-subjected than in rested fibers. These results indicate that an acute bout of ECC potentiates titin-based passive force, maximum active force at long SLs, and length-dependent activation and suggest that this potentiation may resist muscle fatigue in the muscles of the exercising body.NEW & NOTEWORTHY It remains unclear whether eccentric contraction of skeletal muscle affects titin stiffness-related contractile properties. Here, we provide evidence that an acute bout of eccentric contraction can potentiate titin-based passive force, maximum active force at long sarcomere lengths, and length-dependent activation. This potentiation may resist muscle fatigue in the muscles of the exercising body.

Mechanisms of eccentric contraction-induced muscle damage and nutritional supplementations for mitigating it.

Eccentric contraction (ECC) often results in large and long-lasting force deficits accompanied by muscle soreness, primarily due to muscle damage. In this sense, exercises that involve ECC are less desirable. Paradoxically, exercise training that includes a substantial eccentric phase leads to a more powerful activation of the genes responsible for skeletal muscle remodeling (e.g., hypertrophy) than other types of training that emphasize a concentric or isometric phase. Therefore, effective strategies that lessen ECC-induced muscle damage will be of interest and importance to many individuals. The purpose of this brief review is to highlight the published literature on the effects of ECC and/or nutritional supplementations on proteins, lipids, metabolic and ionic changes, and enzyme activities in skeletal muscles subjected to an acute bout of ECC. First, we discuss the potential mechanisms by which ECC causes muscle damage. Previous findings implicate a Ca2+ overload-oxidative modification pathway as one possible mechanism contributing to muscle damage. Thereafter, the efficacy of two nutritional supplementations, i.e., L-arginine and antioxidant, is discussed because L-arginine and antioxidant would be expected to ameliorate the adverse effects of Ca2+ overload and oxidative modification, respectively. Of these, L-arginine ingestion before ECC seems likely to be the effective strategy for mitigating ECC-related proteolysis. More studies are needed to establish the effectiveness of antioxidant ingestion. The application of effective strategies against muscle damage may contribute to improvements in health and fitness, muscle function, and sports performance.

[How to evaluate skeletal muscle function: suggestion from studies on skeletal muscle fatigue].

In studies on skeletal muscle, an in vitro force measurement has been widely used to evaluate its function. However, it is recently suggested that in some cases, the results obtained by such measurement do not necessarily reflect the force in vivo, because the measurement has some disadvantages. For example, the muscles are contracted under different conditions from in vivo and there is no blood flow. To resolve this issue, we have developed an experimental system, in which muscles are contracted in vivo and the organelle function is subsequently estimated by an in vitro force measurement using a mechanically skinned fiber technique. This experimental system makes it possible to examine not only the muscle force in vivo but also the mechanisms of changes in the force at organelle levels. In this review, we depict the advantages and disadvantages of the in vitro and in vivo measurements of force and then discuss the effectiveness of our experimental system.

Effects of vigorous isometric muscle contraction on titin stiffness-related contractile properties in rat fast-twitch muscles.

This study was conducted to examine the effects of an acute bout of vigorous isometric contractions on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ until the force was reduced to ∼50% of the initial force. Immediately after cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. The stimulation resulted in a decrease in the titin-based passive force. The amounts of fragmented titin were unchanged by the stimulation. Protein kinase Cα-treatment increased the passive force in stimulated fibers to resting levels. The stimulation had no effect on the maximum Ca2+-activated force (max Ca2+ force) at a sarcomere length (SL) of 2.4 µm and decreased myofibrillar (my)-Ca2+ sensitivity at 2.6-µm SL. Stretching the SL to 3.0 µm led to the augmentation of the max Ca2+ force and my-Ca2+ sensitivity in both rested and stimulated fibers. For the max Ca2+ force, the extent of the increase was smaller in stimulated than in rested fibers, whereas for my-Ca2+ sensitivity, it was higher in stimulated than in rested fibers. These results suggest that vigorous isometric contractions decrease the titin-based passive force, possibly because of a reduction in phosphorylation by protein kinase Cα, and that the decreased titin stiffness may contribute, at least in part, to muscle fatigue.

Orthograde signal of dihydropyridine receptor increases Ca2+ leakage after repeated contractions in rat fast-twitch muscles in vivo.

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.

Fatigue-induced change in T-system excitability and its major cause in rat fast-twitch skeletal muscle in vivo.

KEY POINTS: Using mechanically skinned rat muscle fibres, we investigated (i) transverse tubular-system (T-system) excitability after high-intensity contractions, and (ii) the mechanisms underlying the fatigue-induced alteration of the T-system excitability. T-system excitability estimated by using skinned fibres, which is highly regulated by T-system Na+ -K+ -ATPase, was decreased after muscle contractions, but was fully restored by treatment with dithiothreitol. The S-glutathionylation of Na+ -K+ -ATPase in whole muscle was increased after muscle contractions and also occurred under very low ATP conditions in rested but not stimulated fibres. In conclusion, T-system excitability was decreased after high-intensity exercise due at least in part to the S-glutathionylation of Na+ -K+ -ATPase, which may be enhanced by contraction-induced ATP depression. ABSTRACT: The purpose of this study was to investigate transverse tubular system (T-system) excitability after skeletal muscle contractions in vivo, and the contribution of S-glutathionylation of Na+ -K+ -ATPase. T-system excitability was estimated by measuring the repriming period (RP) required for double action potentials in mechanically skinned fibres where the sarcolemma was removed but the T-system still functioned. The RP under partially depolarized conditions was highly dependent on the function of Na+ -K+ -ATPase. Rat gastrocnemius (GAS) muscles were subjected to repetitive contractions until the force was decreased to ∼50% of initial force, and then the muscles were very quickly excised and used for skinned fibre and biochemical experiments. The RP under partially depolarized conditions was increased in stimulated fibres (5.9 ± 1.0 ms in rested vs. 8.0 ± 1.5 ms in stimulated); however, this increase in RP was reversed by sequential treatment with dithiothreitol. The skinned fibres from rested muscles exhibited slower repriming only when treated with oxidized glutathione (GSSG) under very low ATP (≤1 mm) conditions, whereas the RP in stimulated fibres was not altered after GSSG treatment without ATP. In line with this, the α2- and ß-subunits of Na+ -K+ -ATPase were more S-glutathionylated in stimulated than in rested muscles. These results suggest that (i) T-system excitability is decreased during contractions, in part due to a downregulation of T-system Na+ -K+ -ATPase, (ii) S-glutathionylation contributes to the fatigue-induced decline of the T-system Na+ -K+ -ATPase function, and (iii) ATP depression throughout contractions may enhance S-glutathionylation of T-system Na+ -K+ -ATPase.

Effects of high-intensity interval exercise on muscle fatigue and SR function in rats: a comparison with moderate-intensity continuous exercise.

In this study, we compared muscle fatigue induced by high-intensity interval exercise (HIIE) and moderate-intensity continuous exercise (MICE), with a focus on changes in the function of sarcoplasmic reticulum (SR) and myofibril. To achieve the aim of this study with mechanically skinned fibers with sealed transverse tubules and intact SR membrane, myofibrillar Ca2+ sensitivity, depolarization-induced force, and action potential-induced force were evaluated. Rat gastrocnemius muscles were subjected to HIIE-mimicking or MICE-mimicking stimulation in situ. The number of contractions was the same for MICE- and HIIE-mimicking stimulation (total of 360 contractions). Three hours after cessation of stimulation, the superficial regions of gastrocnemius muscles were dissected and used for biochemical and skinned fiber analyses. At 3 h of recovery, forces at 20 and 100 Hz in whole muscles had returned to resting levels in MICE but not HIIE muscles. The reduced glutathione content was decreased only in HIIE muscles. Both MICE- and HIIE-mimicking stimulation resulted in an increase in myofibrillar Ca2+ sensitivity in skinned fibers. Only HIIE-mimicking stimulation led to a decrease in the ratio of force at 1 Hz to that at 50 Hz and the ratio of depolarization-induced force to the maximum Ca2+-activated force. These results reflect the properties of type IIX and IIB fibers (the latter is not expressed in human skeletal muscles) and suggest that HIIE requires longer recovery periods than those normally used with MICE, which is ascribable to long-lasting depressions in SR Ca2+ release.NEW & NOTEWORTHY Over the past decade, high-intensity interval exercise (HIIE) training has received attention as a more efficient training to improve endurance capacity. It is unclear, however, whether the extent of acute exercise-related muscle fatigue differs between HIIE and moderate-intensity continuous exercise, traditional endurance training. Here we provide evidence that restoration of force production takes a longer time after HIIE, which is ascribable to long-lasting depressions in Ca2+ release of the sarcoplasmic reticulum.

Effects of reduced muscle glycogen on excitation-contraction coupling in rat fast-twitch muscle: a glycogen removal study.

The aim of this study was to investigate the effects of an enzymatic removal of glycogen on excitation-contraction coupling in mechanically skinned fibres of rat fast-twitch muscles, with a focus on the changes in the function of Na+-K+-pump and ryanodine receptor (RyR). Glycogen present in the skinned fibres and binding to microsomes was removed using glucoamylase (GA). Exposure of whole muscle to 20 U mL-1 GA for 6 min resulted in a 72% decrease in the glycogen content. Six minutes of GA treatment led to an 18 and a 22% reduction in depolarization- and action potential-induced forces in the skinned fibres, respectively. There was a minor but statistically significant increase in the repriming period, most likely because of an impairment of the Na+-K+-pump function. GA treatment exerted no effect on the maximum Ca2+ release rate from the RyR in the microsomes and the myofibrillar Ca2+ sensitivity in the skinned fibres. These results indicate that reduced glycogen per se can decrease muscle performance due to the impairment of SR Ca2+ release and suggest that although Na+-K+-pump function is adversely affected by reduced glycogen, the extent of the impairment is not sufficient to reduce Ca2+ release from the sarcoplasmic reticulum. This study provides direct evidence that glycogen above a certain amount is required for the preservation of the functional events preceding Ca2+ release from the sarcoplasmic reticulum.

Treatment with EUK-134 improves sarcoplasmic reticulum Ca2+ release but not myofibrillar Ca2+ sensitivity after fatiguing contraction of rat fast-twitch muscle.

Skeletal muscles undergoing vigorous activity can enter a state of prolonged low-frequency force depression (PLFFD). This study was conducted to examine whether antioxidant treatment is capable of accelerating the recovery from PLFFD, with a focus on the function of the sarcoplasmic reticulum (SR) and myofibril. One hour before fatiguing stimulation (FS) was administered, rats received an intraperitoneal injection of Eukarion (EUK-134), which mimics the activities of superoxide dismutase and catalase. Intact muscles of the hindlimbs were electrically stimulated via the sciatic nerve until the force was reduced to ~50% of the initial force (FS). Thirty minutes after cessation of FS, the superficial regions of gastrocnemius muscles were dissected and used for biochemical and skinned-fiber analyses. Whole muscle analyses revealed that antioxidant alleviated the FS-induced decrease in the reduced glutathione content. Skinned-fiber analyses showed that the antioxidant did not affect the FS-induced decrease in the ratio of force at 1 Hz to that at 50 Hz. However, the antioxidant partially inhibited the FS-mediated decrease in the ratio of depolarization-induced force to the maximum Ca2+-activated force. Furthermore, the antioxidant completely suppressed the FS-induced increase in myofibrillar Ca2+ sensitivity. These results suggest that antioxidant treatment is ineffective in facilitating the restoration of PLFFD, probably due to its negative effect on myofibrillar Ca2+ sensitivity, which supersedes its positive effect on SR Ca2+ release.

Ingestion of soy protein isolate attenuates eccentric contraction-induced force depression and muscle proteolysis via inhibition of calpain-1 activation in rat fast-twitch skeletal muscle.

OBJECTIVE: Eccentric contraction (ECC) is a contraction in which skeletal muscles are stretched while contracting. The aim of this study was to determine how ingestion of soy protein isolate (SPI) or animal-based proteins affect force deficit, calpain activation, and proteolysis of calcium ion (Ca2+)-regulatory proteins in rat fast-twitch muscles subjected to ECC. METHODS: In the first experiment, male Wistar rats were randomly assigned to a control and an SPI group, which were fed a 20% casein and a 20% SPI diet, respectively, for 28 d before the ECC protocol. Anterior crural muscles underwent 200 repeated ECCs and were excised 3 d later. In the second experiment, half of the SPI rats were given water containing NG-nitro-l-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase, for 3 d of recovery after ECC. RESULTS: SPI ingestion attenuated ECC-induced force deficit, proteolysis of Ca2+-regulatory proteins, and autolysis of calpain-1. Co-ingestion of L-NAME inhibited SPI-associated increases in nitrite and nitrate levels and negated the force recovery effects of SPI. CONCLUSION: These results suggest that SPI ingestion inhibits ECC-elicited force deficit and proteolysis of Ca2+ regulatory proteins, which is caused by inhibited activation of calpain-1 via increased nitric oxide production.

Thermal pretreatment facilitates recovery from prolonged low-frequency force depression in rat fast-twitch muscle.

The aim of this study was to examine whether thermal pretreatment can accelerate recovery from prolonged low-frequency force depression. The hindlimbs of thermal treated (T-treated) rats were immersed in water heated to 42.0°C for 20 min (thermal pretreatment). The thermal pretreatment was performed once a day for 5 days before fatiguing stimulation. Intact gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected immediately [recovery 0 (REC0)] or 60 min [recovery 60 (REC60)] following the cessation of stimulation. Using skinned fiber prepared from the superficial region, the ratio of force at 1 Hz to that at 50 Hz (low-to-high force ratio), the ratio of depolarization (depol)-induced force to maximum Ca2+ -activated force (depol/max Ca2+ force ratio), the steepness of force-Ca2+ concentration curves, and myofibrillar Ca2+ sensitivity were measured. At REC0, the low-to-high force ratio and depol/max Ca2+ force ratio decreased in stimulated muscles from both non- and thermal-treated rats. At REC60, these two parameters remained depressed in non-treated rats, whereas they reverted to resting levels in T-treated rats. Thermal pretreatment exerted no effect on myofibrillar Ca2+ sensitivity. The present results reveal that thermal pretreatment can facilitate recovery of submaximum force after vigorous contraction, which is mediated via a quick return of Ca2+ release from the sarcoplasmic reticulum to resting levels.

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.

Electrical Stimulation Prevents Preferential Skeletal Muscle Myosin Loss in Steroid-Denervation Rats.

Severe muscle weakness concomitant with preferential depletion of myosin has been observed in several pathological conditions. Here, we used the steroid-denervation (S-D) rat model, which shows dramatic decrease in myosin content and force production, to test whether electrical stimulation (ES) treatment can prevent these deleterious changes. S-D was induced by cutting the sciatic nerve and subsequent daily injection of dexamethasone for 7 days. For ES treatment, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. Plantarflexor in situ isometric torque, muscle weight, skinned muscle fiber force, and protein and mRNA expression were measured after the intervention period. ES treatment partly prevented the S-D-induced decreases in plantarflexor in situ isometric torque and muscle weight. ES treatment fully prevented S-D-induced decreases in skinned fiber force and ratio of myosin heavy chain (MyHC) to actin, as well as increases in the reactive oxygen/nitrogen species-generating enzymes NADPH oxidase (NOX) 2 and 4, phosphorylation of p38 MAPK, mRNA expression of the muscle-specific ubiquitin ligases muscle ring finger-1 (MuRF-1) and atrogin-1, and autolyzed active calpain-1. Thus, ES treatment is an effective way to prevent muscle impairments associated with loss of myosin.

l-arginine ingestion inhibits eccentric contraction-induced proteolysis and force deficit via S-nitrosylation of calpain.

It has been shown that calpains are involved in the proteolysis of muscle proteins that occurs with eccentric contraction (ECC) and that exogenously applied nitric oxide decreases the calpain-mediated proteolysis. The aim of this study was to examine the effects of ingestion of l-arginine (ARG), a nitric oxide precursor, on ECC-related calpain activation. In the first and second experiments, male Wistar rats were given ARG in water for 7 days starting from 3 days before the ECC protocol (average ingestion, ~600 mg kg-body wt-1  day-1 ). Tibialis anterior muscles underwent 200 repeated ECCs and, subsequently, were excised 3 days later. Whole muscle analyses (the first experiment) revealed that ARG attenuated ECC-induced force deficit and autolysis of calpain-1, and increased the amounts of S-nitrosylated calpain-1. Regarding ryanodine receptor (RyR) and dihydropyridine receptor (DHPR), ECC-induced proteolysis was completely inhibited by ARG, whereas the inhibition was partial for junctophilin-1 (JP1). Skinned fiber analyses (the second experiment) showed that ARG also inhibited ECC-elicited reductions in the ratio of depolarization-induced to maximum Ca2+ -activated force. In the third experiment, homogenates of rested muscles were treated with S-nitrosylating agent, S-nitrosoglutathione (GSNO), and/or high Ca2+ concentration ([Ca2+ ]). Treatment with high [Ca2+ ] and without GSNO produced proteolysis of RyR, DHPR, and JP1. On the other hand, treatment with high [Ca2+ ] and GSNO caused complete inhibition of RyR and DHPR proteolysis and partial inhibition of JP1 proteolysis. These results indicate that ARG ingestion can attenuate ECC-induced proteolysis of Ca2+ regulatory proteins and force deficit by decreasing calpain activation via S-nitrosylation.