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We investigated whether time-of-day dependent changes in the rat soleus (SOL) muscle size, after eccentric exercises, operate via the mechanistic target of rapamycin (mTOR) signaling pathway. For our first experiment, we assigned 9-week-old male Wistar rats randomly into four groups: light phase (zeitgeber time; ZT6) non-trained control, dark phase (ZT18) non-trained control, light phase-trained, and dark phase-trained. Trained animals performed 90 min of downhill running once every 3 d for 8 weeks. The second experiment involved dividing 9-week-old male Wistar rats to control and exercise groups. The latter were subjected to 15 min of downhill running at ZT6 and ZT18. The absolute (+12.8%) and relative (+9.4%) SOL muscle weights were higher in the light phase-trained group. p70S6K phosphorylation ratio was 42.6% higher in the SOL muscle of rats that had exercised only in light (non-trained ZT6). Collectively, the degree of muscle hypertrophy in SOL is time-of-day dependent, perhaps via the mTOR/p70S6K signaling., Competing Interests: The authors have declared that no competing interests exist.

Purpose: We aimed to investigate the hypothesis that type I collagen plays a role in increasing bone mineral density (BMD) and muscle stiffness, leading to low and high risks of fatigue fracture and muscle injury, respectively, in athletes. As a potential mechanism, we focused on the effect of the type I collagen alpha 1 chain gene (COL1A1) variant associated with transcriptional activity on bone and skeletal muscle properties., Methods: The association between COL1A1 rs1107946 and fatigue fracture/muscle injury was evaluated in Japanese athletes. Effects of the polymorphism on tissue properties (BMD and muscle stiffness) and type I collagen α1/α2 chain ratios in muscles were examined in Japanese nonathletes., Results: The C-allele carrier frequency was greater in female athletes with fatigue fracture than in those without (odds ratio = 2.44, 95% confidence interval [CI] = 1.17-5.77) and lower in female athletes with muscle injury than in those without (odds ratio = 0.46, 95% CI = 0.24-0.91). Prospective validation analysis confirmed that in female athletes, muscle injury was less frequent in C-allele carriers than in AA genotype carriers (multivariable-adjusted hazard ratio = 0.27, 95% CI = 0.08-0.96). Among female nonathletes, the C-allele of rs1107946 was associated with lower BMD and lower muscle stiffness. Muscle biopsy revealed that C-allele carriers tended to have a larger type I collagen α1/α2 chain ratio than AA genotype carriers (2.24 vs 2.05, P = 0.056), suggesting a higher proportion of type I collagen α1 homotrimers., Conclusion: The COL1A1 rs1107946 polymorphism exerts antagonistic effects on fatigue fracture and muscle injury among female athletes by altering the properties of these tissues, potentially owing to increased levels of type I collagen α1 chain homotrimers., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Sports Medicine.)

Recent studies of the ketogenic diet, an extremely high-fat diet with extremely low carbohydrates, suggest that it changes the energy metabolism properties of skeletal muscle. However, ketogenic diet effects on muscle metabolic characteristics are diverse and sometimes countervailing. Furthermore, ketogenic diet effects on skeletal muscle performance are unknown. After male Wistar rats (8 weeks of age) were assigned randomly to a control group (CON) and a ketogenic diet group (KD), they were fed for 4 weeks respectively with a control diet (10% fat, 10% protein, 80% carbohydrate) and a ketogenic diet (90% fat, 10% protein, 0% carbohydrate). After the 4-week feeding period, the extensor digitorum longus (EDL) muscle was evaluated ex vivo for twitch force, tetanic force, and fatigue. We also analyzed the myosin heavy chain composition, protein expression of metabolic enzymes and regulatory factors, and citrate synthase activity. No significant difference was found between CON and KD in twitch or tetanic forces or muscle fatigue. However, the KD citrate synthase activity and the protein expression of Sema3A, citrate synthase, succinate dehydrogenase, cytochrome c oxidase subunit 4, and 3-hydroxyacyl-CoA dehydrogenase were significantly higher than those of CON. Moreover, a myosin heavy chain shift occurred from type IIb to IIx in KD. These results demonstrated that the 4-week ketogenic diet improves skeletal muscle aerobic capacity without obstructing muscle contractile function in sedentary male rats and suggest involvement of Sema3A in the myosin heavy chain shift of EDL muscle., Competing Interests: The authors have declared that no competing interests exist.

Males and females of many species, including humans, exhibit different muscle responses and adaptations to exercise stress; however, the molecular mechanisms that underlie these changes are poorly understood. Therefore, the present study assessed sex-related differences in intracellular signaling pathway responses to bouts of horizontal or downhill running in rat soleus muscles. Age-matched male and female Wistar rats (10 weeks old, n = 18/group) were either rested (control group) or subjected to an either a bout of horizontal (22 m/min, 20 min, 0° incline) or downhill (16 m/min, 10 min, - 16% incline) treadmill running. Soleus muscle samples were collected both prior to and immediately after exercise (n = 6/group). Intramuscular signaling responses to each type of exercise were determined via real-time (RT) PCR and western blot analyses. Although mTOR signaling (mTOR/S6K1/S6) responses to both horizontal and downhill exercise were found to be similar in both sexes, ERK phosphorylation levels were found to be significantly higher in male than in female rats after downhill exercise. Similarly, heat shock protein (Hsp) 72 and myostatin protein expression levels were both found to be significantly altered after downhill exercise: Hsp levels increased in male and decreased in female rats, whereas myostatin increased in female but decreased in male rats. Thus, the results of the present study suggest that downhill exercise may elicit sex-specific differential changes to Hsp72 expression, ERK phosphorylation, and myostatin-signaling activation in female compared with those in male rat soleus muscles. Further study is required to confirm these findings and to determine the way in which they impact sex-specific differences in exercise-induced muscle adaptations.

Changes in histone acetylation and methylation status with aging affect gene expression and phenotype in several tissues; however, age-related changes in histone modification in the skeletal muscle have not been elucidated yet. This study investigated age-related global changes in histone modification in rat gastrocnemius muscle. Male Wistar rats (n = 28) were assigned to one of four age groups (n = 7 per group) corresponding to different life stages: 3 months old (3-mo; young), 6 months old (adult), 12 months old (12-mo; middle-aged), and 24 months old (24-mo; old). The gastrocnemius muscle was removed and global histone modification (acetylation and tri-methylation) at K9 and K27 was evaluated by western blotting. Relative muscle mass decreased in the 12- and 24-mo rats accompanied with reduction in type IIb myosin heavy chain isoforms and Myh4 (MHC IIB) mRNA expression. Histone H3 acetylation decreased in an age-dependent manner, with lower levels in 12- and 24-mo groups than in the 3-mo group. K9 and K27 acetylation decreased with age. Although there was no significant change in K27 tri-methylation, K9 tri-methylation showed an age-dependent decline. Histone modification status (acetylation at K9 and K27 and tri-methylation at K9) was positively associated with relative gastrocnemius muscle weight, the percentage of type IIb myosin heavy chain isoform, myosin heavy chain type IIb protein expression, and the level of Myh4 mRNA. Thus, global histone H3 methylation and acetylation decrease with age, and the latter might be associated with age-related muscle atrophy of rat gastrocnemius muscle., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The mechanisms involved in unloading-induced skeletal muscle loss may be age-specific, and the evidence for exercise preconditioning-induced protection against disuse muscle atrophy in aged rats is limited. Therefore, in this study, we investigated age-related differences in the activation of the HDAC4/Gadd45α pathway following hindlimb unloading (HU). We also assessed the protective effect of preconditioning exercise on this pathway in young and old rat gastrocnemius muscle. Three-month-old (young, n = 18) and 24-month-old (old, n = 18) male Wistar rats were assigned to the following groups: control group (n = 6), seven days of HU group (n = 6), and a bout of exercise preconditioning prior to HU (Ex+HU) group (n = 6). Rats of both ages in the Ex + HU group ran continuously on a motor-driven treadmill (0° slope, 20 m/min, 15 min) prior to HU. The gastrocnemius muscles were removed after 7 days of HU and analyzed for protein content and mRNA expression. Gastrocnemius muscle weight was significantly higher in the Ex+HU group than in the HU group of old rats, but not in young rats. Levels of HDAC4 protein and mRNA were significantly increased in the old HU group. However, the increase was significantly suppressed in the old Ex+HU group. Moreover, the protective effect of exercise preconditioning had a positive effect on Gadd45α mRNA and protein levels only in the old Ex+HU group. No exercise preconditioning-related protection was observed in the young rats. Our data indicated that a single bout of preconditioning exercise prior to HU may exert a protective effect in disuse muscle atrophy in old rats and that these effects may be partially mediated by the HDAC4/Gadd45α axis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We tested the hypothesis that there are sex differences in hindlimb unloading-induced activation of the forkhead box subfamily O3a (FoxO3a) signaling pathway in rat soleus muscle. Age-matched male and female Wistar rats were subjected to hindlimb unloading, and the soleus muscle was removed before or 1 or 7 days after unloading. Female rats showed greater percent changes in relative soleus muscle weight than males. FoxO3a phosphorylation was lower in females than in males and was associated with higher levels of protein ubiquitination 7 days after unloading. Heat shock protein 72 (Hsp72) levels were lower in female rats and increased in males during unloading. Female rats showed slightly higher myostatin levels, which showed a non-significant decline in male rats following unloading. Thus, males and females show different responses to the FoxO3a/ubiquitin-proteasome pathway following hindlimb unloading in rat soleus muscle, which may be associated with differences in Hsp72 expression and myostatin signaling.

Type 2 diabetes is characterized by reduced contractile force production and increased fatigability of skeletal muscle. While the maintenance of Ca 2+ homeostasis during muscle contraction is a requisite for optimal contractile function, the mechanisms underlying muscle contractile dysfunction in type 2 diabetes are unclear. Here, we investigated skeletal muscle contractile force and Ca 2+ flux during contraction and pharmacological stimulation in type 2 diabetic model mice ( db/db mice). Furthermore, we investigated the effect of treadmill exercise training on muscle contractile function. In male db/db mice, muscle contractile force and peak Ca 2+ levels were both lower during tetanic stimulation of the fast-twitch muscles, while Ca 2+ accumulation was higher after stimulation compared with control mice. While 6 wk of exercise training did not improve glucose tolerance, exercise did improve muscle contractile dysfunction, peak Ca 2+ levels, and Ca 2+ accumulation following stimulation in male db/db mice. These data suggest that dysfunctional Ca 2+ flux may contribute to skeletal muscle contractile dysfunction in type 2 diabetes and that exercise training may be a promising therapeutic approach for dysfunctional skeletal muscle contraction. NEW & NOTEWORTHY The purpose of this study was to examine muscle contractile function and Ca 2+ regulation as well as the effect of exercise training in skeletal muscle in obese diabetic mice ( db/db). We observed impairment of muscle contractile force and Ca 2+ regulation in a male type 2 diabetic animal model. These dysfunctions in muscle were improved by 6 wk of exercise training.

This study examined the effect of changes in body temperature during exercise on signal transduction-related glucose uptake in the skeletal muscle of type 2 diabetic rats. Otsuka Long-Evans Tokushima Fatty rats (25 weeks of age), which have type 2 diabetes, were divided into the following four weight-matched groups; control (CON, n = 6), exercised under warm temperature (WEx, n = 8), exercised under cold temperature (CEx, n = 8), and heat treatment (HT, n = 6). WEx and CEx animals were subjected to running on a treadmill at 20 m/min for 30 min under warm (25°C) or cold (4°C) temperature. HT animals were exposed to single heat treatment (40-41°C for 30 min) in a heat chamber. Rectal and muscle temperatures were measured immediately after exercise and heat treatment, and the gastrocnemius muscle was sampled under anesthesia. Rectal and muscle temperatures increased significantly in rats in the WEx and HT, but not the CEx, groups. The phosphorylation levels of Akt, AS160, and TBC1D1 (Thr590) were significantly higher in the WEx and HT groups than the CON group (p < 0.05). In contrast, the phosphorylation levels of AMP-activated protein kinase, ACC, and TBC1D1 (Ser660) were significantly higher in rats in the WEx and CEx groups than the CON group (p < 0.05) but did not differ significantly between rats in the WEx and CEx groups. Body temperature elevation by heat treatment did not activate the AMPK signaling. Our data suggest that body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of rats with type 2 diabetic rats., Competing Interests: The authors have declared that no competing interests exist.

Extended periods of skeletal muscle disuse results in muscle atrophy and weakness. Currently, no therapeutic treatment is available for the prevention of this problem. Nonetheless, growing evidence suggests that prevention of disuse-induced oxidative stress in inactive muscle fibers can delay inactivity-induced muscle wasting. Therefore, this study tested the hypothesis that dietary supplementation with the antioxidant astaxanthin would protect against disuse muscle atrophy, in part, by prevention of myonuclear apoptosis. Wistar rats (8 weeks old) were divided into control (CT, n = 9), hindlimb unloading (HU, n = 9), and hindlimb unloading with astaxanthin (HU + AX, n = 9) groups. Following 2 weeks of dietary supplementation, rats in the HU and HU + AX groups were exposed to unloading for 7 days. Seven-day unloading resulted in reduced soleus muscle weight and myofiber cross-sectional area (CSA) by ~30 and ~47 %, respectively. Nonetheless, relative muscle weights and CSA of the soleus muscle in the HU + AX group were significantly greater than those of the HU group. Moreover, astaxanthin prevented disuse-induced increase in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei. We conclude that astaxanthin supplementation prior to and during hindlimb unloading attenuates soleus muscle atrophy, in part, by suppressing myonuclear apoptosis.

The study described herein aimed to examine changes in HDAC4 and its downstream targets in immobilization-induced rat skeletal muscle atrophy. Eleven male Wistar rats were used, and one hindlimb was immobilized in the plantar flexion position using a plaster cast. The contralateral, non-immobilized leg served as an internal control. After 10 days, the gastrocnemius muscles were removed from both hindlimbs. Ten days of immobilization resulted in a significant reduction (-27.3 %) in gastrocnemius muscle weight. A significant decrease in AMPK phosphorylation was also observed in nuclear fractions from immobilized legs relative to the controls. HDAC4 expression was significantly increased in immobilized legs in both the cytoplasmic and nuclear fractions. Moreover, Myogenin and MyoD mRNA levels were upregulated in immobilized legs, resulting in increased Atrogin-1 mRNA expression. Our data suggest that nuclear HDAC4 accumulation is partly related to immobilization-induced muscle atrophy.

Purpose: Protein ingestion after resistance exercise increases muscle protein synthesis (MPS) in a dose-dependent manner. However, the molecular mechanism(s) for the dose-dependency of MPS remains unclear. This study aimed to determine the dose response of mammalian target of rapamycin (mTOR) signaling in muscle with ingestion of protein after resistance exercise., Methods: Fifteen male subjects performed four sets of six unilateral isokinetic concentric knee extensions. Immediately after exercise, eight subjects consumed water only. The other seven subjects, in a randomized-order crossover design, took either a 10 [3.6 g essential amino acids (EAA)] or 20 g (7.1 g EAA) solution of whey protein. Muscle biopsies from the vastus lateralis muscle were taken 30 min before and 1 h after resistance exercise. Phosphorylation of Akt (Ser473), mTOR (Ser2448), 4E-BP1 (Thr37/46), and S6K1 (Thr389) was measured by western blotting., Results: Concentric knee extension exercise alone did not increase phosphorylation of Akt and mTOR 1 h after exercise, but ingesting protein after exercise significantly increased the phosphorylation of Akt and mTOR in a dose-dependent manner (P < 0.05). 4E-BP1 phosphorylation significantly decreased after resistance exercise (P < 0.05), but subjects who took 10 or 20 g of protein after exercise showed increased 4E-BP1 from post-exercise dephosphorylation (P < 0.05). S6K1 phosphorylation significantly increased after resistance exercise (P < 0.05), and 20 g of protein further increased S6K1 phosphorylation compared with ingestion of 10 g (P < 0.05)., Conclusions: These findings suggest that whey protein intake after resistance exercise activates mTOR signaling in a dose-dependent manner in untrained men.

Knowledge of the effects of training volume on upper limb muscular strength and hypertrophy is rather limited. In this study, both arms of the same subject were trained in a crossover-like design with different training volumes (1 or 3 sets) to eliminate the effects of genetic variation and other individual differences. The purpose of this study was to investigate the effects of training volume on muscular strength and hypertrophy in sedentary, untrained young Japanese men. Eight subjects (age, 25.0 ± 2.1 years; body mass, 64.2 ± 7.9 kg; height, 171.7 ± 5.1 cm) were recruited. The subjects trained their elbow flexor muscles twice per week for 12 consecutive weeks using a seated dumbbell preacher curl. The arms were randomly assigned to training with 1 or 3 sets. The training weight was set at 80% of 1 repetition maximum for all sets. The 3-set protocol increased cross-sectional area significantly more than did 1 set (1 set, 8.0 ± 3.7%; 3 sets, 13.3 ± 3.6%, p < 0.05). Furthermore, gains in strength with the 3-set protocol tended to be greater than those with 1 set (1 set, 20.4 ± 21.6%; 3 sets, 31.7 ± 22.0%, p = 0.076). Based on the results, the authors recommend 3 sets for sedentary untrained individuals. However, this population should incorporate light training days of 1 set into their training program to prevent overtraining and ensure adherence. The findings are relevant for the sedentary, untrained young male population and must be interpreted within the context of this study.

This study investigated the effect of heat stress (HS) on mammalian target of rapamycin (mTOR) signaling involved in translation initiation after resistance exercise in human skeletal muscle. Eight young male subjects performed four sets of six maximal repetitions of knee extension exercises, with or without HS, in a randomized crossover design. HS was applied to the belly of the vastus lateralis by using a microwave therapy unit prior to and during exercise. Muscle biopsies were taken from the vastus lateralis before, immediately after, and 1 h after exercise. HS significantly increased the phosphorylation of Akt/PKB, mTOR, and ribosomal protein S6 at 1 h after exercise (P < 0.05), and the 4E-BP1 phosphorylation level, which had initially decreased with exercise, had recovered by 1 h after exercise with HS. In addition, the phosphorylation of ribosomal S6 kinase 1 was significantly increased immediately after exercise with HS (P < 0.05). These results indicate that HS enhances mTOR signaling after resistance exercise in human skeletal muscle.

Alpha (alpha)-actinin-3 is located in the skeletal muscle Z-line and forms actin-actin crosslinks. An interesting property of alpha-actinin-3 is its expression pattern, which is restricted to fast type II skeletal muscle fibers. However, little is known about the response of alpha-actinin-3 levels to changes in skeletal muscle such as fiber type transformation. This study examined alpha-actinin-3 levels in the soleus muscles of rats subjected to hindlimb unloading, which causes a slow-to-fast fiber transformation in the soleus muscle. After unloading, type II myosin heavy chain (MyHC) and fast myosin levels increased significantly (P<0.0001 for type II MyHC, P<0.005 for fast myosin). Along with these increases in fast fibers, alpha-actinin-3 expression levels increased significantly (P<0.0007) and dramatically. These results indicate that alpha-actinin-3 levels increase concomitantly with increases in skeletal muscle fast fibers.