Genome-Wide Association Study Identifies CDKN1A as a Novel Locus Associated with Muscle Fiber Composition.

Muscle fiber composition is associated with physical performance, with endurance athletes having a high proportion of slow-twitch muscle fibers compared to power athletes. Approximately 45% of muscle fiber composition is heritable, however, single nucleotide polymorphisms (SNP) underlying inter-individual differences in muscle fiber types remain largely unknown. Based on three whole genome SNP datasets, we have shown that the rs236448 A allele located near the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene was associated with an increased proportion of slow-twitch muscle fibers in Russian (n = 151; p = 0.039), Finnish (n = 287; p = 0.03), and Japanese (n = 207; p = 0.008) cohorts (meta-analysis: p = 7.9 × 10−5. Furthermore, the frequency of the rs236448 A allele was significantly higher in Russian (p = 0.045) and Japanese (p = 0.038) elite endurance athletes compared to ethnically matched power athletes. On the contrary, the C allele was associated with a greater proportion of fast-twitch muscle fibers and a predisposition to power sports. CDKN1A participates in cell cycle regulation and is suppressed by the miR-208b, which has a prominent role in the activation of the slow myofiber gene program. Bioinformatic analysis revealed that the rs236448 C allele was associated with increased CDKN1A expression in whole blood (p = 8.5 × 10−15) and with greater appendicular lean mass (p = 1.2 × 10−5), whereas the A allele was associated with longer durations of exercise (p = 0.044) reported amongst the UK Biobank cohort. Furthermore, the expression of CDKN1A increased in response to strength (p < 0.0001) or sprint (p = 0.00035) training. Accordingly, we found that CDKN1A expression is significantly (p = 0.002) higher in the m. vastus lateralis of strength athletes compared to endurance athletes and is positively correlated with the percentage of fast-twitch muscle fibers (p = 0.018). In conclusion, our data suggest that the CDKN1A rs236448 SNP may be implicated in the determination of muscle fiber composition and may affect athletic performance.

High-throughput muscle fiber typing from RNA sequencing data.

BACKGROUND: Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. METHODS: By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). RESULTS: The correlation between the sequencing-based method and the other two were rATPas = 0.44 [0.13-0.67], [95% CI], and rmyosin = 0.83 [0.61-0.93], with p = 5.70 × 10-3 and 2.00 × 10-6, respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of ~ 10,000 paired-end reads. CONCLUSIONS: This new method ( https://github.com/OlaHanssonLab/PredictFiberType ) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.

Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles.

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.

The MOTS-c K14Q polymorphism in the mtDNA is associated with muscle fiber composition and muscular performance.

Human skeletal muscle fiber is heterogenous due to its diversity of slow- and fast-twitch fibers. In human, slow-twitched fiber gene expression is correlated to MOTS-c, a mitochondria-derived peptide that has been characterized as an exercise mimetic. Within the MOTS-c open reading frame, there is an East Asian-specific m.1382A>C polymorphism (rs111033358) that changes the 14th amino acid of MOTS-c (i.e., K14Q), a variant of MOTS-c that has less biological activity. Here, we examined the influence of the m.1382A>C polymorphism causing MOTS-c K14Q on skeletal muscle fiber composition and physical performance. The myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, and MHC-IIx) as an indicator of muscle fiber composition were assessed in 211 Japanese healthy individuals (102 men and 109 women). Muscular strength was measured in 86 physically active young Japanese men by using an isokinetic dynamometer. The allele frequency of the m.1382A>C polymorphism was assessed in 721 Japanese athletes and 873 ethnicity-matched controls. The m.1382A>C polymorphism genotype was analyzed by TaqMan SNP Genotyping Assay. Individuals with the C allele of the m.1382A>C exhibited a higher proportion of MHC-IIx, an index of fast-twitched fiber, than the A allele carriers. Men with the C allele of m.1382A>C exhibited significantly higher peak torques of leg flexion and extension. Furthermore, the C allele frequency was higher in the order of sprint/power athletes (6.5%), controls (5.1%), and endurance athletes (2.9%). Additionally, young male mice were injected with the MOTS-c neutralizing antibody once a week for four weeks to mimic the C allele of the m.1382A>C and assessed for protein expression levels of MHC-fast and MHC-slow. Mice injected with MOTS-c neutralizing antibody showed a higher expression of MHC-fast than the control mice. These results suggest that the C allele of the East Asian-specific m.1382A>C polymorphism leads to the MOTS-c K14Q contributes to the sprint/power performance through regulating skeletal muscle fiber composition.

Female Athletes Genetically Susceptible to Fatigue Fracture Are Resistant to Muscle Injury: Potential Role of COL1A1 Variant.

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.

Long-term physical inactivity exacerbates hindlimb unloading-induced muscle atrophy in young rat soleus muscle.

This study investigated the effects of long-term physical inactivity in adolescent on subsequent hindlimb unloading-induced muscle atrophy in rat soleus muscle. First, 3-wk-old male Wistar rats were assigned to an age-matched control (n = 6) or a physical inactivity (n = 8) group. Rats in the physical inactivity group were housed in narrow cages with approximately half the usual floor space for 8 wk to limit range of movement. Whole body energy consumption was measured, and the blood, organs, femoral bone, and hindlimb muscles were removed. We found that long-term physical inactivity did not affect the metabolic and physiological characteristics of growing rats. Then, fifty-six 3-wk-old male Wistar rats were assigned randomly into control (n = 28) and physical inactivity (n = 28) groups. After 8 wk, the rats in both groups underwent hindlimb unloading. The soleus muscles were removed before unloading (0 day), and 1, 3, and 7 days after unloading (n = 7 for each). Although the soleus muscle weight was significantly decreased after 7 days of hindlimb unloading in both groups, the decrease was drastic in the inactive group. A significant interaction between inactivity and unloading (P < 0.01) was observed according to the 4-hydroxynonenal-conjugated protein levels and the histone deacetylase 4 (HDAC4) and NF-κB protein levels. HDAC4 and NF-κB p65 protein levels in the physical inactivity group increased significantly 1 day after hindlimb unloading, along with the mRNA levels of their downstream targets myogenin and muscle RING finger protein 1 (MuRF1). Subsequent protein ubiquitination was upregulated by long-term physical inactivity (P < 0.05).NEW & NOTEWORTHY Long-term physical inactivity exacerbates hindlimb unloading-induced disuse muscle atrophy in young rat soleus muscles, possibly mediated by oxidative stress-induced protein ubiquitination via HDAC4- and NF-κB p65-induced MuRF1 mRNA upregulation.

Genotype Score for Iron Status Is Associated with Muscle Fiber Composition in Women.

Human muscle fiber composition is heterogeneous and mainly determined by genetic factors. A previous study reported that experimentally induced iron deficiency in rats increases the proportion of fast-twitch muscle fibers. Iron status has been reported to be affected by genetic factors. As the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms are strongly associated with iron status in humans, we hypothesized that the genotype score (GS) based on these polymorphisms could be associated with the muscle fiber composition in humans. Herein, we examined 214 Japanese individuals, comprising of 107 men and 107 women, for possible associations of the GS for iron status with the proportion of myosin heavy chain (MHC) isoforms (I, IIa, and IIx) as markers of muscle fiber composition. No statistically significant correlations were found between the GS for iron status and the proportion of MHC isoforms in all participants. When the participants were stratified based on sex, women showed positive and negative correlations of the GS with MHC-IIa (age-adjusted p = 0.020) and MHC-IIx (age-adjusted p = 0.011), respectively. In contrast, no correlation was found in men. In women, a 1-point increase in the GS was associated with 2.42% higher MHC-IIa level and 2.72% lower MHC-IIx level. Our results suggest that the GS based on the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms for iron status is associated with muscle fiber composition in women.

Are Genome-Wide Association Study Identified Single-Nucleotide Polymorphisms Associated With Sprint Athletic Status? A Replication Study With 3 Different Cohorts.

PURPOSE: To replicate previous genome-wide association study identified sprint-related polymorphisms in 3 different cohorts of top-level sprinters and to further validate the obtained results in functional studies. METHODS: A total of 240 Japanese, 290 Russians, and 593 Brazilians were evaluated in a case-control approach. Of these, 267 were top-level sprint/power athletes. In addition, the relationship between selected polymorphisms and muscle fiber composition was evaluated in 203 Japanese and 287 Finnish individuals. RESULTS: The G allele of the rs3213537 polymorphism was overrepresented in Japanese (odds ratio [OR]: 2.07, P = .024) and Russian (OR: 1.93, P = .027) sprinters compared with endurance athletes and was associated with an increased proportion of fast-twitch muscle fibers in Japanese (P = .02) and Finnish (P = .041) individuals. A meta-analysis of the data from 4 athlete cohorts confirmed that the presence of the G/G genotype rather than the G/A+A/A genotypes increased the OR of being a sprinter compared with controls (OR: 1.49, P = .01), endurance athletes (OR: 1.79, P = .001), or controls + endurance athletes (OR: 1.58, P = .002). Furthermore, male sprinters with the G/G genotype were found to have significantly faster personal times in the 100-m dash than those with G/A+A/A genotypes (10.50 [0.26] vs 10.76 [0.31], P = .014). CONCLUSION: The rs3213537 polymorphism found in the CPNE5 gene was identified as a highly replicable variant associated with sprinting ability and the increased proportion of fast-twitch muscle fibers, in which the homozygous genotype for the major allele (ie, the G/G genotype) is preferable for performance.

Maintenance of contractile force and increased fatigue resistance in slow-twitch skeletal muscle of mice fed a high-fat diet.

Consumption of a high-fat diet (HFD) significantly increases exercise endurance performance during treadmill running. However, whether HFD consumption increases endurance capacity via enhanced muscle fatigue resistance has not been clarified. In this study, we investigated the effects of HFDs on contractile force and fatigue resistance of slow-twitch dominant muscles. The soleus (SOL) muscle of male C57BL/6J mice fed an HFD (60% kcal from fat) or a low-fat diet (LFD) for 12 wk was analyzed. Muscle contractile force was measured under resting conditions and during fatigue induced by repeated tetanic contractions (100 Hz, 50 contractions, and 2-s intervals). Differences in muscle twitch or tetanic force were not evident between HFD and LFD groups, whereas fatigue resistance was higher in the HFD groups. The SOL muscle of HFD-fed mice showed increased levels of markers related to oxidative capacity such as succinate dehydrogenase (SDH) and citrate synthase (CS) activity. In addition, electron microscopy analyses indicated that the total number of mitochondria and mitochondrial volume density increased in the SOL muscle of the HFD groups. These findings suggest that HFD consumption induces increased muscle fatigue resistance in slow-twitch dominant muscle fibers. This effect of HFD may be related to elevated oxidative enzyme activity, high mitochondrial content, or both.NEW & NOTEWORTHY In this study, we examined the effects of HFDs on muscle contractile force and fatigue resistance of slow-twitch dominant muscles ex vivo. We found that contractile function was comparable between the HFD groups and the LFD group, whereas fatigue resistance was higher in the HFD groups. This effect of HFD may be related to elevated oxidative enzyme activity, high mitochondrial content, or both.

Ketogenic diet feeding improves aerobic metabolism property in extensor digitorum longus muscle of sedentary male rats.

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.

PPARGC1A rs8192678 and NRF1 rs6949152 Polymorphisms Are Associated with Muscle Fiber Composition in Women.

PPARGC1A rs8192678 G/A (Gly482Ser) and NRF1 rs6949152 A/G polymorphisms have been associated with endurance athlete status, endurance performance phenotypes, and certain health-related markers of different pathologies such as metabolic syndrome, diabetes, and dyslipidemia. We hypothesized that they could be considered interesting candidates for explaining inter-individual variations in muscle fiber composition in humans. We aimed to examine possible associations of these polymorphisms with myosin heavy-chain (MHC) isoforms as markers of muscle fiber compositions in vastus lateralis muscle in a population of 214 healthy Japanese subjects, aged between 19 and 79 years. No significant associations were found in men for any measured variables. In contrast, in women, the PPARGC1A rs8192678 A/A genotype was significantly associated with a higher proportion of MHC-I (p = 0.042) and with a lower proportion of MHC-IIx (p = 0.033), and the NRF1 rs6949152 AA genotype was significantly associated with a higher proportion of MHC-I (p = 0.008) and with a lower proportion of MHC IIx (p = 0.035). In women, the genotype scores of the modes presenting the most significant results for PPARGC1A rs8192678 G/A (Gly482Ser) and NRF1 rs6949152 A/G polymorphisms were significantly associated with MHC-I (p = 0.0007) and MHC IIx (p = 0.0016). That is, women with combined PPARGC1A A/A and NRF1 A/A genotypes presented the highest proportion of MHC-I and the lowest proportion of MHC-IIx, in contrast to women with combined PPARGC1A GG+GA and NRF1 AG+GG genotypes, who presented the lowest proportion of MHC-I and the highest proportion of MHC-IIx. Our results suggest possible associations between these polymorphisms (both individually and in combination) and the inter-individual variability observed in muscle fiber composition in women, but not in men.

A chronic high-fat diet exacerbates contractile dysfunction with impaired intracellular Ca2+ release capacity in the skeletal muscle of aged mice.

Obesity and aging reduce skeletal muscle contractile function. However, it remains unclear whether obesity additively promotes muscle contractile dysfunction in the setting of aging. In this study, we investigated skeletal muscle contractile function ex vivo and intracellular Ca2+ release in male C57BL/6J mice fed a low-fat diet (LFD) or a high-fat diet (HFD) for 4 or 20 mo. Tetanic force production in the extensor digitorum longus muscle was decreased by aging or HFD feeding, and the further reduction was observed in aged HFD mice. The 20-mo HFD-fed mice, not the 20-mo LFD-fed mice or 4-mo HFD-fed mice, showed reduced intracellular Ca2+ peak levels by high concentration of caffeine (25 mM) compared with 4-mo LFD mice. Aging and HFD feeding additively increased intramyocellular lipid (IMCL) levels and were associated with the degree of impaired muscle contractile force and peak Ca2+ level. These data suggest that impairment in the contractile force in aged muscle is aggravated by HFD, which may be due, at least in part, to dysfunction in intracellular Ca2+ release. The IMCL level may be a marker for impaired muscle contractile force caused by aging and HFD.NEW & NOTEWORTHY The aim of this study was to examine the effect of high-fat diet (HFD)-induced obesity on contractile function and Ca2+ release capacity in aged skeletal muscle. Not only were the force production and peak Ca2+ levels decreased by aging and HFD feeding, respectively, but also, these interventions had an additive effect in aged HFD-fed mice. These data suggest that the impairment in the contractile force in aged muscle is aggravated by a HFD, which may be due to synergistic dysfunction in intracellular Ca2+ release.

JAK-STAT-dependent regulation of scavenger receptors in LPS-activated murine macrophages.

In atherosclerosis progression, atherosclerotic plaques develop upon accumulated foam cells derived from macrophages that take up modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Lipopolysaccharide (LPS) exacerbates atherosclerosis by enhancing the expression of scavenger receptors and thus increasing the uptake of modified LDL into macrophages. However, the signaling pathways that mediate LPS and scavenger receptor expression have not been fully elucidated. We used mouse bone marrow-derived macrophages and investigated the effects of LPS in vitro. LPS enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-1 (STAT-1). Inhibitors of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) pathway (U0126 and PD0325901) suppressed the uptake of acetylated-LDL (Ac-LDL) and the expression of CD204 but not CD36 in LPS-activated macrophages. Inhibitors of the Janus tyrosine kinase (JAK)-STAT pathway (ruxolitinib and tofacitinib) suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. We next injected LPS into the peritoneal cavity of mice and analyzed the effects of LPS. MEK inhibitor U0126 suppressed the uptake of Ac-LDL and the expression of CD204 but not CD36 in LPS-activated macrophages. JAK inhibitor ruxolitinib suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. These results suggest that scavenger receptors in LPS-activated mouse macrophages are regulated through a JAK-STAT-dependent pathway. Although further evaluation is necessary, JAK-STAT inhibition could be useful in atherosclerosis therapy, at least for atherosclerosis exacerbated by LPS.

Sex-specific differences in rat soleus muscle signaling pathway responses to a bout of horizontal and downhill running.

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.

Age-related changes in histone modification in rat gastrocnemius muscle.

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.

Exercise preconditioning attenuates hind limb unloading-induced gastrocnemius muscle atrophy possibly via the HDAC4/Gadd45 axis in old rats.

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.

Sex differences in forkhead box O3a signaling response to hindlimb unloading in rat soleus muscle.

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.

Dysfunction of muscle contraction with impaired intracellular Ca2+ handling in skeletal muscle and the effect of exercise training in male db/db mice.

Type 2 diabetes is characterized by reduced contractile force production and increased fatigability of skeletal muscle. While the maintenance of Ca2+ 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 Ca2+ 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 Ca2+ levels were both lower during tetanic stimulation of the fast-twitch muscles, while Ca2+ 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 Ca2+ levels, and Ca2+ accumulation following stimulation in male db/db mice. These data suggest that dysfunctional Ca2+ 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 Ca2+ 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 Ca2+ regulation in a male type 2 diabetic animal model. These dysfunctions in muscle were improved by 6 wk of exercise training.

Effect of a combination of astaxanthin supplementation, heat stress, and intermittent reloading on satellite cells during disuse muscle atrophy.

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.

Body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of type 2 diabetic rats.

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.