Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

Vibration acceleration enhances proliferation, migration, and maturation of C2C12 cells and promotes regeneration of muscle injury in male rats.

Vibration acceleration (VA) using a whole-body vibration device is beneficial for skeletal muscles. However, its effect at the cellular level remains unclear. We aimed to investigate the effects of VA on muscles in vitro and in vivo using the C2C12 mouse myoblast cell line and cardiotoxin-induced injury in male rat soleus muscles. Cell proliferation was evaluated using the WST/CCK-8 assay and proportion of Ki-67 positive cells. Cell migration was assessed using wound-healing assay. Cell differentiation was examined by the maturation index in immunostained cultured myotubes and real-time polymerase chain reaction. Regeneration of soleus muscle in rats was assessed by recruitment of satellite cells, cross-sectional area of regenerated muscle fibers, number of centrally nucleated fibers, and conversion of regenerated muscle from fast- to slow-twitch. VA at 30 Hz with low amplitude for 10 min promoted C2C12 cell proliferation, migration, and myotube maturation, without promoting expression of genes related to differentiation. VA significantly increased Pax7-stained satellite cells and centrally nucleated fibers in injured soleus muscles on Day 7 and promoted conversion of fast- to slow-twitch muscle fibers with an increase in the mean cross-sectional area of regenerated muscle fibers on Day 14. VA enhanced the proliferation, migration, and maturation of C2C12 myoblasts and regeneration of injured rat muscles.

Exercise-induced histone H3 trimethylation at lysine 27 facilitates the adaptation of skeletal muscle to exercise in mice.

Histone H3 trimethylation at lysine 27 (H3K27me3) is known to act as a transcriptionally repressive histone modification via heterochromatin formation. In skeletal muscle, it was also reported that H3K27me3 was enriched at the sites transcriptionally activated by exercise, although the role of H3K27me3 in adaptation to exercise is unknown. In this study, using mouse tibialis anterior muscle, we initially determined the genome-wide enrichment of RNA polymerase II and histone H3 trimethylation at lysine 4 (H3K4me3) and H3K27me3 using chromatin immunoprecipitation, followed by sequencing analysis. The loci that were transcriptionally upregulated by a single bout of running exercise were marked by both H3K27me3 and H3K4me3, which were also correlated with the distribution of RNA polymerase II. The genes that were not responsive to exercise exhibited high H3K4me3 occupancy, similar to the upregulated genes but with less H3K27me3. Next, we tested the effects of GSK343, a specific inhibitor of enhancer of zeste homologue 2 (EZH2). Unexpectedly, GSK343 administration enhanced the H3K27me3 occupancy at the target loci, leading to the upregulation of gene responses to acute exercise. Administration of GSK343 also facilitated the phenotypic transformation of type IIb to type IIa fibres and the upregulation of AMPK phosphorylation and levels of heat shock protein 70, pyruvate dehydrogenase kinase 4, peroxisome proliferator-activated receptor Î³ coactivator-1α and muscle RING finger 1. Furthermore, in contrast to the accelerated adaptation to exercise by GSK343, administration of the EZH1/2 dual inhibitor valemetostat prevented the changes in the aforementioned parameters after exercise training. These results indicate that exercise-induced H3K27me3 plays a key role in inducing exercise-related effects in the skeletal muscle. KEY POINTS: Exercise mediates histone H3 trimethylation at lysine 27 (H3K27me3) at transcriptionally upregulated loci in skeletal muscle, but the role of H3K27me3 in the adaptation of skeletal muscle to exercise training is unclear. Chromatin immunoprecipitation followed by sequencing analysis demonstrated that H3K27me3, in addition to H3K4me3 modifications, is the hallmark of sites showing higher responses to acute exercise. GSK343, a selective inhibitor of the enhancer of zeste homologue 2 (EZH2), enhanced the gene responses to a single bout of exercise and accelerated the adaptive changes during exercise training in association with myonuclear H3K27me3 accumulation. Administration of valemetostat, an EZH1/2 dual inhibitor, repressed myonuclear H3K27me3 accumulation during training and caused a failure of adaptive changes. Exercise-induced H3K27me3 might play a key role in inducing exercise-related effects in skeletal muscle.

ATP spreads inflammation to other limbs through crosstalk between sensory neurons and interneurons.

Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron-interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.

Responses of neuromuscular properties to unloading and potential countermeasures during space exploration missions.

We reviewed the responses of the neuromuscular properties of mainly the soleus and possible mechanisms. Sensory nervous activity in response to passive shortening and/or active contraction, associated with plantar-flexion or dorsi-flexion of the ankle joints, may play an essential role in the regulation of muscle properties. Passive shortening of the muscle fibers and sarcomeres inhibits the development of tension, electromyogram (EMG), and afferent neurogram. Remodeling of the sarcomeres, which decreases the total sarcomere number in a single muscle fiber causing recovery of the length in each sarcomere, is induced in the soleus following chronic unloading. Although EMG activity and tension development in each sarcomere are increased, the total tension produced by the whole muscle is still less owing to the lower sarcomere number. Therefore, muscle atrophy continues to progress. Moreover, walking or slow running by rear-foot strike landing with the application of greater ground reaction force, which stimulates soleus mobilization, could be an effective countermeasure. Periodic, but not chronic, passive stretching of the soleus may also be effective.

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth.

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Chronic exercise training activates histone turnover in mouse skeletal muscle fibers.

Epigenetic regulation of skeletal muscle adaptation to exercise is a recent topic for which there is limited information. This study investigated whether exercise training activates histone turnover in the skeletal muscle fibers of mice. Experiments using a tetracycline-inducible H2B-GFP expression model demonstrated that 4 weeks of running training, but not 2 weeks of training, significantly promoted the incorporation of H2B-GFP into nucleosomes and the dissociation of histone H3.3 at both transcriptionally upregulated and nonresponsive loci. Muscle-specific PGC-1α-b-overexpressing mice crossed with H2B-GFP mice showed a slight increase in H2B-GFP incorporation at transcriptionally active loci, but not in the dissociation of H3.3 from nucleosomes. Gene expression responses to a single bout of running were significantly enhanced in 4-week trained mice when compared with those in 2-week trained mice. The most drastic increase in the gene response was found in the expression of Hspa1a and Hspa1b, in which the magnitude of upregulation in response to running was significantly enhanced from 8-fold in 2 week trained mice to 97- and 121-fold in 4 week trained mice, respectively. It was also found that the HSP70 level increased during the training period. In a myonuclear immunohistochemical analysis of chromatin remodelers, we further found that the level of SPT16, an H2A-H2B-specific chaperone, was upregulated after running training. These results revealed that 4 weeks of running training activated histone turnover in skeletal muscle fibers. They also suggested that histone turnover led to loosening of the nucleosomes and enhanced gene responses to exercise.

Early high-fat feeding improves histone modifications of skeletal muscle at middle-age in mice.

The purpose of the present study was to investigate how the effects of high-fat diet feeding on the skeletal muscle persisted during aging using mice. Post-weaned male mice were fed a high-fat diet between 1- and 3-mo-old followed by return to supply a normal diet until 13-mo-old. Monthly physical tests demonstrated that age-related glucose intolerance that was generally developed after 10-mo-old in the control mice was significantly improved in mice fed a high-fat diet. Interestingly, mRNA expressions of Pdk4, Ucp3, and Zmynd17 were up-regulated by high-fat feeding and persisted in the tibialis anterior muscle until 13-mo-old. At Pdk4 and Ucp3 loci, enhanced distributions of active histone modifications were noted in the high-fat-fed mice at 13-mo-old. In contrast, age-related accumulation of histone variant H3.3 at these loci was suppressed. These results indicated that epigenetic modifications caused by early nutrition mediated the changes in skeletal muscle gene expression during aging.

Adaptive responses of histone modifications to resistance exercise in human skeletal muscle.

Exercise training causes epigenetic changes in skeletal muscle, although it is unclear how resistance exercise (RE) affects histone modifications. The present study was carried out to investigate the effects of acute RE and RE training on gene expression profiles and histone modifications in human skeletal muscle. Healthy male adults were assigned to acute RE (n = 9, age = 20.5±4.3yr, BMI = 28.0±6.8kg/m2) or RE training (n = 21, age = 23.7±2.5yr, BMI = 24.2±2.7kg/m2) groups. Biopsy samples were obtained from the vastus lateralis muscle before and three hours after a single bout of acute RE, or 3-days after 10 weeks of RE training. RNA sequencing analysis revealed that 153 genes with GO terms including muscle development, stress response, metabolism, cell death, and transcription factor were significantly up-regulated (+291% vs. pre-acute RE) upon acute RE. Expressions of these genes were also greater (+9.6% vs. pre-RE training, p<0.05) in RE trained subjects. Significant up-regulation of acetylated histone 3 (H3) (+235%) and H3 mono-methylated at lysine 4 (+290%) and tri-methylated at lysine 27 (+849%), whereas down-regulation of H3.3 variant (-39%) distributions relative to total H3 were observed at transcriptionally activated loci after acute RE compared to pre-acute RE levels. Interestingly, the distribution of acetylated H3 was found to be up-regulated as compared to the level of total H3 after RE training (+40%, p<0.05). These results indicate that a single bout of RE drastically alters both gene expressions and histone modifications in human skeletal muscle. It is also suggested that enhanced histone acetylation is closely related to up-regulation of gene expressions after RE training.

Muscle type-specific RNA polymerase II recruitment during PGC-1α gene transcription after acute exercise in adult rats.

Epigenetic regulation of gene expression differs between fast- and slow-twitch skeletal muscles in adult rats, although the precise mechanisms are still unknown. The present study investigates the differences in responses of RNA polymerase II (Pol II) and histone acetylation during transcriptional activation in the plantaris and soleus muscles of adult rats after acute treadmill running. We targeted the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) gene to analyze epigenomic changes by chromatin immunoprecipitation. The mRNA expression of the PGC-1α-b isoform was significantly upregulated in both plantaris and soleus muscles 2 h after acute running, although the magnitude of the upregulation was more pronounced in the plantaris muscle. The sequences of proximal exons of the PGC-1α locus were expressed more in the plantaris muscle after acute running. Accumulation of Pol II was noted near the alternative exon 1 in both plantaris and soleus muscles in association with the enhanced distribution of acetylated histone 3. Accumulation of Pol II was also observed at the transcription start site, exon 2, and exon 3 in the plantaris muscle, but not the soleus muscle. It was noted that in the soleus muscle, acetylation of histone 3 at lysine 27 was enhanced throughout the PGC-1α locus in response to transcriptional activation, suggesting that elongating Pol II was capable of traveling through to the end of the locus. These results indicate that the mobility of Pol II during PGC-1α transcription differed between fast- and slow-twitch skeletal muscles, affecting the strength of the transcriptional activity.NEW & NOTEWORTHY Fast- and slow-twitch skeletal muscles have distinct characteristics in both force production and metabolism. Epigenetic regulations also largely differ in these muscles. Here we show that RNA polymerase II is distributed extensively at the proximal regions downstream of transcription start site during the transcriptional activation of PGC-1α in fast-twitch muscles, but it accumulates at the first exon in slow-twitch muscles. These findings will provide a basis to understand type-specific mechanisms in skeletal muscle.

Amount of daily exercise is an essential stimulation to alter the epigenome of skeletal muscle in rats.

Long-term running training causes epigenetic changes in the skeletal muscles. Here we tested the effects of the total amount or duration of running training on the distribution of histones in the rat plantaris muscle. Post-weaned young rats were assigned to 3 different training groups: Run-1, 30 min/day running exercise for 8 wk using an animal treadmill at 24 m/min; Run-2, 15 min/day for 8 wk; and Run-3, 60 min/day for 4 wk. Citrate synthase activity was not significantly changed by running training, although the slight increase was observed in Run-3. Genes that were previously defined as showing the typical responses to running training were targeted to measure the distribution of histones using chromatin immunoprecipitation. The distribution of acetylated histone 3 was elevated in Run-2 and Run-3, but not in Run-1. Incorporation of H3.3 into the nucleosome was stimulated in Run-1, whereas H3.3 distribution was unchanged in Run-2 or downregulated in Run-3. Significant downregulation of H3.3 expression was also detected in Run-3. We further checked the responses of the target genes during acute running. Target genes were transcriptionally activated and histone acetylation was stimulated at the loci in response to acute running. These results suggested that the exchange of the histone component to H3.3 was stimulated by running training, inhibiting the accumulation of acetylated histones in Run-1. Additionally, it was further suggested that the enhanced daily amount of running caused changes in the H3.3 expression, affecting the rate of the histone exchange in Run-3. NEW & NOTEWORTHY Chromatin remodeling in the skeletal muscle is a potent mechanism preventing disuse atrophy in later life that can be acquired via long-term exercise training. Here we demonstrated in rats that daily exercise amount is a key factor in the development of epigenetic changes in the skeletal muscle. To acquire a health benefit, our research suggests the importance of considering the time endurance for daily exercise bouts.

Leucine supplementation after mechanical stimulation activates protein synthesis via L-type amino acid transporter 1 in vitro.

Branched-chain amino acid supplements consumed following exercise are widely used to increase muscle mass. Although both exercise (ie, mechanical stimulation) and branched-chain amino acid leucine supplementation have been reported to stimulate muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway independently, the mechanisms underlying their synergistic effects are largely unknown. Utilizing cultured differentiated C2C12 myotubes, we established a combination treatment model in which the cells were subjected to cyclic uniaxial mechanical stretching (4 h, 15%, 1 Hz) followed by stimulation with 2 mM leucine for 45 min. Phosphorylation of p70 S6 kinase (p70S6K), an mTOR-regulated marker of protein translation initiation, was significantly increased following mechanical stretching alone but returned to the baseline after 4 h. Leucine supplementation further increased p70S6K phosphorylation, with a greater increase observed in the stretched cells than in the non-stretched cells. Notably, the expression of L-type amino acid transporter 1 (LAT1), a stimulator of the mTOR pathway, was also increased by mechanical stretching, and siRNA-mediated knockdown partially attenuated leucine-induced p70S6K phosphorylation. These results suggest that mechanical stretching promotes LAT1 expression and, consequently, amino acid uptake, leading to enhanced leucine-induced activation of protein synthesis. LAT1 has been demonstrated to be a point of crosstalk between exercise- and nutrition-induced skeletal muscle growth.

Running training experience attenuates disuse atrophy in fast-twitch skeletal muscles of rats.

Responsiveness to physiological stimuli, such as exercise and muscular inactivation, differs in individuals. However, the mechanisms responsible for these individual differences remain poorly understood. We tested whether a prior experience of exercise training affects the responses of skeletal muscles to unloading. Young rats were assigned to perform daily running training with a treadmill for 8 wk. After an additional 8 wk of normal habitation, the rats were hindlimb unloaded by tail suspension for 1 wk. Fast-twitch plantaris, gastrocnemius, and tibialis anterior muscles did not atrophy after unloading in rats with training experience, although soleus muscle lost weight similar to sedentary rats. We also analyzed the transcriptome in plantaris muscle with RNA sequencing followed by hierarchical clustering analysis and found that a subset of genes that were generally upregulated in sedentary rats after unloading were less responsive in rats with training experience. The distribution of histone 3 was diminished at the loci of these genes during the training period. Although the deposition of histone 3 was restored after an additional period of normal habitation, the incorporation of H3.3 variant was promoted in rats with training experience. This remodeling of nucleosomes closely correlated to the conformational changes of chromatin and suppressed gene expression in response to unloading. These results suggest that exercise training stimulated the early turnover of histone components, which may alter the responsiveness of gene transcription to physiological stimuli.NEW & NOTEWORTHY The present study demonstrates that disuse atrophy was suppressed in fast-twitch skeletal muscles of rats with training experience in early life. We also found a subset of genes that were less responsive to unloading in the muscle of rats with training experience. It was further determined that exercise training caused an early turnover of nucleosome components, which may alter the responsiveness of genes to stimulus in later life.

Vibration acceleration promotes bone formation in rodent models.

All living tissues and cells on Earth are subject to gravitational acceleration, but no reports have verified whether acceleration mode influences bone formation and healing. Therefore, this study was to compare the effects of two acceleration modes, vibration and constant (centrifugal) accelerations, on bone formation and healing in the trunk using BMP 2-induced ectopic bone formation (EBF) mouse model and a rib fracture healing (RFH) rat model. Additionally, we tried to verify the difference in mechanism of effect on bone formation by accelerations between these two models. Three groups (low- and high-magnitude vibration and control-VA groups) were evaluated in the vibration acceleration study, and two groups (centrifuge acceleration and control-CA groups) were used in the constant acceleration study. In each model, the intervention was applied for ten minutes per day from three days after surgery for eleven days (EBF model) or nine days (RFH model). All animals were sacrificed the day after the intervention ended. In the EBF model, ectopic bone was evaluated by macroscopic and histological observations, wet weight, radiography and microfocus computed tomography (micro-CT). In the RFH model, whole fracture-repaired ribs were excised with removal of soft tissue, and evaluated radiologically and histologically. Ectopic bones in the low-magnitude group (EBF model) had significantly greater wet weight and were significantly larger (macroscopically and radiographically) than those in the other two groups, whereas the size and wet weight of ectopic bones in the centrifuge acceleration group showed no significant difference compared those in control-CA group. All ectopic bones showed calcified trabeculae and maturated bone marrow. Micro-CT showed that bone volume (BV) in the low-magnitude group of EBF model was significantly higher than those in the other two groups (3.1±1.2mm3 v.s. 1.8±1.2mm3 in high-magnitude group and 1.3±0.9mm3 in control-VA group), but BV in the centrifuge acceleration group had no significant difference compared those in control-CA group. Union rate and BV in the low-magnitude group of RFH model were also significantly higher than those in the other groups (Union rate: 60% v.s. 0% in the high-magnitude group and 10% in the control-VA group, BV: 0.69±0.30mm3 v.s. 0.15±0.09mm3 in high-magnitude group and 0.22±0.17mm3 in control-VA group). BV/TV in the low-magnitude group of RFH model was significantly higher than that in control-VA group (59.4±14.9% v.s. 35.8±13.5%). On the other hand, radiographic union rate (10% in centrifuge acceleration group v.s. 20% in control-CA group) and micro-CT parameters in RFH model were not significantly different between two groups in the constant acceleration studies. Radiographic images of non-union rib fractures showed cartilage at the fracture site and poor new bone formation, whereas union samples showed only new bone. In conclusion, low-magnitude vibration acceleration promoted bone formation at the trunk in both BMP-induced ectopic bone formation and rib fracture healing models. However, the micro-CT parameters were not similar between two models, which suggested that there might be difference in the mechanism of effect by vibration between two models.

Muscle-specific deletion of BDK amplifies loss of myofibrillar protein during protein undernutrition.

Branched-chain amino acids (BCAAs) are essential amino acids for mammals and play key roles in the regulation of protein metabolism. However, the effect of BCAA deficiency on protein metabolism in skeletal muscle in vivo remains unclear. Here we generated mice with lower BCAA concentrations by specifically accelerating BCAA catabolism in skeletal muscle and heart (BDK-mKO mice). The mice appeared to be healthy without any obvious defects when fed a protein-rich diet; however, bolus ingestion of BCAAs showed that mTORC1 sensitivity in skeletal muscle was enhanced in BDK-mKO mice compared to the corresponding control mice. When these mice were fed a low protein diet, the concentration of myofibrillar protein was significantly decreased (but not soluble protein) and mTORC1 activity was reduced without significant change in autophagy. BCAA supplementation in drinking water attenuated the decreases in myofibrillar protein levels and mTORC1 activity. These results suggest that BCAAs are essential for maintaining myofibrillar proteins during protein undernutrition by keeping mTORC1 activity rather than by inhibiting autophagy and translation. This is the first report to reveal the importance of BCAAs for protein metabolism of skeletal muscle in vivo.

Prenatal myonuclei play a crucial role in skeletal muscle hypertrophy in rodents.

Multinucleated muscle fibers are formed by the fusion of myogenic progenitor cells during embryonic and fetal myogenesis. However, the role of prenatally incorporated myonuclei in the skeletal muscle fibers of adult animals is poorly understood. We demonstrated, using muscle-specific reporter mice, that the prenatal myonuclei remained in the adult soleus muscle, although cardiotoxin injection caused the loss of prenatal myonuclei. Overloading by the tendon transection of synergists failed to induce compensatory hypertrophy in regenerated soleus muscle fibers of adult rats, whereas unloading by tail suspension normally induced the fiber atrophy. Loss of hypertrophying function correlated with the lowered histone acetylation at the transcription start site of Igf1r gene, which was one of the genes that did not respond to the overloading. These parameters were improved by the transplantation of cells harvested from the juvenile soleus muscles of neonatal rats in association with enhanced histone acetylation of Igf1r gene. These results indicated that the presence of prenatal myonuclei was closely related to the status of histone acetylation, which could regulate the responsiveness of muscle fibers to physiological stimuli.

Differences in histone modifications between slow- and fast-twitch muscle of adult rats and following overload, denervation, or valproic acid administration.

Numerous studies have reported alterations in skeletal muscle properties and phenotypes in response to various stimuli such as exercise, unloading, and gene mutation. However, a shift in muscle fiber phenotype from fast twitch to slow twitch is not completely induced by stimuli. This limitation is hypothesized to result from the epigenetic differences between muscle types. The main purpose of the present study was to identify the differences in histone modification for the plantaris (fast) and soleus (slow) muscles of adult rats. Genome-wide analysis by chromatin immunoprecipitation followed by DNA sequencing revealed that trimethylation at lysine 4 and acetylation of histone 3, which occurs at transcriptionally active gene loci, was less prevalent in the genes specific to the slow-twitch soleus muscle. Conversely, gene loci specific to the fast-twitch plantaris muscle were associated with the aforementioned histone modifications. We also found that upregulation of slow genes in the plantaris muscle, which are related to enhanced muscular activity, is not associated with activating histone modifications. Furthermore, silencing of muscle activity by denervation caused the displacement of acetylated histone and RNA polymerase II (Pol II) in 5' ends of genes in plantaris, but minor effects were observed in soleus. Increased recruitment of Pol II induced by forced acetylation of histone was also suppressed in valproic acid-treated soleus. Our present data indicate that the slow-twitch soleus muscle has a unique set of histone modifications, which may relate to the preservation of the genetic backbone against physiological stimuli.

Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells.

Cellular protein synthesis is believed to be antagonistically regulated by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways. In the present study, we examined the relationship between mTOR/p70 S6 kinase (p70S6K) and AMPK in response to mechanical stretch. C2C12 myoblasts were grown on a silicone elastomer chamber to confluence and further cultured in differentiation medium for 4 days to form multinucleated myotubes. Cells were subjected to 15% cyclic uniaxial stretch for 4 h at a frequency of 1 Hz. Phosphorylation of p70S6K at threonine 389 and AMPK at threonine 172 of the catalytic α subunit were concomitantly increased by mechanical stretch. Stimulation of the mTOR pathway by adding leucine and insulin increased the phosphorylation of p70S6K without inactivation of AMPK. In contrast, addition of compound C, a pharmacological inhibitor of AMPK, increased the phosphorylation of p70S6K in stretched cells. Activation of AMPK by the addition of 5-amino-4-imidazolecarboxamide ribonucleoside reduced the phosphorylation of p70S6K in response to mechanical stretch. In conclusion, crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or nutrients. However, pharmacological modulation of AMPK influenced the mTOR/p70S6K signaling pathway.

Responses of skeletal muscles to gravitational unloading and/or reloading.

Adaptation of morphological, metabolic, and contractile properties of skeletal muscles to inhibition of antigravity activities by exposure to a microgravity environment or by simulation models, such as chronic bedrest in humans or hindlimb suspension in rodents, has been well reported. Such physiological adaptations are generally detrimental in daily life on earth. Since the development of suitable countermeasure(s) is essential to prevent or inhibit these adaptations, effects of neural, mechanical, and metabolic factors on these properties in both humans and animals were reviewed. Special attention was paid to the roles of the motoneurons (both efferent and afferent neurograms) and electromyogram activities as the neural factors, force development, and/or length of sarcomeres as the mechanical factors and mitochondrial bioenergetics as the metabolic factors.

Retardation of C2C12 myoblast cell proliferation by exposure to low-temperature atmospheric plasma.

As the first step in evaluating the possibility of low-temperature atmospheric plasma for clinical applications in the treatment of rhabdomyosarcoma (RMS), we determined the effects of plasma exposure on C2C12 myoblasts. The low-temperature atmospheric plasma was generated through an electrical discharge in argon gas. One minute of plasma exposure every 24 h inhibited the cell proliferation, whereas myoblast differentiation was not affected. Plasma exposure increased the phosphorylation of ERK and JNK at 30 min after the exposure, but the phosphorylation of both was decreased to less than control levels at 1 and 4 h after the exposure. Plasma exposure increased the percentage of cells in the G2/M phase at 8 h after the exposure. In conclusion, plasma exposure retarded the proliferation of C2C12 myoblasts by G2/M arrest. Therefore, plasma exposure can be a possible treatment for the anti-proliferative effects of malignant tumors, such as RMS, without affecting differentiated skeletal muscle cells.