Fuminori Kawano

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.

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 (4u2009h, 15%, 1u2009Hz) followed by stimulation with 2u2009mM leucine for 45u2009min. 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 4u2009h. 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.

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.

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 up-regulated in both plantaris and soleus muscles 2 h after acute running, although the magnitude of the up-regulation 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-1αtranscription differed between fast- and slow-twitch skeletal muscles, affecting the strength of the transcriptional activity.