Human skeletal muscle methylome after low-carbohydrate energy-balanced exercise.

We aimed to investigate the human skeletal muscle (SkM) DNA methylome after exercise in low-carbohydrate (CHO) energy-balance (with high-fat) conditions compared with exercise in low-CHO energy-deficit (with low-fat) conditions. The objective was to identify novel epigenetically regulated genes and pathways associated with "train-low sleep-low" paradigms. The sleep-low conditions included nine males that cycled to deplete muscle glycogen while reaching a set energy expenditure. Postexercise, low-CHO meals (protein matched) completely replaced (using high fat) or only partially replaced (low fat) the energy expended. The following morning, resting baseline biopsies were taken and the participants then undertook 75 minutes of cycling exercise, with skeletal muscle biopsies collected 30 minutes and 3.5 hours postexercise. Discovery of genome-wide DNA methylation was undertaken using Illumina EPIC arrays, and targeted gene expression analysis was conducted by quantitative RT-PCR. At baseline, participants under energy balance (high fat) demonstrated a predominantly hypermethylated (60%) profile across the genome compared to energy-deficit low-fat conditions. However, postexercise performed in energy balance (with high fat) elicited a more prominent hypomethylation signature 30 minutes postexercise in gene regulatory regions important for transcription (CpG islands within promoter regions) compared with exercise in energy-deficit (with low-fat) conditions. Such hypomethylation was enriched within pathways related to IL6-JAK-STAT signaling, metabolic processes, p53/cell cycle, and oxidative/fatty acid metabolism. Hypomethylation within the promoter regions of the genes; histone deacetylase 2 (HDAC2), MECR, IGF2, and c13orf16 were associated with significant increases in gene expression in the postexercise period in energy balance compared with an energy deficit. Furthermore, HDAC11 was oppositely regulated at the gene expression level compared with family member HDAC2, where HDAC11 was hypomethylated yet increased in energy-deficit compared with energy-balance conditions. Overall, we identify some novel epigenetically regulated genes associated with train-low sleep-low paradigms.NEW & NOTEWORTHY We identify novel epigenetically regulated genes associated with train-low sleep-low paradigms. Exercise under low-carbohydrate (CHO) energy-balance (high-fat) conditions elicited a more prominent DNA hypomethylation signature 30 minutes postexercise compared with low-CHO energy-deficit (low-fat) conditions. This was enriched within IL6-JAK-STAT signaling, metabolic processes, p53, cell cycle, oxidative phosphorylation, and fatty acid metabolism. Histone deacetylase (HDAC) family members 2, 4, 10, and 11 demonstrated hypomethylation, with HDAC2 and HDAC11 possessing alternative regulation of gene expression in energy balance versus deficit conditions.

Aerobic exercise training resets the human skeletal muscle methylome 10 years after breast cancer treatment and survival.

Cancer survivors suffer impairments in skeletal muscle in terms of reduced mass and function. Interestingly, human skeletal muscle possesses an epigenetic memory of earlier stimuli, such as exercise. Long-term retention of epigenetic changes in skeletal muscle following cancer survival and/or exercise training has not yet been studied. We, therefore, investigated genome-wide DNA methylation (methylome) in skeletal muscle following a 5-month, 3/week aerobic-training intervention in breast cancer survivors 10-14 years after diagnosis and treatment. These results were compared to breast cancer survivors who remained untrained and to age-matched controls with no history of cancer, who undertook the same training intervention. Skeletal muscle biopsies were obtained from 23 females before(pre) and after(post) the 5-month training period. InfiniumEPIC 850K DNA methylation arrays and RT-PCR for gene expression were performed. The breast cancer survivors displayed a significant retention of increased DNA methylation (i.e., hypermethylation) at a larger number of differentially methylated positions (DMPs) compared with healthy age-matched controls pre training. Training in cancer survivors led to an exaggerated number of DMPs with a hypermethylated signature occurring at non-regulatory regions compared with training in healthy age-matched controls. However, the opposite occurred in important gene regulatory regions, where training in cancer survivors elicited a considerable reduction in methylation (i.e., hypomethylation) in 99% of the DMPs located in CpG islands within promoter regions. Importantly, training was able to reverse the hypermethylation identified in cancer survivors back toward a hypomethylated signature that was observed pre training in healthy age-matched controls at 300 (out of 881) of these island/promoter-associated CpGs. Pathway enrichment analysis identified training in cancer survivors evoked a predominantly hypomethylated signature in pathways associated with cell cycle, DNA replication/repair, transcription, translation, mTOR signaling, and the proteosome. Differentially methylated region (DMR) analysis also identified genes: BAG1, BTG2, CHP1, KIFC1, MKL2, MTR, PEX11B, POLD2, S100A6, SNORD104, and SPG7 as hypermethylated in breast cancer survivors, with training reversing these CpG island/promoter-associated DMRs toward a hypomethylated signature. Training also elicited a largely different epigenetic response in healthy individuals than that observed in cancer survivors, with very few overlapping changes. Only one gene, SIRT2, was identified as having altered methylation in cancer survivors at baseline and after training in both the cancer survivors and healthy controls. Overall, human skeletal muscle may retain a hypermethylated signature as long as 10-14 years after breast cancer treatment/survival. Five months of aerobic training reset the skeletal muscle methylome toward signatures identified in healthy age-matched individuals in gene regulatory regions.

The Comparative Methylome and Transcriptome After Change of Direction Compared to Straight Line Running Exercise in Human Skeletal Muscle.

The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g., high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in five male sport team members using a repeated measures design of: (1) change of direction (COD) versus; (2) straight line (ST) running exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 min and 24 h post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed × distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) exertion measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 min and 24 h post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 min post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We also confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via COD exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.

Mechanical loading of bioengineered skeletal muscle in vitro recapitulates gene expression signatures of resistance exercise in vivo.

Understanding the role of mechanical loading and exercise in skeletal muscle (SkM) is paramount for delineating the molecular mechanisms that govern changes in muscle mass. However, it is unknown whether loading of bioengineered SkM in vitro adequately recapitulates the molecular responses observed after resistance exercise (RE) in vivo. To address this, the transcriptional and epigenetic (DNA methylation) responses were compared after mechanical loading in bioengineered SkM in vitro and after RE in vivo. Specifically, genes known to be upregulated/hypomethylated after RE in humans were analyzed. Ninety-three percent of these genes demonstrated similar changes in gene expression post-loading in the bioengineered muscle when compared to acute RE in humans. Furthermore, similar differences in gene expression were observed between loaded bioengineered SkM and after programmed RT in rat SkM tissue. Hypomethylation occurred for only one of the genes analysed (GRIK2) post-loading in bioengineered SkM. To further validate these findings, DNA methylation and mRNA expression of known hypomethylated and upregulated genes post-acute RE in humans were also analyzed at 0.5, 3, and 24 h post-loading in bioengineered muscle. The largest changes in gene expression occurred at 3 h, whereby 82% and 91% of genes responded similarly when compared to human and rodent SkM respectively. DNA methylation of only a small proportion of genes analyzed (TRAF1, MSN, and CTTN) significantly increased post-loading in bioengineered SkM alone. Overall, mechanical loading of bioengineered SkM in vitro recapitulates the gene expression profile of human and rodent SkM after RE in vivo. Although some genes demonstrated differential DNA methylation post-loading in bioengineered SkM, such changes across the majority of genes analyzed did not closely mimic the epigenetic response to acute-RE in humans.

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.