Molecular responses to acute exercise and their relevance for adaptations in skeletal muscle to exercise training.

Repeated, episodic bouts of skeletal muscle contraction undertaken frequently as structured exercise training are a potent stimulus for physiological adaptation in many organs. Specifically, in skeletal muscle, remarkable plasticity is demonstrated by the remodeling of muscle structure and function in terms of muscular size, force, endurance, and contractile velocity as a result of the functional demands induced by various types of exercise training. This plasticity, and the mechanistic basis for adaptations to skeletal muscle in response to exercise training, are underpinned by activation and/or repression of molecular pathways and processes in response to each individual acute exercise session. These pathways include the transduction of signals arising from neuronal, mechanical, metabolic, and hormonal stimuli through complex signal transduction networks, which are linked to a myriad of effector proteins involved in the regulation of pre- and posttranscriptional processes, and protein translation and degradation processes. This review therefore describes acute exercise-induced signal transduction and the molecular responses to acute exercise in skeletal muscle including emerging concepts such as epigenetic pre- and posttranscriptional regulation and the regulation of protein translation and degradation. A critical appraisal of methodological approaches and the current state of knowledge informs a series of recommendations offered as future directions in the field.

A multi-epigenomic map of endurance exercise training.

The Molecular Transducers of Physical Activity Consortium (MoTrPAC) aims to comprehensively map molecular alterations in response to acute exercise and chronic training. In one of a recent series of papers from MoTrPAC, Nair et al. provide the first multi-epigenomic and transcriptomic integration across eight tissues in both sexes following adaptation to endurance exercise training (EET).

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.

Skeletal muscle memory.

Skeletal muscle memory is an exciting phenomenon gaining significant traction across several scientific communities, among exercise practitioners, and the public. Research has demonstrated that skeletal muscle tissue can be "primed" by earlier positive encounters with exercise training that can enhance adaptation to later retraining, even following significant periods of exercise cessation or detraining. This review will describe and discuss the most recent research investigating the underlying mechanisms of skeletal muscle memory: 1) "cellular" muscle memory and, 2) "epigenetic" muscle memory, as well as emerging evidence of how these theories may work in synergy. We will discuss both "positive" and "negative" muscle memory and highlight the importance of investigating muscle memory for optimizing exercise interventions and training programs as well as the development of therapeutic strategies for counteracting muscle wasting conditions and age-related muscle loss. Finally, important directions emerging in the field will be highlighted to advance the next generation of studies in skeletal muscle memory research into the future.

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.

Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle.

Resistance training (RT) dynamically alters the skeletal muscle nuclear DNA methylome. However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older, Caucasian untrained males (65 ± 7 y.o.) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 h following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Biochemical assays were also performed, and older male data were compared to younger trained males (22 ± 2 y.o., n = 7, n = 6 Caucasian & n = 1 African American). RT increased whole-body lean tissue mass (p = .017), VL thickness (p = .012), and knee extensor torque (p = .029) in older males. RT also affected the mtDNA methylome, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p < .05). Several mtDNA sites presented a more "youthful" signature in older males after RT in comparison to younger males. The 1.12 kilobase mtDNA D-loop/control region, which regulates replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and complex III/IV protein levels were also observed (p < .05). While limited to a shorter-term intervention, this is the first evidence showing that RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT evokes mitochondrial methylome profiles to mimic younger men. The significance of these findings relative to broader RT-induced epigenetic changes needs to be elucidated.

Ubiquitin Ligases in Longevity and Aging Skeletal Muscle.

The development and prevalence of diseases associated with aging presents a global health burden on society. One hallmark of aging is the loss of proteostasis which is caused in part by alterations to the ubiquitin-proteasome system (UPS) and lysosome-autophagy system leading to impaired function and maintenance of mass in tissues such as skeletal muscle. In the instance of skeletal muscle, the impairment of function occurs early in the aging process and is dependent on proteostatic mechanisms. The UPS plays a pivotal role in degradation of misfolded and aggregated proteins. For the purpose of this review, we will discuss the role of the UPS system in the context of age-related loss of muscle mass and function. We highlight the significant role that E3 ubiquitin ligases play in the turnover of key components (e.g., mitochondria and neuromuscular junction) essential to skeletal muscle function and the influence of aging. In addition, we will briefly discuss the contribution of the UPS system to lifespan. By understanding the UPS system as part of the proteostasis network in age-related diseases and disorders such as sarcopenia, new discoveries can be made and new interventions can be developed which will preserve muscle function and maintain quality of life with advancing age.

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.

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.

Graded reductions in pre-exercise glycogen concentration do not augment exercise-induced nuclear AMPK and PGC-1α protein content in human muscle.

NEW FINDINGS: What is the central question of this study? What is the absolute level of pre-exercise glycogen concentration required to augment the exercise-induced signalling response regulating mitochondrial biogenesis? What is the main finding and its importance? Commencing high-intensity endurance exercise with reduced pre-exercise muscle glycogen concentrations confers no additional benefit to the early signalling responses that regulate mitochondrial biogenesis. ABSTRACT: We examined the effects of graded muscle glycogen on the subcellular location and protein content of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and mRNA expression of genes associated with the regulation of mitochondrial biogenesis and substrate utilisation in human skeletal muscle. In a repeated measures design, eight trained male cyclists completed acute high-intensity interval (HIT) cycling (8 × 5 min at 80% peak power output) with graded concentrations of pre-exercise muscle glycogen. Following initial glycogen-depleting exercise, subjects ingested  2 g kg-1  (L-CHO), 6 g kg-1 (M-CHO) or 14 g kg-1 (H-CHO) of carbohydrate during a 36 h period, such that exercise was commenced with graded (P < 0.05) muscle glycogen concentrations (mmol (kg dw)-1 : H-CHO, 531 ± 83; M-CHO, 332 ± 88; L-CHO, 208 ± 79). Exercise depleted muscle glycogen to <300 mmol (kg dw)-1 in all trials (mmol (kg dw)-1 : H-CHO, 270 ± 88; M-CHO, 173 ± 74; L-CHO, 100 ± 42) and induced comparable increases in nuclear AMPK protein content (∼2-fold) and PGC-1α (∼5-fold), p53 (∼1.5-fold) and carnitine palmitoyltransferase 1 (∼2-fold) mRNA between trials (all P < 0.05). The magnitude of increase in PGC-1α mRNA was also positively correlated with post-exercise glycogen concentration (P < 0.05). In contrast, neither exercise nor carbohydrate availability affected the subcellular location of PGC-1α protein or PPAR, SCO2, SIRT1, DRP1, MFN2 or CD36 mRNA. Using a sleep-low, train-low model with a high-intensity endurance exercise stimulus, we conclude that pre-exercise muscle glycogen does not modulate skeletal muscle cell signalling.

The Interplay Between Exercise Metabolism, Epigenetics, and Skeletal Muscle Remodeling.

We explore work from within the field of skeletal muscle and across the broader field of molecular biology, to propose that the link between exercise and skeletal muscle adaptation lies in the interplay between metabolism and epigenetics. Future investigations into such an interaction are crucial to advance our understanding of the beneficial effects of exercise on performance and health.

Post-exercise carbohydrate and energy availability induce independent effects on skeletal muscle cell signalling and bone turnover: implications for training adaptation.

KEY POINTS: Reduced carbohydrate (CHO) availability before and after exercise may augment endurance training-induced adaptations of human skeletal muscle, as mediated via modulation of cell signalling pathways. However, it is not known whether such responses are mediated by CHO restriction, energy restriction or a combination of both. In recovery from a twice per day training protocol where muscle glycogen concentration is maintained within 200-350 mmol kg-1 dry weight (dw), we demonstrate that acute post-exercise CHO and energy restriction (i.e. < 24 h) does not potentiate potent cell signalling pathways that regulate hallmark adaptations associated with endurance training. In contrast, consuming CHO before, during and after an acute training session attenuated markers of bone resorption, effects that are independent of energy availability. Whilst the enhanced muscle adaptations associated with CHO restriction may be regulated by absolute muscle glycogen concentration, the acute within-day fluctuations in CHO availability inherent to twice per day training may have chronic implications for bone turnover. ABSTRACT: We examined the effects of post-exercise carbohydrate (CHO) and energy availability (EA) on potent skeletal muscle cell signalling pathways (regulating mitochondrial biogenesis and lipid metabolism) and indicators of bone metabolism. In a repeated measures design, nine males completed a morning (AM) and afternoon (PM) high-intensity interval (HIT) (8 × 5 min at 85% V̇O2peak ) running protocol (interspersed by 3.5 h) under dietary conditions of (1) high CHO availability (HCHO: CHO ∼12 g kg-1 , EA∼ 60 kcal kg-1 fat free mass (FFM)), (2) reduced CHO but high fat availability (LCHF: CHO ∼3 (-1 , EA∼ 60 kcal kg-1 FFM) or (3), reduced CHO and reduced energy availability (LCAL: CHO ∼3 g kg-1 , EA∼ 20 kcal kg-1 FFM). Muscle glycogen was reduced to ∼200 mmol kg-1  dw in all trials immediately post PM HIT (P < 0.01) and remained lower at 17 h (171, 194 and 316 mmol kg-1  dw) post PM HIT in LCHF and LCAL (P < 0.001) compared to HCHO. Exercise induced comparable p38MAPK phosphorylation (P < 0.05) immediately post PM HIT and similar mRNA expression (all P < 0.05) of PGC-1α, p53 and CPT1 mRNA in HCHO, LCHF and LCAL. Post-exercise circulating ßCTX was lower in HCHO (P < 0.05) compared to LCHF and LCAL whereas exercise-induced increases in IL-6 were larger in LCAL (P < 0.05) compared to LCHF and HCHO. In conditions where glycogen concentration is maintained within 200-350 mmol kg-1  dw, we conclude post-exercise CHO and energy restriction (i.e. < 24 h) does not potentiate cell signalling pathways that regulate hallmark adaptations associated with endurance training. In contrast, consuming CHO before, during and after HIT running attenuates bone resorption, effects that are independent of energy availability and circulating IL-6.

UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy.

KEY POINTS: We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining. In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy. We demonstrated that UBR5 was epigenetically altered via DNA methylation during recovery from atrophy. We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy. UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation. Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes. ABSTRACT: We aimed to investigate a novel and uncharacterized E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 vs. well characterized E3-ligases, MuRF1/MAFbx, where, after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated, yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post TTX-induced atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, whereas a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, as well as after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in vitro, and after chronic electrical stimulation-induced hypertrophy in rats in vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 single nucleotide polymorphisms) demonstrated that the A alleles of rs10505025 and rs4734621 single nucleotide polymorphisms in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power vs. endurance/untrained phenotypes. Overall, we suggest that: (i) UBR5 comprises a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx; (ii) UBR5 is epigenetically regulated; and (iii) UBR5 is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.

Mimicking exercise in three-dimensional bioengineered skeletal muscle to investigate cellular and molecular mechanisms of physiological adaptation.

Bioengineering of skeletal muscle in vitro in order to produce highly aligned myofibres in relevant three dimensional (3D) matrices have allowed scientists to model the in vivo skeletal muscle niche. This review discusses essential experimental considerations for developing bioengineered muscle in order to investigate exercise mimicking stimuli. We identify current knowledge for the use of electrical stimulation and co-culture with motor neurons to enhance skeletal muscle maturation and contractile function in bioengineered systems in vitro. Importantly, we provide a current opinion on the use of acute and chronic exercise mimicking stimuli (electrical stimulation and mechanical overload) and the subsequent mechanisms underlying physiological adaptation in 3D bioengineered muscle. We also identify that future studies using the latest bioreactor technology, providing simultaneous electrical and mechanical loading and flow perfusion in vitro, may provide the basis for advancing knowledge in the future. We also envisage, that more studies using genetic, pharmacological, and hormonal modifications applied in human 3D bioengineered skeletal muscle may allow for an enhanced discovery of the in-depth mechanisms underlying the response to exercise in relevant human testing systems. Finally, 3D bioengineered skeletal muscle may provide an opportunity to be used as a pre-clinical in vitro test-bed to investigate the mechanisms underlying catabolic disease, while modelling disease itself via the use of cells derived from human patients without exposing animals or humans (in phase I trials) to the side effects of potential therapies.

Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)-administered to the common peroneal nerve-resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P ≤ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit α1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuse-induced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-surgery controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal activity.-Fisher, A. G., Seaborne, R. A., Hughes, T. M., Gutteridge, A., Stewart, C., Coulson, J. M., Sharples, A. P., Jarvis, J. C. Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction.

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 µM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the 'slow' type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for 'intermediate' and 'faster' IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48-72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring 'slower' Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction.

Whey Protein Augments Leucinemia and Postexercise p70S6K1 Activity Compared With a Hydrolyzed Collagen Blend When in Recovery From Training With Low Carbohydrate Availability.

We examined the effects of whey versus collagen protein on skeletal muscle cell signaling responses associated with mitochondrial biogenesis and protein synthesis in recovery from an acute training session completed with low carbohydrate availability. In a repeated-measures design (after adhering to a 36-hr exercise-dietary intervention to standardize preexercise muscle glycogen), eight males completed a 75-min nonexhaustive cycling protocol and consumed 22 g of a hydrolyzed collagen blend (COLLAGEN) or whey (WHEY) protein 45 min prior to exercise, 22 g during exercise, and 22 g immediately postexercise. Exercise decreased (p < .05) muscle glycogen content by comparable levels from pre- to postexercise in both trials (≈300-150 mmol/kg·dry weight). WHEY protein induced greater increases in plasma branched chain amino acids (p = .03) and leucine (p = .02) than COLLAGEN. Exercise induced (p < .05) similar increases in PGC-1α (fivefold) mRNA at 1.5 hr postexercise between conditions, although no effect of exercise (p > .05) was observed for p53, Parkin, and Beclin1 mRNA. Exercise suppressed (p < .05) p70S6K1 activity in both conditions immediately postexercise (≈25 fmol·min-1·mg-1). Postexercise feeding increased p70S6K1 activity at 1.5 hr postexercise (p < .05), the magnitude of which was greater (p < .05) in WHEY (180 ± 105 fmol·min-1·mg-1) versus COLLAGEN (73 ± 42 fmol·min-1·mg-1). We conclude that protein composition does not modulate markers of mitochondrial biogenesis when in recovery from a training session deliberately completed with low carbohydrate availability. By contrast, whey protein augments postexercise p70S6K activity compared with hydrolyzed collagen, as likely mediated via increased leucine availability.

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation.

Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.

Murine myoblast migration: influence of replicative ageing and nutrition.

Cell migration is central to skeletal muscle repair following damage. Leucine and ß-Hydroxy ß-methylbutyric acid (HMB) are supplements consumed for recovery from muscle damaging exercise in humans, however, their impact on muscle cell migration with age is not yet understood. We hypothesised that replicatively aged ("aged"; P46-P48) myoblasts would be less efficient at basal and supplemented repair versus parental controls ("control"; P12-P16). Aged and control myoblasts were scratch-damaged and migration velocity, directionality and distance assessed over 48 h in the absence and presence of leucine (10 mM) or HMB (10 mM) ± PI3K/Akt (LY294002 10 µM), ERK (PD98059 5 µM) or mTOR (rapamycin 0.5 µM) inhibition. Opposing our hypothesis, aged cells displayed increased velocities, directionality and distance migrated (P < 0.001) versus control. Leucine and HMB significantly increased (P < 0.001) the same parameters in control cells. The supplements were with smaller, albeit significant impact on aged cell velocity (P < 0.001) and in the presence of HMB only, distance (P = 0.041). Inhibitor studies revealed that, PI3K and ERK activation were essential for velocity, directionality and migration distance of aged cells in basal conditions, whereas mTOR was important for directionality only. While PI3K activation was critical for all parameters in control cells (P < 0.001), inhibition of ERK or mTOR improved, rather than reduced, control cell migration distance. Enhanced basal velocity, directionality and distance in aged cells required ERK and PI3K activation. By contrast, in control cells, basal migration was underpinned by PI3K activation, and facilitated by leucine or HMB supplementation, to migration levels seen in aged cells. These data suggest that replicatively aged myoblasts are not anabolically resistant per se, but are capable of efficient repair, underpinned by altered signaling pathways, compared with unaged control myoblasts.

l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc.