DNA methylation is a covalent biochemical modification controlling chromatin structure and gene expression. Exercise elicits gene expression changes that trigger structural and metabolic adaptations in skeletal muscle. We determined whether DNA methylation plays a role in exercise-induced gene expression. Whole genome methylation was decreased in skeletal muscle biopsies obtained from healthy sedentary men and women after acute exercise. Exercise induced a dose-dependent expression of PGC-1α, PDK4, and PPAR-δ, together with a marked hypomethylation on each respective promoter. Similarly, promoter methylation of PGC-1α, PDK4, and PPAR-δ was markedly decreased in mouse soleus muscles 45 min after ex vivo contraction. In L6 myotubes, caffeine exposure induced gene hypomethylation in parallel with an increase in the respective mRNA content. Collectively, our results provide evidence that acute gene activation is associated with a dynamic change in DNA methylation in skeletal muscle and suggest that DNA hypomethylation is an early event in contraction-induced gene activation.

We tested the hypothesis that exercise, a physiological stressor that is known to alter whole body energy and glucose homeostasis, rapidly alters DNA methylation in skeletal muscle. The effects of a single, acute bout of exercise was studied using methylated DNA capture, followed by quantitative PCR (qPCR) and bisulfite sequencing. We provide evidence that exercise-induced gene induction is associated with transient alterations in promoter methylation.

Skeletal muscle is distinguished by a high degree of plasticity in its adaptive response to environmental stressors that challenge the structural and metabolic demands of the tissue. Muscle contraction through physical exercise drives adaptive responses to improve metabolic efficiency, oxidative capacity, and contractile activity by altering gene expression profiles and protein levels (). Exercise increases the messenger RNA (mRNA) expression and protein levels of a plethora of genes regulating mitochondrial function and fuel usage, including PGC-1α, transcription factor A, mitochondrial (TFAM); peroxisome proliferator-activated receptor δ (PPAR-δ); and pyruvate dehydrogenase kinase, isoenzyme 4 (PDK4) (). It remains unknown whether DNA methylation controls these genomic responses. Moreover, the unifying trigger that orchestrates the genomic response to exercise is incompletely defined. Working independently or synergistically, potential mechanisms include changes in calcium flux, the AMP:ATP ratio, or oxidative stress ().

DNA methylation is generally thought to be mitotically stable. Consequently, environmental factors have been disregarded as driving substantial and sustained changes in DNA methylation patterns in adult tissues. However, several studies support the notion that environmentally induced changes in DNA methylation patterns throughout life influence gene-expression signatures. For example, the naturally occurring short-chain fatty acid butyrate acutely alters histone deacetylase activity and DNA methylation status in normal () and cancer-cell lines (). Moreover, acute exposure of cultured human myotubes to either palmitate or oleate increases promoter methylation of the mitochondrial protein peroxisome proliferator-activated receptor gamma, coactivator 1 α (PGC-1α) (). Evidence is emerging that epigenetic modifications through DNA methylation contribute to the increased risk and development of metabolic disease by modifying the expression of genes controlling whole body energy and glucose homeostasis ().

Effects of sodium butyrate on the synthesis and methylation of DNA in normal cells and their transformed counterparts.

The regulation of gene expression is a fundamental process that establishes and impacts the phenotype of each tissue. Although the genetic code is identical in all cells of an organism, each cell type possesses its own gene expression pattern, driven by a specific epigenetic signature. DNA methylation is a major epigenetic modification that suppresses gene expression by modulating the access of the transcription machinery to the chromatin or by recruiting methyl binding proteins ().

The amplitude of gene transcription in skeletal muscle in response to exercise is orchestrated by primary messengers such as changes in the AMP:ATP ratio, calcium release from the endoplasmic reticulum, or the intracellular redox state (). By increasing the elevation of cytoplasmic Calevels, caffeine mimics exercise-induced expression of genes related to mitochondrial function in L6 myotubes (). We performed a time-course study in L6 myotubes to determine whether changes in gene expression are associated with a promoter demethylation. Pgc-1α, Tfam, Mef2a, Cs, and Pdk4 mRNA expression was elevated upon exposure to caffeine ( Figure 4 A ). Caffeine exposure decreased promoter methylation of Pgc-1α, Tfam, Mef2a, Cs, and Pdk4, as measured by methylcytosine capture followed by qPCR. Promoter methylation of Pgc-1α, Tfam, Mef2a, and Cs, but not Pdk4, decreased prior to gene expression changes ( Figure 4 A). Dantrolene blocks caffeine-induced gene expression by inhibiting Carelease from the sarcoplasmic reticulum (). Coincubation of L6 myotubes with caffeine and dantrolene markedly inhibited gene expression and suppressed promoter hypomethylation ( Figure 4 B), implicating the involvement of Carelease. L6 myotubes were also incubated in the presence of the Caionophore ionomycin (1 μM). Consistent with previous results (), ionomycin increased mRNA expression of Pgc-1α, Tfam, Mef2a, and Cs, but not PDK4 ( Figure 4 C). Promoter methylation was unaltered by ionomycin ( Figure 4 C). Activation of AMP kinase (using aminoimidazole-carboxamide-ribonucleiotide [AICAR]) did not affect promoter methylation. Conversely, reactive oxygen species (ROS) production (induced by H) elicited hypermethylation ( Figure S2 ). Thus, calcium release is necessary but not sufficient to induce DNA hypomethylation.

(A–C) Promoter methylation (hatched line) and mRNA levels (solid line) were measured after exposure to 5 mM caffeine (A), 5 mM caffeine (B), and 10 μM dantrolene or 1 μM ionomycin (C). Results are mean ± SE. # P < 0.05 versus BASAL for mRNA. ∗ P < 0.05 versus BASAL for promoter methylation.

Intracellular pathways associated with muscle contraction and exercise-induced gene expression can be activated by neurotransmitters and circulating factors. Thus, we studied isolated mouse soleus muscle to determine if extracellular factors are involved in exercise-induced hypomethylation. Gene expression of Pgc-1α, Ppar-δ, and Pdk4 increased 180 min after ex vivo contraction ( Figure 3 A ). Promoter methylation of Pgc-1α, Ppar-δ, Pdk4, Myod1, and Mef2a decreased 45 min after contraction ( Figure 3 B). Thus, contraction-induced gene expression is associated with promoter-methylation remodeling, independent of exercise-induced changes in neurotransmitters or circulating factors.

(A and B) Ex vivo contracted or rested (REST) soleus muscle was incubated for 45 (+ 45′), 90 (+ 90′) or 180 min (+ 180′). Gene expression (A) and promoter methylation (B) was analyzed. Results are mean ± SEM. ∗ p < 0.05 versus BASAL.

Aerobic exercise intensity drives gene transcription in a dose-dependent manner (). Thus, we determined whether the exercise-induced decrease in promoter methylation was dependent on exercise intensity. A dose-response and time-course analysis of DNA methylation after an acute exercise was performed in biopsies of vastus lateralis skeletal muscle from a separate cohort of eight young healthy sedentary men, described earlier (). Skeletal muscle biopsies were obtained before, immediately after, and 3 hr after an acute exercise bout at either 40% (low-intensity) or 80% (high-intensity) of maximal aerobic capacity, and DNA methylation levels were determined by MeDIP, followed by qPCR. High-intensity exercise markedly reduced promoter methylation of PGC-1α, TFAM, MEF2A, and PDK4 immediately after exercise, whereas PPAR-δ methylation was decreased 3 hr after exercise ( Figure 2 A ). Analysis of the corresponding mRNA expression revealed that decreases in DNA methylation were associated with elevations in relative mRNA levels either at the same or the next time point ( Figures 2 A and 2B). The decrease in DNA methylation, as assessed by MeDIP-PCR, was confirmed by bisulfite sequencing of the PGC-1α promoter ( Figure S1 ). Our results suggest that DNA methylation is a component of the exercise-induced effect on expression of these genes.

(A and B) Promoter methylation (A) and respective mRNA level of genes (B) involved in fuel utilization and mitochondrial biogenesis as measured by MeDIP-qPCR at REST, 0 hr, or 3 hr after low- or high-intensity acute exercise (light or dark bars, respectively); n = 8 subjects. Results are mean ± SEM.p < 0.05 versus REST,p < 0.05 versus low-intensity exercise. mRNA expression of PGC-1α in this cohort has been reported previously () but is shown here for comparative purposes against DNA methylation.

To determine whether exercise-induced alterations in DNA methylation are truly global (i.e., all the genes are affected) or gene-specific, we studied a list of genes exerting different metabolic and structural functions in skeletal muscle. We evaluated the effect of acute exercise on DNA methylation levels of genes previously described to be differentially methylated in type 2 diabetes () and whose transcript abundance is elevated after exercise (PGC-1α, TFAM, PPAR-δ, PDK4, citrate synthase [CS]), as well as factors involved in expression of muscle-specific genes (myocyte enhancer factor 2A [MEF2A], myogenic differentiation 1 [MYOD1]) and one housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]). Using methylated DNA Immunoprecitation (MeDIP) followed by quantitative PCR (qPCR), we found that captured methylated promoters for metabolic genes were lower after acute exercise, whereas muscle-specific transcription factors, including MYOD1 and MEF2A, as well GAPDH ( Figure 1 B), were unchanged. Collectively, our results provide evidence to suggest that acute exercise induces gene-specific DNA hypomethylation in human skeletal muscle.

Although acute exercise alters skeletal muscle mRNA and protein levels of genes involved in fuel utilization and mitochondrial function (), the mechanism remains unknown. To determine the effect of exercise on DNA methylation, we first analyzed global DNA methylation levels in biopsies of vastus lateralis skeletal muscle obtained from 14 healthy, young (25 ± 1 years), sedentary men and women before and after an acute bout of exercise. Clinical characteristics are presented in Table S1 (available online). The luminometric methylation assay (LUMA) interrogates methylation of the inner cytosine of all CCGG sites within the genome (). As assessed by LUMA, global methylation decreased after acute exercise ( Figure 1 A ). Global DNA methylation changes did not correlate with hemoglobin mRNA content (R = 0.032, p = 0.753), excluding the possibility that blood contamination in the skeletal muscle biopsy contributed to the decreased global methylation observed after acute exercise.

(B) Promoter-specific analysis of methylation levels. Methylated DNA Immunoprecipitation followed by quantitative PCR analysis (MeDIP-qPCR) was performed. Ratio between methylated levels at rest and acute exercise is shown. Dashed line symbolically delimitate an equal quantity of methylation at rest and after acute exercise. Results are mean ± SEM for n = 14 subjects. ∗ p < 0.05, ∗∗ p < 0.01.

(A) LUMA analysis of global DNA methylation. Global CpG methylation analysis of DNA extracted from muscle at baseline (REST) or 20 min after acute exercise (ACUTE EXERCISE). Results are mean ± SE. ∗ p < 0.05 versus REST.

Discussion

We determined the effect of exercise on DNA methylation in human skeletal muscle and provide evidence that acute exercise alters promoter methylation of exercise-responsive genes in a dose-dependent manner. Using isolated contracting muscle and cultured myotubes, we show DNA methylation remodeling parallels changes in mRNA expression. Acute gene activation is associated with a dynamic change in DNA methylation in skeletal muscle and suggests that DNA hypomethylation is an early event in contraction-induced gene expression.

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Zierath J.R. Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Bisulfite sequencing revealed that acute exercise mainly altered cytosine residues located within a CpA, CpT, or CpC context. Evidence has emerged to support a physiological role of non-CpG methylation (), notably in stems cells, where drastic remodeling of the epigenome occurs (). We have shown that systemic factors associated with insulin resistance, including elevated levels of free fatty acids or the cytokine TNF-α, acutely induced non-CpG methylation in human myocytes (). Non-CpG methylation may play a specialized role as compared to CpG methylation in mediating transient or rapid methylation.

We found that DNA methylation was unaltered 48 hr after a 3-week exercise training program, whereas RNA expression of PGC-1α and TFAM promoters was elevated (data not shown), further suggesting that DNA hypomethylation is a transient mechanism involved in mRNA synthesis. Changes in DNA methylation after acute exercise are inversely associated with gene activation of some but not all genes studied here ( Table S2 ). For instance, PGC-1α mRNA expression was increased 3 hr after low-intensity exercise, whereas methylation was unchanged. Although we cannot exclude the possibility that changes in promoter methylation at this time point were below the limit of sensitivity of the assay, exercise-induced DNA hypomethylation is an unlikely prerequisite for the activation of transcription. Our findings that ionomycin, AICAR, or ROS production increased mRNA expression without altering promoter methylation may support the notion that DNA methylation does not exclusively control exercise-induced gene expression. The exact role of exercise-induced DNA hypomethylation in vivo may be tested by selectively blocking the enzymatic machinery involved but the precise demethylation machinery is currently unknown, thus limiting further investigation into this potential mechanism. Our finding of gene-specificity in exercise-induced DNA hypomethylation suggests that methylation may serve as a selective mechanism to orchestrate the activation of a subset of genes but, clearly, other mechanisms, such as transcription factor activation and recruitment to the chromatin, are likely to be involved.

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Nair K.S. Impact of aerobic exercise training on age-related changes in insulin sensitivity and muscle oxidative capacity. Regular physical activity reduces the risk for cardiovascular diseases, type 2 diabetes, multiple cancers, depression, obesity, and musculoskeletal diseases (). Muscle contraction causes a plethora of intracellular perturbations that disturb metabolic homeostasis that, in turn, promote adaptive responses, including changes in mRNA and protein levels. The increase in cytosolic Cafollowing muscle contraction is an early intracellular signal that provokes changes in mRNA and the adaptive response to exercise training. We provide evidence that caffeine exposure decreased promoter methylation of Pgc-1α, Tfam, Mef2a, Cs, and Pdk4 in cultured myotubes, and this effect was blocked by dantrolene, an inhibitor of Carelease. Ionomycin, a calcium release activator, induced gene expression without altering promoter hypomethylation, suggesting that Carelease is necessary but not sufficient to promote DNA hypomethylation. Consequently, caffeine may also induce Caindependent mechanisms that participate in promoter hypomethylation. Notably, neither AICAR-induced AMPK activation nor ROS production were involved in promoter hypomethylation. Although mechanical stress, neural input, or circulating hormones alter skeletal muscle gene expression after exercise (), disturbances in intracellular homeostasis are sufficient to induce DNA methylation remodeling.

In conclusion, acute exercise leads to transient changes in DNA methylation in adult skeletal muscle. Our finding that the patterns of DNA methylation change in differentiated nondividing somatic cells provides further evidence that the epigenetic marks across the genome are subject to more dynamic variations than previously appreciated.