Background Knowledge of age‐related DNA methylation changes in skeletal muscle is limited, yet this tissue is severely affected by ageing in humans. Methods We conducted a large‐scale epigenome‐wide association study meta‐analysis of age in human skeletal muscle from 10 studies (total n = 908 muscle methylomes from men and women aged 18–89 years old). We explored the genomic context of age‐related DNA methylation changes in chromatin states, CpG islands, and transcription factor binding sites and performed gene set enrichment analysis. We then integrated the DNA methylation data with known transcriptomic and proteomic age‐related changes in skeletal muscle. Finally, we updated our recently developed muscle epigenetic clock (https://bioconductor.org/packages/release/bioc/html/MEAT.html). Results We identified 6710 differentially methylated regions at a stringent false discovery rate <0.005, spanning 6367 unique genes, many of which related to skeletal muscle structure and development. We found a strong increase in DNA methylation at Polycomb target genes and bivalent chromatin domains and a concomitant decrease in DNA methylation at enhancers. Most differentially methylated genes were not altered at the mRNA or protein level, but they were nonetheless strongly enriched for genes showing age‐related differential mRNA and protein expression. After adding a substantial number of samples from five datasets (+371), the updated version of the muscle clock (MEAT 2.0, total n = 1053 samples) performed similarly to the original version of the muscle clock (median of 4.4 vs. 4.6 years in age prediction error), suggesting that the original version of the muscle clock was very accurate. Conclusions We provide here the most comprehensive picture of DNA methylation ageing in human skeletal muscle and reveal widespread alterations of genes involved in skeletal muscle structure, development, and differentiation. We have made our results available as an open‐access, user‐friendly, web‐based tool called MetaMeth (https://sarah-voisin.shinyapps.io/MetaMeth/).

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ABSTRACTBackgroundThe interaction between the muscle methylome and transcriptome is understudied during ageing and periods of resistance training in young, but especially older adults. In addition, more information is needed on the role of retained methylome training adaptations in muscle memory to understand muscle phenotypical and molecular restoration after inactivity or disuse.MethodsWe measured CpG methylation (microarray) and RNA expression (RNA sequencing) in young (n = 5; age = 22 ± 2 yrs) and older (n = 6; age = 65 ± 5 yrs) vastus lateralis muscle samples, taken at baseline, after 12 weeks of resistance training, after training interruption (2 weeks of leg immobilization in young men, 12 weeks of detraining in older men) and after 12 weeks of retraining to identify muscle memory-related adaptations and rejuvenating effects of training.ResultsWe report that of the 427 differentially expressed genes with advanced age, 71 % contained differentially methylated (dm)CpGs in older versus young muscle. The more dmCpGs within a gene, the clearer the inverse methylation-expression relationship. Around 73 % of the age-related dmCpGs approached younger methylation levels when older muscle was trained for 12 weeks. A second resistance training period after training cessation increased the number of hypomethylated CpGs and upregulated genes in both young and older muscle. We found indication for an epi-memory within pro-proliferating AMOTL1 in young muscle and mechanosensing-related VCL in older muscle. For the first time, we integrate muscle methylome and transcriptome data in relation to both ageing and training/inactivity-induced responses and identify focal adhesion as an important pathway herein.ConclusionPreviously trained muscle is more responsive to training than untrained muscle at methylome and transcriptome level and recurrent resistance training can partially restore ageing-induced methylome alterations.

BackgroundThe interaction between the muscle methylome and transcriptome is understudied during ageing and periods of resistance training in young, but especially older adults. More information is needed on the role of retained methylome training adaptations in muscle memory to understand muscle phenotypical and molecular restoration after inactivity or disuse. Methods We measured CpG methylation (microarray) and RNA expression (RNA sequencing) in young (n = 5; age = 22 ± 2 years) and older (n = 6; age = 65 ± 5 years) vastus lateralis muscle samples, taken at baseline, after 12 weeks of resistance training, after training interruption (2 weeks of leg immobilization in young men, 12 weeks of detraining in older men) and after 12 weeks of retraining to identify muscle memory-related adaptations and rejuvenating effects of training. ResultsWe report that of the 427 differentially expressed genes with advanced age (FDR < 0.1), 71% contained differentially methylated (dm)CpGs in older versus young muscle (FDR < 0.1, M-value difference >0.4). The more dmCpGs within a gene, the clearer the inverse methylation-expression relationship. Around 73% of the age-related dmCpGs approached younger methylation levels when older muscle was trained for 12 weeks. A second resistance training period after training cessation increased the number of hypomethylated CpGs and upregulated genes in both young and older muscle. We found indication for an epi-memory within pro-proliferating AMOTL1 in young muscle and mechanosensing-related VCL in older muscle. For the first time, we integrate muscle methylome and transcriptome data in relation to both ageing and training-induced/inactivity-induced responses and identify focal adhesion as an important pathway herein. Conclusions This preliminary evidence indicates that previously trained muscle is more responsive to training than untrained muscle at methylome and transcriptome level and recurrent resistance training can partially restore ageing-induced methylome alterations.

Exercise training prevents age‐related decline in muscle function. Targeting epigenetic aging is a promising actionable mechanism and late‐life exercise mitigates epigenetic aging in rodent muscle. Whether exercise training can decelerate, or reverse epigenetic aging in humans is unknown. Here, we performed a powerful meta‐analysis of the methylome and transcriptome of an unprecedented number of human skeletal muscle samples (n = 3176). We show that: (1) individuals with higher baseline aerobic fitness have younger epigenetic and transcriptomic profiles, (2) exercise training leads to significant shifts of epigenetic and transcriptomic patterns toward a younger profile, and (3) muscle disuse “ages” the transcriptome. Higher fitness levels were associated with attenuated differential methylation and transcription during aging. Furthermore, both epigenetic and transcriptomic profiles shifted toward a younger state after exercise training interventions, while the transcriptome shifted toward an older state after forced muscle disuse. We demonstrate that exercise training targets many of the age‐related transcripts and DNA methylation loci to maintain younger methylome and transcriptome profiles, specifically in genes related to muscle structure, metabolism, and mitochondrial function. Our comprehensive analysis will inform future studies aiming to identify the best combination of therapeutics and exercise regimes to optimize longevity.