DNA methylation across the genome in aged human skeletal muscle tissue and stem cells: The role of HOX genes and physical activity

Abstract

Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle stem cells and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged muscle stem cells over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; ‘pathways-in-cancer’ (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), ‘rap1-signaling’, ‘axon-guidance’ and ‘hippo-signalling’. Aged muscle stem cells also demonstrated a hypermethylated profile in pathways; ‘axon-guidance’, ‘adherens-junction’ and ‘calcium-signaling’, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the ‘focal adhesion’ and ‘PI3K-AKT signalling’ pathways. While the methylomes were vastly different between muscle tissue and isolated muscle stem cells, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle and tissue and stem cells (on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and muscle stem cells and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Recurrent training rejuvenates and enhances transcriptome and methylome responses in young and older human muscle

“…(Figure 3a). Promoterassociated dmCpGs in CpG islands were underrepresented compared to CpGs in the whole genome ( Figure 3b, File S1b), which is a confirmation of the results of Zykovich et al 8 , but in contrast to the findings of Turner et al 9 , where most dmCpGs were located in CpG islands. Finally, we found a significant overlap of 2,334 CpGs between the age-related dmCpGs of the present study and the age-related dmCpGs of Zykovich et al (p = 2.2e-16) (Figure 3c, File S1d).…”

“…However, the investigators were unable to find methylation changes after 12 weeks of RT, probably due to the low sample size, the strict FDR adjustment and the limited number of CpGs tested. We hypothesized that training would reverse the ageing-induced hypermethylation, predominantly found in older muscle tissue in literature 8,9 , based on the hypomethylating effect of aerobic exercise [10][11][12][13] , lifelong physical activity 15 and RT in young to middle-aged adults 13,81 . However, both in young and older muscle tissue we observed that a first training period induced both hypomethylation and hypermethylation.…”

“…5,6 Age-related methylation changes in muscle tissue, on the other hand, appear distinct compared to other tissues 7 . Recently, two genome-wide DNA methylation analyses identified 5,518 8 and 17,994 9 differentially methylated CpGs (dmCpGs) between old and young skeletal muscle with most of them hypermethylated in older adults. The integration between age-related muscle methylome and transcriptome changes remains yet to be studied and is a crucial step in understanding the molecular mechanism behind muscle ageing.…”

Meta-analysis of genome-wide DNA methylation and integrative OMICs in human skeletal muscle

“…Previous studies on the overall pattern of age-related DNA methylation changes in muscle showed mixed results, three reporting more hypermethylation with age 7,8,11 and one finding slightly more hypomethylation with age 10 .…”

“…Age-related DNA methylation changes in skeletal muscle may be one of the molecular mechanisms underlying sarcopenia, but the full picture is fragmentary. To date, four epigenome-wide association studies (EWAS) 7,8,10,11 have probed age-related DNA methylation changes in the muscle methylome, and both relied on relatively small sample sizes (n=10-50). Studies relying on a small sample size fail to detect small effect sizes and can be prone to large error, so larger initiatives are needed to identify the comprehensive list of CpG loci that change in methylation with age in human skeletal muscle.…”