Despite decades of research demonstrating the transcriptional regulatory roles in SkM adaptation following mechanical loading/RE (as described in chapters 3, 4 and 5 of this thesis ; Goldberg & Goodman, 1969; Goldberg et al., 1975; Egan & Zierath, 2012), the importance of epigenetics, specifically DNA methylation has only more recently begun to emerge. Indeed, it was not until 2014 that researchers first showed that RE could indeed modulate the human DNA methylome. In this study, Rowlands and colleagues subjected middle-aged (49 ± 5 yrs) type II diabetic male and female humans to chronic RE (6-8 reps × 2-3 sets × 8 full body exercise, 3 days/week for 16 weeks) and demonstrated differential DNA methylation across 450K CpG sites at post- versus pre-exercise (Rowlands et al., 2014). Interestingly, the authors reported more genes being hypomethylated versus hypermethylated, and that these differences were in genes associated with ‘cellular assembly and organization, cellular development, tissue morphology, and cardiovascular system development and function’ following pathway enrichment analysis (Rowlands et al., 2014). It is also worth noting that the same study also demonstrated a global reduction in DNA methylation at post- versus pre-chronic endurance cycling exercise (40–60 min steady-state cycling exercise at 65%–85% of heart rate max, 3 days/week for 16 weeks) with pathway enrichment analysis suggesting that genes were associated with ‘lipid/carbohydrate metabolism, metabolic disease, cell death and survival, cardiovascular system development/function, and haematological system development/function’ (Rowlands et al., 2014). This study therefore demonstrated for the first time that DNA methylation is regulated by mechanical loading/RE in-vivo which was influenced by the mode of exercise performed, evidenced by the different enriched pathways in RE vs. endurance exercise. Two years after, an Italian group of researchers reported a significant reduction in leukocyte-specific global DNA methylation following 12 weeks of chronic progressive RE (3 days/week, 10-12 reps × 3-4 sets at 70% 1RM which was assessed
via leg extension testing every 2 weeks) in male and female elderly humans (Dimauro et al., 2016). Shortly after, DNA methylation in leukocytes was further investigated using genome- wide array technology (450K CpG sites) to permit gene-specific methylation analysis following 8 weeks of chronic RE (3 days/week, 10-12 reps × 3-4 sets at 80% 1RM ensuring 3 s contractions) in humans (Denham et al., 2016). In support of previous findings, RE also evoked hypomethylation of genes which were associated with ‘cancer, axon guidance, diabetes’. Interestingly, a number of anabolic signaling-related genes were also hypomethylated including insulin-like growth factor I receptor (IGF-IR), growth hormone- releasing hormone (GHRH) and fibroblast growth factor 1 (FGF1), with the latter two genes also displaying corresponding increases in mRNA expression, further supporting the notion that epigenetic modifications are associated with changes in gene expression (Denham et al., 2016). A year on, Robinson and colleagues analysed DNA methylation (450K array) profiles in young and old human SkM tissue following 12 weeks of either chronic RE, high-intensity interval training (HIIT) exercise or combined/concurrent exercise (Robinson et al., 2017). Interestingly, DNA methylation did not significantly change (<10%), regardless of exercise type or age group (Robinson et al., 2017). It is important to note however that only gene promoter DNA methylation was reported. Conversely, a study published within the same year demonstrated that RE was able to negate increased genome-wide hypermethylation induced by short term high fat feeding (consisting of >77% of total calorie intake) following bisulfite sequencing analysis (Laker et al., 2017). Using more recent genome-wide technology that permits greater CpG methylation coverage (850K CpG sites), work by our group also reported significant total and gene-specific DNA hypomethylation in response to acute and 7 weeks of chronic RE (Seaborne et al., 2018a, 2018b). Interestingly, a large number of these hypomethylated genes also demonstrated corresponding changes in mRNA expression whereby hypomethylation determined by genome wide analysis, with follow up analysis of
candidate genes at the gene expression level following chronic exercise, were associated with increases in mRNA expression. For some genes, hypomethylation was retained during 7 weeks of detraining/unloading where muscle mass returned to baseline/pre-exercise levels of which further decreased following 7 week of retraining (where the greatest increase in muscle mass occurred) which also corresponded with the greatest increase in mRNA expression (Seaborne
et al., 2018a). Suggestive of an ‘epigenetic memory’ (Sharples et al., 2016b) at the DNA level
since earlier exercise encounters resulted in an retained response during future RE. Another subset of genes in this study also demonstrated an inverse relationship between mRNA expression and DNA methylation whereby expression and methylation increased and decreased, respectively after chronic RE. Following detraining however, both gene expression and DNA methylation returned back to baseline levels after which the increased mRNA and decreased methylation was enhanced after reloading, further supporting the notion that DNA methylation may indeed regulate gene expression in response to exercise. Specific details of these regulated gene such as names and their gene expression profiles are reported within chapter 5 of this thesis where the transcriptional and epigenetic regulation of these genes are also assessed following acute and chronic loading/RE in in bioengineered/rodent SkM. Although Seaborne et al., (2018) also assessed the genome-wide epigenetic responses to acute RE, there is still limited research that have studied the effects on DNA methylation after acute exercise/loading. Indeed, Zierath’s group demonstrated that acute endurance exercise (cycling exercise at 40 or 80% VO2peak until 1,674 KJ) in humans induced promoter associated hypomethylation of key metabolic genes, PPAR-δ, PGC1-α and PDK4, all of which also displayed increased mRNA expression (Barrès et al., 2012). It is also important to note however that altered DNA methylation of these genes only occurred after performing high intensity exercise (80% VO2peak) and that exercise-induced alterations in DNA methylation may therefore be load/intensity dependent.