Frequency of genome-wide hypomethylation is the largest after reloading induced hypertrophy where lean muscle mass is enhanced

We aimed to investigate an epigenetic memory of earlier hypertrophy in adult human skeletal muscle using a within measures design, by undertaking: (1) resistance exercise induced muscle growth (loading), followed by; (2) cessation of resistance exercise, to return muscle back towards baseline levels (unloading), and; (3) a subsequent later period of resistance exercise induced muscle hypertrophy (reloading). We first confirmed that we were able to elicit an increase in lean mass of the lower limbs after 7 weeks loading, that returned back to baseline levels after 7 weeks unloading, with 7 weeks reloading evoking the largest increase in lean mass. Interestingly, after DNA methylation analysis of over 850,000 CpG sites, we identified the largest frequency of hypomethylation (18,816 CpG sites) occurred after reloading where the largest lean mass occurred. Previous studies have suggested that hypermethylation of over 6,500 genes are retained, after an more acute stress of high fat intake (for 5 days) 8 weeks later despite removal of the high fat diet14, and hypermethylation occurs following early life inflammatory stress in muscle cells and is maintained for over 30 cellular divisions11. The present study also suggested hypomethylation was maintained during unloading (8,891 CpG sites) where muscle mass returned to baseline having being subjected to an earlier period of load induced muscle growth (9,153 CpG sites), then upon reloading the frequency of hypomethylation was enhanced in association with the largest increases in lean mass. Furthermore, bioinformatic analysis of the PI3K/AKT pathway across loading, unloading and reloading conditions, supports the findings of an enhanced hypomethylated state upon secondary exposure to resistance stimulus. Importantly, this pathway is identified as critical for cell proliferation/differentiation, muscle protein synthesis and therefore muscle hypertrophy27, and therefore, it is plausible that the enhanced hypomethylated state of the genes in this pathways would lead to enhanced gene expression and protein levels. However, further analysis is required to investigate the total protein or activity of these pathways in this model. Nonetheless, collectively, these results provide initial evidence for a maintenance/memory of universal hypomethylation. The only other study to demonstrate a memory of prior hypertrophy in skeletal muscle was in rodents following earlier encounters with testosterone administration, where a retention of myonuclei occurred even during testosterone withdrawal and a return of muscle to baseline levels13, suggesting a memory at the cellular level. However, these are the first studies to demonstrate that a memory occurs at the epigenetic level within skeletal muscle tissue.

Hypomethylation is maintained from earlier load induced hypertrophy even during unloading where muscle mass returns back towards baseline and is inversely associated with gene expression

Following the frequency analysis of hypo/hypermethylated sites mentioned above, closer analysis of the top 500 most significantly differentially modified CpG sites across all conditions, identified two epigenetically modified clusters of interest (named Cluster A&B). Cluster B supported the frequency analysis above and demonstrated hypomethylation after load induced hypertrophy that was then maintained following unloading where muscle returned to baseline levels and this hypomethylation was then also maintained after reload induced hypertrophy. This maintenance of hypomethylation during unloading, suggested that the muscle ‘remembered’ the epigenetic modifications that occurred after an earlier period of load induced muscle hypertrophy. As reduced DNA methylation of genes generally leads to enhanced gene expression due to the removal of methylation allowing improved access of the transcriptional machinery and RNA polymerase that enable transcription, and also creating permissive euchromatin19,28,29,30, this would be suggestive that the earlier period of hypertrophy leads to increased gene expression of this cluster of genes that is then retained during unloading to enable enhanced muscle growth in the later reloading period. To confirm this, in a separate analysis we identified the top 100 most significantly differentially modified CpG sites across all conditions and cross referenced these with the most frequently occurring CpG modifications in all pairwise comparisons of experimental conditions. From this we identified 48 genes that were frequently occurring in all pairwise comparisons and examined gene expression by rt-qRT-PCR. Interestingly, we identified two clusters of genes with distinct temporal expression after loading, unloading and reloading. One of the clusters included AXIN1, GRIK2, CAMK4, TRAF1. Importantly, the majority of these genes demonstrated a mirror/inverse relationship with DNA methylation of the CpG sites within the same genes. Where DNA methylation reduced after loading and remained low into unloading and reloading, gene expression accumulated, demonstrating the highest expression after reloading where the largest increase in lean mass was also demonstrated. Overall, this suggested that these genes were hypomethylated and switched on after the earlier period of load induced hypertrophy, maintained during unloading due to methylation of these genes remaining low, and then upon exposure to a later period of reload induced hypertrophy, these genes were switched on to an even greater extent. Overall, this demonstrates that the methylation and collective responsiveness of these genes are important epigenetic regulators of skeletal muscle memory.

Interestingly, AXIN1 is a component of the beta-catenin destruction complex, where in skeletal muscle cells AXIN1 has been shown to inhibit WNT/β-catenin signalling and enable differentiation31, where treatment with the canonical WNT ligand suppresses differentiation32. Other studies suggested that AXIN2 not AXIN1 is increased after differentiation, however confirmed that the absence of AXIN1 reduced proliferation and myotube formation32. Therefore, together with the present data perhaps suggest an important epigenetic regulation of AXIN1 involved in human skeletal muscle memory and hypertrophy at the tissue level, perhaps due to inhibition of WNT/β-catenin signaling. GRIK2 (glutamate ionotropic receptor kainate type subunit 2, a.k.a. GluK2) belongs to the kainate family of glutamate receptors, which are composed of four subunits and function as ligand-activated ion channels33. Although reportedly expressed in skeletal muscle, its role in muscle growth or cellular function has not been determined. CAMK4 is calcium/calmodulin-dependent protein kinase, that via phosphorylation, triggers the CaMKK-CaMK4 signaling cascade and activates several transcription factors, such as MEF234. MEF2 has been previously associated with a switch to slow fibre types after exercise35 and is hypomethylated after 6 months aerobic exercise36. While resistance exercise has been show to preferentially increase the size of type II faster fibres, chronic innervation even at higher loads can lead to an overall slowing in phenotype [reviewed in ref.37) and therefore this epigenetically regulated gene, although not usally studied during hypertrophy maybe important in fibre type changes in the present study. However, it is unknown how DNA methylation affects the protein levels of CAMK4, and with its role in phosphorylation, would be important to ascertain in the future. Furthermore, fibre type properties were not analyzed in the present study and therefore require further investigation. TRAF1 is the TNF receptor-associated factor 1 and together with TRAF2 form the heterodimeric complex required for TNF-α activation of MAPKs, JNK and NFκB38. In skeletal muscle, acute TNF exposure activates proliferation via activation of MAPKs such as ERK and P38 MAPK39,40,41. Therefore, acutely elevated systemic TNF-α following damaging exercise such as resistance exercise correlates positively with satellite cell activation in-vivo after damaging exercise42,43, yet chronic administration in-vitro inhibits differentiation, promotes myotube atrophy40,44 and muscle wasting in-vivo44. Indeed, exposure to early life TNF-α during an early proliferative age in mouse C2C12s results in maintenance of hypermethylation in the myoD promoter after 30 divisions and an increased susceptibility to reduced differentiation and myotube atrophy when muscle cells encounter TNF-α in later proliferative life11. Suggesting a role for DNA methylation in retention of memory following earlier periods of high inflammation. Because resistance exercise evokes increases in TNF-α in the systemic circulation and has been shown increase locally in muscle at the protein level (discussed above), these data collectively suggest an interesting epigenetic role for TNF and TRAF1 in the epigenetic memory of earlier load induced muscle hypertrophy.

Identification of novel genes with the largest hypomethylation during reloading that are associated with enhanced gene expression

The second DNA methylation cluster determined in the top 500 differentially modified CpG sites across all conditions, identified a cluster of genes (named Cluster A) that was methylated at baseline and also became hypomethylated after loading (similar to Cluster B above), then, upon unloading, genes reverted back to a methylated state, and after reloading switched back to hypomethylated. Therefore, while not demonstrating an epigenetic memory per se, if hypomethylation was further enhanced and was associated with enhanced gene expression in reloading versus loading would also support an epigenetic memory. Further gene expression analysis identified a cluster of genes that demonstrated a mirror/inverse temporal pattern of gene expression versus their DNA methylation pattern. These genes included RPL35a, C12orf50, BICC1, ZFP2, UBR5, HEG1, PLA2G16, SETD3 and ODF2, that demonstrated hypomethylation of DNA after load induced growth and an increase in gene expression. Subsequently, then both DNA methylation and gene expression returned back to baseline levels (in opposite directions) and after reload induced muscle growth DNA was hypomethylated again with an associated increase in gene expression. Importantly, during reloading, gene expression was further enhanced versus loading, suggesting that an earlier period of load induced growth was enough to produce enhanced gene expression when reload induced muscle growth was encountered later, again suggesting a skeletal muscle memory at both the epigenetic and resultant transcript level. Statistical analysis identified the genes RPL35a, UBR5, SETD3 and PLA2G16 as having significantly enhanced expression upon reloading. Importantly, these four genes, plus HEG1, displayed significant correlations between their gene expression and the percentage change in lean mass, suggesting for the first time, a role for these four genes in regulating adult human load induced skeletal muscle growth. Interestingly, SET Domain Containing 3 (SETD3) is a H3K4/H3K36 methyltransferase, is abundant in skeletal muscle, and has been shown to be recruited to the myogenin promoter, with MyoD, to promote its expression45. Furthermore, overexpression of SETD3 in C2C12 murine myoblasts, evokes increases in myogenin, muscle creatine kinase, and Myf6 (or MRF4) gene expression. Inhibition via shRNA in a myoblasts also impairs muscle cell differentiation45, suggesting a role for SETD3 in regulating skeletal muscle regeneration. However, less is known regarding the role of PLA2G16 in skeletal muscle. PLA2G16 is a member of the superfamily of phospholipase A enzymes, whose predominant localization is in adipose tissue. PLA2G16 is known to regulate adipocyte lipolysis in an autocrine/paracrine manner, via interactions with prostaglandin and EP3 in a G-protein-mediated pathway46. Indeed, ablation of PLA2G16 (referred to as Adpla), prevents obesity during periods high fat feeding in mouse models, indicated via significantly less adipose tissue and triglyceride content, compared to relevant controls46. However, to date no known research has elucidated the role of PLA2G16 in skeletal muscle and therefore, this requires future experimentation. Finally, HEG homology 1 (HEG1), initially reported as the heart of glass gene, is recognised for its role in regulating the zebrafish heart growth. HEG1 is a transmembrane receptor that has been reported to be fundamental in the development of both the heart and blood vessels47. However, a recent study reported a distinct role for HEG1 in regulating malignant cell growth48. Tsuji, et al.48 and colleagues reported that gene silencing of HEG1 in human MPM cell line, a cell linage that develop mesothelioma tumours, significantly reduced the survival and proliferation of mesothelioma cells, suggesting a role for HEG1 in regulating cellular growth. However, no known research has examined the role of HEG1 in regulating adult skeletal muscle growth.

In the present study UBR5 displayed the most distinctive inverse relationship between DNA hypomethylation and increased gene expression following loading and reloading. With the largest increase in hypomethylation and gene expression after reloading where the largest increase in lean mass was observed. UBR5 is a highly conserved homologue of the drosophila tumour suppressor hyperplastic discs (HYD), and in the mammalian genome refers to a protein that is a member of the HECT-domain E3 ubiquitin-ligase family49. E3 ubiquitin ligases play an integral role in the ubiquitin - proteasome pathway, providing the majority of substrate recognition for the attachment of ubiquitin molecules onto targeted proteins, preferentially modifying them for targeted autophagy/breakdown50. Indeed, extensive work has identified a distinct role of a number of E3 ubiquitin ligases such as MuRF1, MAFbx and MUSA1 in muscle atrophy51,52. Furthermore, we have recently demonstrated that reduced DNA methylation and increased gene expression of MuRF1 and MAFbx are associated with disuse atrophy in rats following nerve silencing of the hind limbs via tetrodotoxin exposure17. A process that is reversed upon a return to habitual physical activity and a partial recovery of skeletal muscle mass17, suggesting a role for DNA methylation in regulating the transcript behavior of a number of ubiquitin ligases during periods of skeletal muscle atrophy and recovery. However, there have been no studies that the authors are aware of, exmaining the role of UBR5 in skeletal muscle atrophy or growth. Given the role of ubiquitin ligases in skeletal muscle, counterintuitively, we report that the expression of the E3 ubiquitin ligase, UBR5, is increased during earlier periods of skeletal muscle hypertrophy and are even further enhanced in later reload induced muscle growth. We further report that the methylation profile of this E3 ubiquitin ligase portrays an inversed relationship with gene expression, supporting a role for DNA epigenetic modifications in regulating its expression, as previously suggested17. However, in support of its role in positively impacting on muscle, UBR5 has also been shown to promote smooth muscle differentiation through its ability to stabilize myocardin proteins53. While myocardin is only expressed in smooth and cardiac muscle, it is considered the master regulator of smooth muscle gene expression54 and a known transcription factor that upregulates smooth muscle myosin heavy chains (MYHCs), actin and desmin. It therefore possesses a similar role to the myogenic regulatory factors during early differentiation (Mrf5 and MyoD), during fusion (myogenin) and during myotube hypertrophy (adult MYHC’s). Interestingly, it has previously been observed that myocardin-related transcription factors (MRTF) interact with the myogenic regulatory factor, MyoD, to activate skeletal muscle specific gene expression55, suggesting a potential cross-talk between muscle specific regulatory factors, enabling skeletal muscle adaptations55,56. Therefore, UBR5′s expression throughout the time course of skeletal muscle cell differentiation, its role in myotube hypertrophy are required in-vitro as well as mammalian overexpression and knock-out of UBR5 to confirm its importance in-vivo. Further work is needed to characterize UBR5, as well as other HECT-domain E3 ubiquitin ligase protein members identified in this work via pathway analysis of the ubiquitin mediated proteolysis pathway, in the development of muscle growth to better understand its role in facilitating skeletal muscle hypertrophy.

A single bout of acute resistance exercise evokes hypomethylation of genes that have enhanced gene expression in later reload induced hypertrophy: Novel acutely exercise sensitive DNA methylation biomarkers

Finally, we identified genes BICC1, STAG1, GRIK2 and TRAF1 were hypomethylated after a single bout of acute resistance exercise that were maintained as hypomethylated during loading (as identified above) and reloading and demonstrated an enhanced gene expression after later reloading. Previous studies have suggested that acute aerobic exercise hypomethylates important genes in metabolic adaptation and mitochondrial biogenesis such as PGC-1α, mitochondrial transcription factor A (TFAM) and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) post exercise, and reduces PPAR-δ methylation (hypomethylates) 3 hours post exercise16, with corresponding increases in gene expression (3 hrs post exercise for PGC-1α, PDK4 and PPAR-δ, immediately post for TFAM)16. Interestingly, hypermethylation of PGC 1α and reduced gene expression, observed in skeletal muscle of the offspring of obese murine mothers, was reversed (hypomethylated) by exercise in the mothers4. These data support the role for aerobic exercise in hypomethylating candidate genes. We also identify in the present study that hypomethylation (10,284 CpG sites) is favoured over hypermethylation (7,600 CpG sites) across the genome 30 minutes post an acute bout of resistance exercise, yet without changes in gene expression at this time point. Interestingly, however, hypomethylation of BICC1, STAG1, GRIK2 and TRAF1 after acute RE that was maintained after 7 weeks loading and reloading induced hypertrophy, resulted in significantly enhanced gene expression 22 weeks later. This suggested that DNA methylation of these genes after a single bout of resistance exercise were more sensitive biomarkers than their acutely corresponding gene expression for later load induced hypertrophy. BICC1 is an RNA binding protein that has an undermined role in adult skeletal muscle. It has been identified as differentially expressed during prenatal muscle development between two different pig breads57. RNA binding proteins in general are important in post transcriptional modifications, suggesting that perhaps reduced DNA methylation and increased gene expression may indicate an increase in post-transcriptional modification after reloading, however this requires further investigation to confirm. STAG1 (Cohesin subunit SA-1) is fundamental in cell division and part of the cohesin complex, which is required for the cohesion of sister chromatids after DNA replication58. However, to the authors knowledge there is no specific role for STAG1 identified in adult skeletal muscle hypertrophy. GRIK2 and TRAF2 were also identified as being hypomethylated after loading and reloading together with enhanced gene expression. As suggested above, GRIK2′s role in skeletal muscle is not well defined. However, TRAF1 has been widely implicated in skeletal muscle cell proliferation and differentiation, as discussed above, and hypomethylation of TRAF1 appears to be both sensitive to acute RE, as well as maintained following repeated loading and reloading induced hypertrophy that resulted in the largest increase in gene expression after reloading, 22 weeks after being detected as hypomethylated after acute RE. Overall, suggesting an important role for TRAF2 in skeletal muscles epigenetic memory of hypertrophy.