Muscle ageing and (re-)training responses: role of myonuclear density & DNA methylation in muscle memory

Muscle ageing is characterized by a vicious circle of inflammation, oxidative stress and impaired muscle functioning, which ultimately leads to muscle atrophy and reduced strength. At very old age, low muscle mass and strength, termed sarcopenia, reduce functional capacity and increase the risk of chronic age-related diseases, thereby endangering quality of life and survival chances. Harmful lifestyle choices can contribute to this process. Not only directly, but also indirectly by influencing epigenetic mechanisms. CpG methylation, the most studied mechanism, involves the binding of a methyl group to a CpG and can cause gene silencing by altering the accessibility of transcription factors to DNA. The ageing methylome has been studied in various tissues and has been linked to aberrant expression profiles. Which methylome alterations exactly occur in muscle tissue during ageing and how this affects transcription is poorly understood. Identifying the mechanisms behind muscle ageing is, however, of great importance to be able to reverse it. A first aim of this thesis was therefore to characterize muscle ageing on the methylome and transcriptome level. The expanding elderly population urges health care policies to find and implement strategies that can act upon the intrinsic and extrinsic causes underlying sarcopenia. Resistance training may be an excellent candidate for this. To restore muscle deterioration, resistance exercises need to be implemented weekly, as muscle adaptations are gradually lost upon training cessation. Fortunately, some of these adaptations may be 'remembered' for a long time, which could be of value for individuals facing training interruptions. Some neural adaptations, for example, are well preserved, allowing for a fast restoration of muscle strength performance to a previous training level. Interestingly, also muscle mass seems to increase faster during a second training period. The mechanism behind this phenomenon has been challenging researchers for the past 10 years and meanwhile several suggestions have been made. The first 'muscle memory' theory lies within the number of myonuclei and proposes that hypertrophy-acquired myonuclei are not lost during muscle wasting, allowing for a rapid increase in muscle mass upon a second hypertrophic stimulus by skipping the myonuclei recruitment phase. This theory has been supported by rodent studies using mechanically- or surgically-induced muscle atrophy, but lacks proof in humans. The second aim of this thesis was, therefore, to study myonuclear behaviour during more meaningful physiological conditions of muscle atrophy in humans and link this to the muscle memory theory. The second theory involves lasting epigenetic alterations. Retained muscle methylation modifications after a metabolic, catabolic or anabolic stimulus have already been related to a changed expression profile upon a second encounter with the stimulus. More evidence is, however, needed in humans to integrate this 'epi-memory' in the muscle memory theory. The third aim of this thesis was, therefore, to characterize (re-)training adaptations in young and older muscles at the methylome and transcriptome level and link this to the epi-memory theory. In our first study, we recruited five young men for a 12-week resistance training, 2-week leg immobilization and 12-week retraining intervention. At the start and after each intervention phase, we measured muscle strength and power and collected muscle biopsies from the vastus lateralis to investigate changes in fibre size, satellite cell content and myonuclear density. We stained myonuclei with PCM1, a new and more reliable marker for objective identification of myonuclei. We found that training-induced increases in type II fibre myonuclear number did not return to pre-training level, while muscle strength and power, type II fibre size and satellite cell number increases did. Despite these additional myonuclei, we only observed a steeper increase in muscle strength and not in fibre regrowth following retraining. Our results indicated that myonuclei are indeed not lost upon mechanical unloading in young men, although replication studies with a larger sample size are needed to support our result. To test whether this theory also holds for long-term training cessation in elderly, we recruited 40 older men for a second study, which we divided in an exercise group and smaller control group. After 12 weeks of resistance training and after detraining and retraining of equal length, strength and power were measured in all 40 subjects and muscle biopsies were taken from a small subset to measure the same parameters as in the first study. Opposed to our results in young men, the same training protocol was unable to induce myofibre growth and myonuclear addition in older men. Therefore, we were unable to test our primary aim. Observing the individual tracks, satellite cell and myonuclear number seemed to follow changes in fibre size, which was not in support of our initial hypothesis. Although recent studies in mice and older humans found similar results, we advise for further investigations, as a mechanism for myonuclear apoptosis has not yet been found and may not exist. However, we did confirm and extend previous training and detraining studies with regards to muscle strength and power adaptations to training and detraining. Detraining of equal length as the training period did not revert static strength, dynamic strength and power to untrained levels. This allowed for a fast recovery of dynamic one-repetition-maximum performance upon retraining and additional gains compared to post-training. For strength parameters not directly related to the training program, strength gains seemed to level out more rapidly. Interestingly, we also observed that retraining was able to induce type II fibre growth, satellite cell proliferation and myonuclear addition. This might imply that some cell-intrinsic adaptations occurred during the first training period that were retained during detraining and primed the muscle growth response to retraining. Whether these cell-intrinsic adaptations could be epigenetic modifications, was tested in a third study, where the muscle samples of both previous studies were used to measure DNA methylation and RNA expression at genome-wide level. Firstly, we characterized the muscle ageing signature and found 302 genes with altered expression and methylation levels with advanced age. Promoter-associated CpG islands were predominantly hypermethylated, whereas other gene regions were mainly hypomethylated. The more differentially methylated CpGs within a gene, the clearer the inverse methylation-expression relationship. We found indications of, among other, inflammation, insulin resistance and altered circadian clock and nitric oxide signalling in aged muscle. Furthermore, focal adhesion and axon guidance were the most affected pathways by age. Around 73 % of the methylome differences between young and old were no longer found when older men trained for 12 weeks. Comparable to the results of the second study, previously trained older muscles displayed an enhanced retraining response with more hypomethylated CpGs, enriched pathways, upregulated genes and transcriptome-methylome interactions compared to training. Some of these altered genes were previously mentioned in literature. In addition, we found (de/re)training responsive genes involved in muscle structure, remodelling, energy metabolism and inflammation. In young men, besides restoring many of the hypermethylated and downregulated genes due to immobilization, we also observed more hypomethylated CpGs, upregulated genes and transcriptome-methylome interactions following retraining compared to training. The top 100 genes most affected by retraining, displayed the same expression direction as other resistance training studies and the opposite direction as disuse studies. Importantly, one of the main questions was whether retained methylation modifications could have been behind the enhanced retraining response. We reported that 19 % and 11 % of the methylation changes were retained following detraining in older men and following immobilization in younger men, respectively. By the end of retraining, this was only 11 % and 6 %, respectively. Although these retained methylation levels might have contributed to the enhanced retraining response, we found that only VCL in older muscle and AMOTL1 in young muscle displayed the hypothesized epi-memory pattern at both methylation and expression level. Interestingly, these genes play a role in focal adhesion and satellite cell proliferation, respectively. Within the limitations of our project, this thesis provides novel insights in the muscle ageing mechanism at the methylome and transcriptome level, as well as in myonuclear, methylome and transcriptome adaptations during training interruption and resumption in young and older men.

Jaar van publicatie:2020

Toegankelijkheid:Open