Telomere, DNA Methylation and Gene Expression changes caused by exercise training
- Authors: Denham, Joshua
- Date: 2016
- Type: Text , Thesis , PhD
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- Description: Exercise training is one of the few therapeutic interventions that improves health span by delaying the onset of age-related diseases and preventing early death. Despite the clear benefits to health conferred by exercise training, our understanding of the underlying molecular mechanisms remain crude. The primary purpose of this thesis is to determine and analyse the molecular biology changes that occur with strenuous aerobic exercise. Specifically, the main objectives were to investigate the impact of strenuous aerobic exercise training on structural DNA modifications, measured in context with cardiovascular health and fitness adaptations. In the first part of this thesis I investigated the influence of endurance exercise training on leukocyte telomere length and cardiovascular health. Leukocyte telomere length reflects biological age. Indeed, excessively short leukocyte telomeres are associated with age-related chronic diseases. Epidemiological studies indicate endurance athletes live longer than people from the general public who do not engage in extensive aerobic exercise training. In Chapter 2, my literature review on the subject of exercise and telomere biology suggested that, at the time of this study, the impact of exercise training on leukocyte telomere length was equivocal. Therefore, to determine whether strenuous aerobic exercise training influences biological ageing (assessed by leukocyte telomere length), I conducted two cross-sectional studies on leukocyte telomere length differences between endurance athletes and healthy controls. The first study (Chapter 3) was a cross-sectional analysis of leukocyte telomere length between athletes and controls, determined by quantitative polymerase chain reaction (qPCR). This is a relative measurement of telomere length expressed as a telomere (T) to single copy gene (S) ratio. Relative to the healthy controls (n = 56), the ultra-marathon runners (n = 67) possessed 11% longer leukocyte telomeres in age-adjusted analysis (ultra-marathon runners vs controls; average T/S ratio: 3.56 vs 3.16, p = 1.4 × 10-4) and the difference was not explained by the favourable cardiovascular health profile exhibited by the athletes (p = 2.2 × 10-4). The difference in leukocyte telomere length indicated the athletes had reduced their biological age by 16.2 years. To elucidate the potential mechanism for the longer leukocyte telomeres observed in endurance athletes, I recruited another cohort of athletes and controls and measured leukocyte telomere length and gene expression of genes involved in telomere length regulation. In the second study (Chapter 4), I describe data replicating the finding that endurance athletes possess longer leukocyte telomeres compared to healthy controls (athletes v controls mean T/S ratio ± SE: 3.64 ± 0.06 vs 3.38 ± 0.06, p = 0.002). This difference was associated with a concomitant increased activity of two important telomere regulating genes, telomerase reverse transcriptase (TERT) and adrenocortical dysplasia homolog (TPP1) (2- fold and 1.3-fold, respectively, both p < 0.05). The difference in leukocyte telomere length and leukocyte telomere-regulating gene (TERT and TPP1 mRNA) expression was ameliorated after adjusting for maximal oxygen uptake and resting heart rate (all p > 0.05). This finding indicates that cardiorespiratory fitness is an important determinant of telomere biology. Together, these two cross-sectional studies suggest that regular endurance exercise training is associated with longer leukocytes telomeres and that this is likely achieved through higher TPP1 and TERT mRNA expression gained through improved cardiorespiratory fitness. The findings in Chapters 3 and 4 provide evidence for extensive endurance exercise training as an effective lifestyle strategy to attenuate biological ageing. In parallel to telomere length changes, epigenetic modifications (e.g. DNA methylation) caused by environmental factors alter the transcriptomic milieu of cells. My thorough literature review (Chapter 5) revealed that exercise training seems to rearrange chromatin by modifying the DNA methylome in a variety of cells and that the extent is dictated by exercise duration and intensity. Therefore, in the second part of my thesis, I investigated the DNA methylation changes in leukocytes (which are somatic cells) and sperm (male germ cells) from healthy men before and after sprint interval training (SIT). Unlike traditional, long duration training at moderate intensity training, SIT involves short, intense (>85% VO2max to supra-maximal) efforts followed by periods of rest (3–4 min), typically repeated 3–8 times. It is an effective type of training that improves cardiorespiratory fitness quicker than traditional long slow distance training. Thus, to establish the DNA methylome changes associated with SIT, I conducted two training studies and analysed the leukocyte and sperm methylomes using the Infinium HumanMethylation450 BeadChip (Illumina). My third study (Chapter 6) provides the first evidence showing an association between DNA methylation changes paralleled with improvements to lipid profile and cardiorespiratory fitness in humans. Twelve young men (18–24 years) undertook SIT (thrice weekly) for four weeks. Resting blood samples were obtained and whole-blood leukocytes were isolated by red blood cell lysis. Genome-wide DNA methylation was assessed using the 450K BeadChip (Illumina). Cardiorespiratory fitness, determined by maximal oxygen uptake, was improved by 2.1 ml.kg-1.min-1 and low-density lipo-protein cholesterol was decreased by 3.9% after SIT (p < 0.05). Notably, the leukocyte methylome was significantly affected by SIT, in regions throughout the genome in relation to CpG islands – CpG islands, North shores, N shelves, South shores and South shelve – and the nearest genes – 3’ untranslated region (UTR), 5’ UTR, exonic, intergenic, intronic, non-coding and promoter regions (all p < 0.001). Genes with differentially methylated CpG sites (q < 0.005) after SIT were enriched for cardiovascular gene ontology (GO) terms that included metabolic activity, biological adhesion and antioxidant activity. Similarly, pathway analysis revealed genes involved in focal adhesion, calcium signaling and mitogen activated protein kinase were modulated by SIT-induced DNA methylation changes. Amongst the 205,987 probes relating 32,445 transcripts differentially methylated after SIT (q < 0.05), with methylation changes between 0.1 – 62.8%, the largest and most statistically significant demethylated site was in the epidermal growth factor (EGF) gene, causing decreased mRNA expression. As with EGF, the microRNA-21 and microRNA-210 genes (MIR21 and MIR210, respectively), known for their roles in cardiovascular disease (ischemic heart disease and coronary atherosclerosis), had modest but consistently statistically significant DNA methylation changes at numerous CpG sites, which altered mature microRNA abundance. Together, these data suggest that genome-wide DNA methylation changes occur after short-term intense exercise training concurrently with improvements to blood cholesterol profile and cardiorespiratory fitness. The data presented in this thesis provided evidence that the epigenome of somatic cells is malleable to exercise. There is mounting evidence supporting the premise that environmental perturbations cause DNA methylation changes and these are subsequently transgenerationally inherited, altering phenotypes of future generations. In the current study I also asked the question; can exercise training reconfigure the DNA methylome of male germ cells (sperm)? Therefore, my next study (Chapter 7) entails an analysis of the impact that three months of SIT has on genome-wide DNA methylation of sperm in healthy men. Thirteen subjects undertook twice-weekly SIT for three months, while the controls were asked not to change their current physical activity habits (if any). Sperm samples were donated before and after the three-month intervention. Mature sperm were isolated using density gradient centrifugation and DNA was extracted using the Purelink Genomic DNA Mini Kit (Life Technologies). Global and genome-wide DNA methylation was assessed using an enzyme-linked immunosorbent assay-based kit and the 450K BeadChip (Illumina), respectively. Relative to controls, the cases decreased their resting heart rate and had a higher maximal treadmill speed during exercise testing (both p < 0.05). Cases had decreased global DNA methylation after SIT compared to controls (p < 0.05). Genome-wide DNA methylation analysis revealed numerous modest (0.3 – 6%) methylation changes to 7509 CpG sites, relating to 4602 transcripts (q ≤ 0.1). Differentially methylated CpG sites were in genes associated with developmental biology, which included GO terms, such as developmental process, anatomical structure, embryonic morphogenesis and organ development, together with known pathways regulated by exercise training (MAPK, ErbB and PI3K-Akt signalling). Genes with increased methylation were associated with numerous human diseases, with most overrepresented being psychiatric disorders (schizophrenia, Parkinson’s disease and autism). Notably, paternally imprinted genes associated with other diseases were also differentially methylated after SIT. Therefore, exercise training is associated with the modifications to genome-wide DNA methylation of both somatic and germ cells. In conclusion, the studies presented as a series of peer-reviewed publications, outlines investigations that describe an influence of strenuous exercise training on leukocyte telomere length regulation and the DNA methylome of both leukocytes and germ cells. Both of these molecular changes in leukocytes and sperm provide evidence for novel molecular mechanisms by which exercise improves cardiovascular health and fitness. Future investigations should focus on longitudinal studies determining whether these changes are required for improved health and fitness, and should establish whether exercise-induced DNA methylation changes are transgenerationally inherited, and if so, what impact this has to future generations. Such discoveries could change national physical activity guidelines and policies, by emphasising the benefit of regular exercise both in the present and to future offspring.
- Description: Doctor of Philosophy
Controlled ecological evaluation of an implemented exercise-training programme to prevent lower limb injuries in sport : Population-level trends in hospital-treated injuries
- Authors: Finch, Caroline , Gray, Shannon , Akram, Muhammad , Donaldson, Alex , Lloyd, David , Cook, Jill
- Date: 2019
- Type: Text , Journal article
- Relation: British Journal of Sports Medicine Vol. 53, no. 8 (2019), p. 487-492
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- Description: Objective Exercise-training programmes have reduced lower limb injuries in trials, but their population-level effectiveness has not been reported in implementation trials. This study aimed to demonstrate that routinely collected hospital data can be used to evaluate population-level programme effectiveness. Method A controlled ecological design was used to evaluate the effect of FootyFirst, an exercise-training programme, on the number of hospital-treated lower limb injuries sustained by males aged 16-50 years while participating in community-level Australian Football. FootyFirst was implemented with a € support' (FootyFirst+S) or a € without support' (FootyFirst+NS) in different geographic regions of Victoria, Australia: 22 clubs in region 1: FootyFirst+S in 2012/2013; 25 clubs in region 2: FootyFirst+NS in 2012/2013; 31 clubs region 3: control in 2012, FootyFirst+S in 2013. Interrupted time-series analysis compared injury counts across regions and against trends in the rest of Victoria. Results After 1 year of FootyFirst+S, there was a non-statistically significant decline in the number of lower limb injuries in region 1 (2012) and region 3 (2013); this was not maintained after 2 years in region 1. Compared with before FootyFirst in 2006-2011, injury count changes at the end of 2013 were: region 1: 20.0% reduction (after 2 years support); region 2: 21.5% increase (after 2 years without support); region 3: 21.8% increase (after first year no programme, second year programme with support); rest of Victoria: 12.6% increase. Conclusion Ecological analyses using routinely collected hospital data show promise as the basis of population-level programme evaluation. The implementation and sustainability of sports injury prevention programmes at the population-level remains challenging.