Exercise-induced molecular mechanisms in untrained and life-long highly trained individuals

Abstract: Regular physical activity results in extensive systemic and functional adaptation effects in the human body that contribute to physical performance, such as muscular strength and endurance, and can have beneficial health effects on the cardiorespiratory, vascular and immune systems as well as on bone density and metabolic control. Adaptation to regular exercise requires translation of exercise-related signals into molecular responses, including epigenetic modification and other molecular processes. Over time, such processes result in accumulating cellular and sub-cellular biochemical and structural changes of tissues and organ systems. Exercise-related signals that challenge the body as a functional system include hypoxia, flux of energy rich substrates, changes in body temperature, lactate-induced pH changes, changed abundance of metabolites and mechanical shear stress. To overcome such challenges and improve future preparedness, tissues adapt for example by increased mitochondrial content in skeletal muscle, optimized temperature management and circulation, increased plasma volume and altered cell content in circulating blood, increased vascularization or by structural reinforcement and increased ability to develop force in skeletal muscle. In life-long trained athletes, the adaptations can result in outstanding, sports-specific performance. However, not all contributing mechanisms and sports-specific differences are well understood, especially in the context of elite athletes. The results presented in this thesis are based on five papers in which skeletal muscle biopsies (paper I-V) and blood samples (paper V) were collected at different timepoints around acute (papers I, IV, V) and long-term exercise (papers I, II, III). The subjects in papers I-II were young, healthy, normally active men and women, in papers III-V, the subjects were healthy middle-aged men and women, either with a life-long history of sedentary lifestyle or high-level physical activity in enduranceor resistancebased sports. Five experimental models were used: acute bipedal cycling for 60 minutes (paper I), a 12-week unilateral leg extension endurance training protocol consisting of 4x45min of exercise per week (paper I), a 10-week protocol with unilateral leg press and leg extension resistance training at 70-85% 1RM (paper II), acute bipedal cycling for 30 minutes and acute leg extension at 80% 1RM in a cross-over design (papers IV-V). Paper III consisted of a cross-sectional study design without any acute intervention. Collected samples were analyzed by qPCR, western blot (papers I-II), bisulfite-transformation, pyrosequencing and phosphorylation analysis (paper II), immunohistochemistry, citrate synthase assay, RNA sequencing (papers III-V) and FACS sorting (paper V). The overall aim of this thesis was to investigate molecular mechanisms that support and maintain life-long high-level adaptations to exercise training. In paper I, the translation of the biomechanical impulse from contracting skeletal muscle into downstream molecular signaling was investigated. In brief, it was shown that the previously described STARS signaling pathway, which links biomechanical and molecular effects is upregulated immediately following acute cycling exercise and that longterm training neither blunts nor amplifies such an acute response pattern. Furthermore, for the first time it was shown that there is no difference between men and women in STARS response. In paper II we investigated how these adaptations can be “memorized” after a period of detraining. We found increased levels of phosphorylation of key genes in previously trained muscle and identified differences in gene expression of PGC-1α and other genes important for myogenesis, suggesting potential mechanisms for a “muscle memory”. In a cross-sectional investigation in paper III, using global transcriptome analysis, gene ontology and genome-scale metabolic modelling we show that life-long high-level adaptation to endurance exercise is very different from the adaptation following life-long high-level resistance training, particularly in pathways related to the prevention of metabolic diseases, such as type 2 diabetes, and that differences between resistance training and sedentary behavior is comparably small. Furthermore, we found significant sex differences between untrained men and women and that these differences were markedly smaller comparing long-term trained men and women. We also showed that metabolically impaired individuals who submit to short-term endurance training become more similar to long-term endurance trained subjects and identified potential exercise-responsive genes. In paper IV we identified acute exercise-specific patterns of differential gene expression and identified important transcription factor motifs that contribute to these differences in long-term trained athletes. We showed that acute resistance exercise results in generally larger numbers of differentially expressed genes compared to acute endurance exercise and identified amongst others HIF1A and MYFfamily motifs as highly relevant to endurance and resistance exercise respectively. Furthermore, we identified groups of candidate genes that are especially relevant to these transcription factors and show that these genes are functionally closely connected. We also demonstrate that endurance trained athletes handle the metabolic stress of energy production differently than strength trained athletes and untrained subjects, surprisingly by a largescale downregulation of metabolites and enzymes engaged in energy production processes immediately following acute endurance exercise, confirming the uniqueness of endurance athletes proposed in paper III. In paper V we investigated how high-level long-term training modulates the response of circulating immune cells to acute endurance and resistance exercise. We show that lifelong high-level endurance athletes increase their numbers of circulating monocytes to a significantly larger extent and that the recovery of numbers of macrophages is significantly lower compared to untrained controls. Additionally, we show significant differences between the immune system response to acute endurance or resistance exercise. Furthermore, we cross-referenced immune cell concentrations in circulating plasma with the expression of immune cell marker genes in skeletal muscle and cytokine signaling in blood and demonstrated a higher enrichment of immune cell mobility related functional groups of genes in untrained control subjects compared to long-term trained athletes and a generally higher coordination of these functional groups of genes in response to acute endurance exercise compared to acute resistance exercise across all groups. In conclusion we show that life-long trained endurance athletes handle metabolic challenges in a unique way and have a resting transcriptome largely different to strength trained and control individuals. Furthermore, we suggest the phosphorylation of proteins related to protein synthesis as potential molecular mechanism for a muscle memory effect. Finally, we show, that long-term training does not blunt STARS pathway-based signal translation of mechanical contraction.