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

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

Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

Funder: Vetenskapsrådet; FundRef: http://dx.doi.org/10.13039/501100004359Funder: Cancerfonden; FundRef: http://dx.doi.org/10.13039/501100002794Funder: Barncancerfonden; FundRef: http://dx.doi.org/10.13039/501100006313Funder: Svenska Läkaresällskapet; FundRef: http://dx.doi.org/10.13039/501100007687Funder: Cancer Research UK; FundRef: http://dx.doi.org/10.13039/501100000289Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Exercise has a wide range of systemic effects. In animal models, repeated exertion reduces malignant tumor progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effects of exercise on tumor progression are unclear, as are the cellular actors involved. We show here that in mice, exercise-induced reduction in tumor growth is dependent on CD8+ T cells, and that metabolites produced in skeletal muscle and excreted into plasma at high levels during exertion in both mice and humans enhance the effector profile of CD8+ T-cells. We found that activated murine CD8+ T cells alter their central carbon metabolism in response to exertion in vivo, and that immune cells from trained mice are more potent antitumor effector cells when transferred into tumor-bearing untrained animals. These data demonstrate that CD8+ T cells are metabolically altered by exercise in a manner that acts to improve their antitumoral efficacy

Molecular and structural effects of hypertrophy training and anabolic steroids on skeletal muscle

Anabolic steroids are widely used and have a long history in amateur and professional athletics, especially in disciplines profiting from hypertrophy effects. Not only their detection is an objective of recent research but also the understanding of their underlying mechanisms and overall influence on the cellular signalling cascades. Enhancement of performance based on muscular hypertrophy relies on numerous pathways and a concert of factors which together induce a positive adaption process. This study takes a close up look at selected influential genes and pathways in the process of regeneration, hypertrophy and myogenesis in an animal experimental design in rat. Two key independent variables are applied through interventional treatments of the test subjects - exercise and application of the anabolic steroid Metandienone, both designed to depict the real life situation of a steroid abusing athlete regarding intensity and focus of training and dosage of drug application. As the regulational network for influencing the physical ability athleticism and hypertrophy is a complex web of pathways, numerous biological processes have to be examined. In this thesis these factors cover major mitogens and systemic elements like the Androgen Receptor, IGF-1 and Pax7, several players in the muscle related TGF$\beta$-pathway - the Activin receptor IIB, myostatin, follistatin, Smad3 and Smad7, the myogenic factors MyoD and Myogenin, structural proteins through the embryonic myosin heavy chain and factors of the immune system - Tumor Necrosis Factor $\alpha$, Interleukin 6 and Interleukin 10. These factors are examined by comparative real-time PCR method in rat gastrocnemius muscle, demonstrating the wide array of organismic response to intervention on mRNA level. Furthermore, promising and important factors embryonic myosin heavy chain and MyoD are analyzed on protein level through western blot analysis. Finally, a possible definite structural influence is examined by histological analysis of fat tissue and \textit gastrocnemius} cross sections and evaluated by ATPase fibre typing. Results show the basic influence of intervention regarding body composition and a reduction of body fat parameters as a reaction to exercise. Furthermore a complex modulational effect on the regulational network across several important factors on mRNA level especially on embryonic myosin heavy chain, Myogenin, IL-6, Smad7 and the Androgen Receptor can be observed. These changes follow a specific parameter-dependent pattern in most genes. In protein data of selected gene products several tendencies are confirmed, demonstrating the downstream adaptational effect of the intervention but also revealing no visible effect downstream of other promising results on mRNA level. Finally in histological analysis on structural level the influence reaches minimal extent as no significant change can be observed in muscle fibre size and muscle fibre type composition of gastrocnemius muscle.Stefan Markus ReitznerInnsbruck und Köln, Univ., Master-Arb., 2015(VLID)42115

Dataset for: Expression of Striated Activator of Rho-Signaling (STARS) in human skeletal muscle following acute exercise and long-term training

Aim: The Striated Activator of Rho-Signaling (STARS) protein acts as a link between external stimuli and exercise adaptation such as muscle hypertrophy. However, the acute and long-term adaptational response of STARS is still unclear. This study aimed at investigating the acute and long-term endurance training response on the mRNA and protein expression of STARS and its related upstream and downstream factors in human skeletal muscle. Methods: mRNA and protein levels of STARS and related factors were assessed in skeletal muscle of healthy young men and women following an acute bout of endurance exercise (n=20) or 12 weeks of one-legged training (n=23). Muscle biopsies were obtained before (acute and long-term), at 30 min, 2h and 6h following acute exercise, and at 24 hrs following both acute exercise and long-term training. Results: Following acute exercise, STARS mRNA was significantly elevated 3.9-fold at 30 minutes returning back to baseline 24 hours after exercise. STARS protein levels were were numerically but non-significantly increased 7.2-fold at 24 hours. No changes of STARS or ERRα mRNA or STARS protein expression were seen following long-term training. PGC-1α mRNA increased 1.7-fold following long-term training. MRTF-A mRNA was increased both following acute exercise and long-term training, in contrast to SRF mRNA and protein which did not change. Conclusion: STARS mRNA is acutely upregulated with exercise, but there is no cumulative effect to long-term training as seen in PGC-1α mRNA expression. Exercise intensity might play a role in manifestation of protein expression, suggesting a more complex regulation of STARS

Cytotoxic T-cells mediate exercise-induced reductions in tumor growth.

Exercise has a wide range of systemic effects. In animal models, repeated exertion reduces malignant tumor progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effects of exercise on tumor progression are unclear, as are the cellular actors involved. We show here that in mice, exercise-induced reduction in tumor growth is dependent on CD8+ T cells, and that metabolites produced in skeletal muscle and excreted into plasma at high levels during exertion in both mice and humans enhance the effector profile of CD8+ T-cells. We found that activated murine CD8+ T cells alter their central carbon metabolism in response to exertion in vivo, and that immune cells from trained mice are more potent antitumor effector cells when transferred into tumor-bearing untrained animals. These data demonstrate that CD8+ T cells are metabolically altered by exercise in a manner that acts to improve their antitumoral efficacy.Wellcome Trus