Training-induced gene expression plasticity in cardiac function and neural regulation for ultra-trail runners

This study aims to assess the gene regulatory response from a group of 16 athletes and to observe the plasticity induced by their training regime on the gene expression response after their participation in an 82km race. Blood samples for differential gene expression (DGE) were collected before and after this effort from two groups of runners with different training regimes: elite and active. Analyses only focused on genes annotated as related to cardiac function (CF) and neural regulation (NR) from the KEGG PATHWAY Database. Thus, 13 pathways were considered accounting for a total of 629 genes. Training regime modulated the response to exercise based on a list of 18 ranked genes with significant DGE for elite runners while remained statistically insignificant for active athletes. UQCR11, COX7C and COX4I1 genes, related to mitochondrial respiratory chain, were down-regulated which may indicate mitochondrial function impairment in cardiac muscle. Increased expression levels were obtained for PIK3R2, PLCG2, IRAK3 genes from the positive signaling cascades of neurotrophins pathway, which may reveal an improved heart rate control thanks to a better cardiac sympathetic innervation.Postprint (author's final draft

Endurance races effects in non-elite runners by clinical biomarkers and bioimpedance measurements

Participation in marathons and ultra-endurance races is growing every day, but it is worrisome that these types of races are becoming mass events, where many runners are not sufficiently prepared. For example, in the 2017 Barcelona marathon there were 19.740 runners, whereas in the 2007 marathon there were 7.430_ runners (Zurich Barcelona Marathon). In the age of social media everyone wants to put his/her photo and comment on instagram, facebook, twitter, etc., about the achievement reached, without caríng if his/her healt.h has been compromised. A marathon or longer distance races, require significant exertions and result in impact to our bodies and bring short and long-term consequences for our health. Never befare has running been so fashionable, but to what extent is it done in a healthy manner? The motivation and merit of this thesis líes in the fact of finding the impact of a 42km run on our body and evaluate the risk of muscle and cardiac damage, inflammation and infection, and renal impairment. The results embodied in the different research articles of this thesís, due to the small number of runners are subjective and show a tendency to be validated in other studies with more athletes. More than 80 non-elite runners were studied during 4 years (from 2016 to 2019). The changes evaluated in pre-race conditions {baseline), at the end, and 48h post-race were through blood and saliva biomarkers. Additionally, other variables were assessed before the race, like nutrition balance and sorne specific supplement intake, and it was proved how the body weight, recovery time, and amount of training done can directly affect the runner's performance and health. The highest elevations in biomarkers associated to muscle damage (CK), inflammation (CRP), and cardiovascular health (Hs-TnT, NT-proBNP, ST2) were seen in the runners with the worse performance time and less training hours before-race. Also, the marathon provoked changes in the salivary immunity and increased the risk of developing a respiratory tract infection. In addition, supplementation actions like polysaccharide-based ones, positively affected on the immune response initiated during a 42 km effort. By BIVA methodogly, a correlation can be made with a displacement ofthe vector impedance at 24 h and 48 h post-race on the tolerance ellipses of a European Caucasian reference population with biomarkers of renal damage. Transient values of acute kidney injury (AKI) stage 1, more related to inflammatory factors rather than muscle damage, were found in many marathoners which recovered mostly 48 h post-race. · Regarding diet, a correct mono- and poly-unsaturated fatty acid, potassium, and magnesium intake befare the marathon influenced better performance and better cardiovascular health. But, what are the negative long-term effects of these acute changes in clinical biomarkers and bioimpedance measurements after 42 km? How does it affect an inadequate diet and preparation? This thesis is based on five original publications, three accepted and two under review, separated in three chapters ordered from 2 to 4, starting with cardidVascular biomarkers, immunity, and supplements. In Appendix 1, renal function and bioimpedance vector displacement, and in Appendix 2, diet effects on performance and cardiac health. An introduction in Chapter 1, describes the rationale of the published work and its place within the whole topic of this thesis. As an elite runner for many years and already a veteran, this thesis is my "grain of sand" in the fasciriating world of metabolic responses while running.La participació en maratons i curses d'ultra resistència creix cada dia, però és preocupant que aquest tipus de curses es converteixin en esdeveniments massius, on molts corredors no estan prou preparats. Per exemple, a la marató Barcelona del 2017 hi havía 19.740 corredors, mentre que a la marató del 2007 hi havia 7.430 corredors (Zurich Barcelona Marató). En l'era de les xarxes socials, tots volen posar la seva foto i comentar a lnstagram, Facebook, Twitter, etc., sobre l'assoliment aconseguit, independentment de si la seva salut s'ha vist compromesa. Una marató o cursa de llarga distància, requereix esforços sígnificatius i té un impacte en el nostre cos amb unes conseqüències a curt i llarg termini pera la nostra salut. Mai abans havia¿ estat tan de moda córrer, però fins a quin punt es fa de manera saludable? La motivació i el mèrit d'aquesta tesi rau en el fet de veure !'impacte d'una carrera de 42 km en el nostre cos i avaluar el risc de dany muscular i cardíac, d'inflamació i infecció, i d'insuficiència renal. Els resultats exposats en els diferents articles d'investigació d'aquesta tesi, a causa del petit nombre de corredors, són subjectius i mostren una tendència a ser validats en altres estudis amb més atletes. Es van estudiar més de 80 corredors no elit durant 4 anys (del 2016 al 2019). Els canvis avaluats en les condicions prèvies a la cursa (estat basal), al final i 48 hores després de la cursa, es van realitzar a través de biomarcadors en sang i saliva. A més a més, es van avaluar altres variables abans de la cursa, corn l'equilibri nutricional i una suplementació específica, i es va demostrar com el pes corporal, el temps de recuperació i la quantitat d'entrenament realitzat poden afectar directament el rendiment i la salut del corredor. Les elevacions més altes en els biomarcadors associats amb el dany muscular (CK), la inflamació (CRP) i la salut cardiovascular (Hs-TnT, NT-proBNP, ST2) es van observar en els corredors amb pitjor rendiment en carrera i amb menys hores d'entrenament abans de la cursa. A més, la marató va provocar canvis en la immunitat salival i va augmentar el risc de desenvolupar una infecció del tracte respiratori. Així mateix, les accions de suplementació com les basades en polisacàrids van afectar positivament la resposta immune iniciada durant l'esforç de córrer 42 km. Segons el mètode BIVA, es pot fer una correlació del desplaçament del vector impedància a les 24 h i 48 h després de la carrera sobre les el·lipses de tolerància d'una població de referencia caucàsica europea, amb els biomarcadors de dany renal. Els valors transitoris de la lesió renal aguda (AKI) tipus 1, més relacionats amb factors inflamatoris que amb el dany muscular, es van trobar en molts maratonians que van recuperar a les 48 h després de la cursa. Pel que fa a la dieta, una correcta ingesta d'àcids grassos monoinsaturats i poliinsaturats, potasi i magnesi abans de la marató va influir en un millar rendiment i una millar salut cardiovascular. Però quins són els efectes negatius a llarg termini d'aquests canvis aguts en els bíomarcadors clínics i en les mesures de bioimpedància després de 42 km? Com afecta una dieta i una preparació inadequades? Aquesta tesi es basa en cinc publicacions originals, 3 acceptades i dos en revisió, separades en tres capítols ordenats del 2 al 4, començant amb biomarcadors cardiovasculars, infecció, immunitat i suplementació. En l'Annex 1, la funció renal i el desplaçament del vector de bioimpedància, i l'Annex 2, els efectes de la dieta sobre el rendiment í la salut cardíaca. Una introducció al Capital 1 descriu la justificació del treball publicat i el seu lloc dins el tema general d'aquesta tesi. Com a corredora d'elit durant molts anys i ja veterana, aquesta tesi és el meu "gra de sorra" en el fascinant món de les respostes metabòliques mentre correm.La participación en maratones y carreras de ultra resistencia crece cada día, pero es preocupante que este tipo de carreras se conviertan en eventos masivos, donde muchos corredores no están lo suficientemente preparados. Por ejemplo, en la maratón Barcelona de 2017 había 19.740 corredores, mientras que en la maratón de 2007 había 7.430 corredores (Zurich Barcelona Marathon). En la era de las redes sociales, todos quieren subir su foto y comentar en Instagram, Facebook, Twitter, etc., sobre el logro alcanzado, sin importar si su salud se ha visto comprometida. Una maratón o carrera de larga distancia, requiere esfuerzos significativos y tiene un impacto en nuestro cuerpo con unas consecuencias a corto y largo plazo para nuestra salud. Nunca antes había estado tan de moda correr, pero ¿hasta qué punto se hace de manera saludable? La motivación y el mérito de esta tesis radica en el hecho de ver el impacto de una carrera de 42 km en nuestro cuerpo y evaluar el riesgo de daño muscular, daño cardíaco, inflamación, cambios inmunológicos, infección, e insuficiencia renal. Un total de 234 corredores no elite se estudiaron durante 4 años (del 2016 al 2019). Los artículos presentados en esta tesis se basan en los corredores que participaron en la maratón del 2016. En el artículo “The Dynamics of Cardiovascular Biomarkers in non-Elite Marathon Runners” se analizaron 79 corredores, en el de “Salivary immunity and lower respiratory tract infections in non-elite marathon runners” se analizaron 47 corredores y en el de “Effects of a polysaccharide-based multiingredient supplement on salivary immunity in non-elite marathon runners” se analizaron 41 corredores. Los cambios evaluados en las condiciones previas a la carrera (estado basal), al final y 48 horas después de la carrera, se realizaron a través de biomarcadores de sangre y saliva. Además, se evaluaron otras variables antes de la carrera, como el equilibrio nutricional y una suplementación específica, y se demostró como el peso corporal, el tiempo de recuperación y la cantidad de entrenamiento realizado pueden afectar directamente el rendimiento y la salud del corredor. Las elevaciones más altas en los biomarcadores asociados con el daño muscular (CK), la inflamación (CRP) y la salud cardiovascular (Hs-TnT, NT-proBNP, ST2) se observaron en los corredores con peor rendimiento en carrera y con menos horas de entrenamiento antes de la misma. Además, la maratón provocó cambios en la inmunidad salival y aumentó el riesgo de desarrollar una infección del tracto respiratorio. A su vez, las acciones de suplementación como las basadas en polisacáridos afectaron positivamente la respuesta inmune iniciada durante el esfuerzo de correr 42 km. Según el método BIVA, se puede hacer una correlación del desplazamiento del vector impedancia a las 24 h y 48 h después de la carrera sobre las elipses de tolerancia de una población de referencia caucásica europea, con los biomarcadores de daño renal. Los valores transitorios de la lesión renal aguda (AKI) tipo 1, más relacionados con factores inflamatorios que con el daño muscular, se encontraron en muchos maratonianos que recuperaron a las 48 h después de la carrera. Con respecto a la dieta, una correcta ingesta de ácidos grasos monoinsaturados y poliinsaturados, potasio y magnesio antes de la maratón influyó en un mejor rendimiento y una mejor salud cardiovascular. ¿Pero cuáles son los efectos negativos a largo plazo de estos cambios agudos en los biomarcadores clínicos y las mediciones de bioimpedancia después de 42 km? ¿Cómo afecta una dieta y una preparación inadecuadas? Esta tesis se basa en cinco publicaciones originales, tres aceptadas y dos en revisión, separadas en tres capítulos ordenados del 2 al 4, comenzando con biomarcadores cardiovasculares, infección, inmunidad y suplementación. En el Anexo 2, la función renal y el desplazamiento del vector de bioimpedancia, y en el Anexo 3, los efectos de la dieta sobre el rendimiento y la salud cardíaca. Una introducción en el Capítulo 1 describe la justificación del trabajo publicado y su lugar dentro del tema general de esta tesis. Como corredora de élite durante muchos años y ya veterana, esta tesis es mi "grano de arena" en el fascinante mundo de las respuestas metabólicas que hace nuestro cuerpo mientras corremo

The molecular athlete: exercise physiology from mechanisms to medals

Human skeletal muscle demonstrates remarkable plasticity, adapting to numerous external stimuli including the habitual level of contractile loading. Accordingly, muscle function and exercise capacity encompass a broad spectrum, from inactive individuals with low levels of endurance and strength, to elite athletes who produce prodigious performances underpinned by pleiotropic training-induced muscular adaptations. Our current understanding of the signal integration, interpretation and output coordination of the cellular and molecular mechanisms that govern muscle plasticity across this continuum is incomplete. As such, training methods and their application to elite athletes largely rely on a "trial and error" approach with the experience and practices of successful coaches and athletes often providing the bases for "post hoc" scientific enquiry and research. This review provides a synopsis of the morphological and functional changes along with the molecular mechanisms underlying exercise adaptation to endurance- and resistance-based training. These traits are placed in the context of innate genetic and inter-individual differences in exercise capacity and performance, with special considerations given to the ageing athletes. Collectively, we provide a comprehensive overview of skeletal muscle plasticity in response to different modes of exercise, and how such adaptations translate from "molecules to medals"

Telomere, DNA Methylation and Gene Expression changes caused by exercise training

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.Doctor of Philosoph

Oxidative Stress and Exercise

Although regular moderate-intensity exercise can activate important cell adaptive properties, sporadic and strenuous bouts of exercise may induce oxidative stress due to an augmented production of reactive metabolites of oxygen (ROS) and nitrogen free radical species (RNS). Exercise-induced free radical formation may impair cell function by oxidatively modifying nucleic acids, where DNA damage and insufficient repair may lead to genomic instability. Likewise, lipid and protein damage are significant cellular events that can elicit potentially toxic perturbations in cellular homeostasis. This book focuses on aspects of exercise-induced oxidative stress while taking into consideration the basic mechanisms, consequences and function of ROS production, and whether antioxidants may either support or hinder these responses

The relationship between exercise and cognition in diabetes mellitus

The increasing prevalence and incidence of type 2 diabetes mellitus (T2D) has been referred to as a global epidemic. This thesis aimed to synthesise the evidence base in both animal models and human studies that exercise exposure is related to better cognition in diabetes, via 2 systematic reviews. Secondly, we investigated the efficacy of a novel form of exercise, POWER training (high velocity PRT), for cognitive function in this cohort. We hypothesised that 12 months of high intensity POWER training would significantly improve cognitive function in a cohort of older adults with T2D and multiple co-morbidities. The GREAT2DO study was the first RCT to evaluate the effects of a one-year intervention of POWER training compared to a SHAM exercise control condition on insulin resistance, HbA1c, body composition, physical performance, inflammation, adipokines, cardiovascular health status, and quality of life as well as to explore relationships between these domains in response to the intervention in this cohort. In this GREAT2DO cognitive sub-study, we assessed global cognition and several cognitive domains at baseline in relation to physical and psychological health, fitness and functional performance, as well as changes over time in cognitive outcomes in response to the intervention. We found that cognitive function improved in both POWER and sham exercise groups over time, although unexpectedly without group effect. However, we showed for the first time that there were significant direct relationships between increases in skeletal muscle mass, total muscle strength, total static balance time, and total adiponectin levels and improvements in cognitive function, and that these relationships only existed in the POWER group, as hypothesised. There is need for further study, in particular exploration of the persistence, clinical relevance, and mechanisms underlying attenuation of the rate of cognitive decline and incident dementia in this high-risk cohort

Sport Modalities, Performance and Health

Sport modalities are highly practiced in order to improve many aspects of human beings, including performance and health. The increasing interest in the quantitative and qualitative aspects of sport training is ascribable to the fact that several training systems and new methodologies are appearing in all sport modalities. These methodologies can have different effects on the organism depending on the degree of training.On the other hand, some of the main objectives in sport research are to describe match activity and to detect effective performance indicators. A better knowledge of players' performance adaptations and game dynamics during competition is extremely useful for optimizing the training process. The need to develop training methodologies according to actions occurring during the game is essential for each sport