Purpose: In response to resistance exercise training, it has been shown that individuals with a previous training history acquire muscle volume at an accelerated rate. This phenomenon may be attributed, in part, to the myonuclear enrichment resulting from the proliferation of muscle progenitor cells, which promotes essential protein synthesis following subsequent muscle training. As a highly energy demand tissue, the successful hypertrophy of muscle fiber depends on mitochondrial biogenic progression. Moreover, the majority of genes that encode mitochondrial proteins are within nuclear genome. Therefore, in this study, we investigated the effect of increased number of myonuclei in response to the previous resistance exercise preconditioning on mitochondrial adaptations to subsequent resistance training. Our central hypothesis was that pre-trained muscles would show an accelerated acquisition of training-induced mitochondrial function leading to a greater skeletal muscle hypertrophy compared to previously non-trained muscles and this may be associated with increased number of myonuclei in the pre trained muscles. Methods: Thirty-two Sprague-Dawley rats were randomly assigned to four groups (n=8 per group) which include control, pre-training, training, and retraining group. Resistance exercise training was carried out by ladder climbing with weights attached to the tail at ages of either 8- (pre-training) and 36-week-old (training), or both (retraining). Each training session consisted of 3 sets of 5 repetitions, and the training protocol was performed every third day for 8 weeks. At 44 weeks of age, specific muscle groups were carefully collected and stored at -80 °C until further analyses. 4', 6-Diamidino-2-phenylindole staining, hematoxylin & eosin staining, cytochrome c oxidase and succinate dehydrogenase staining were performed. Western blotting and immunohistochemstry were performed to assess the abundance of mitochondrial regulatory proteins and the mitochondrial content. In complementary in vitro studies, confluent L6 myoblast cells were further grown in differentiation media for 4 days with or without insulin-like growth factor 1 (50 ng/ml) supplementation. Mitochondrial gene expression levels and mitochondrial respiratory function were assessed after 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM), a 5' AMP-activated protein kinase activator, treatment. Results: Myonuclei numbers were higher in training and retraining groups than control group (all, p < 0.05), suggesting that ladder climbing training protocol increased myonuclei number. There was a significantly higher level of myonuclei number in pretraining group compared to the control group indicating that the acquired myonuclei during exercise preconditioning were retained over the 20-week detraining period. Muscle cross-sectional area, mitochondrial content and mitochondrial enzymatic activities (COX and SDH) were significantly greater in retraining group compared to training group (p < 0.01, p < 0.01 and p < 0.05, respectively). In in vitro study, L6 myotubes preconditioned with IGF-1 showed increased myonuclei numbers within each myotube and presented a higher level of mitochondrial gene expression and oxygen consumption rate under AICAR treatment condition. Conclusions: These data provide physiological evidence that pre-trained muscle with more myonuclei make the muscles more responsive to subsequent training in terms of muscle hypertrophy and mitochondrial remodeling. Furthermore, this study provides a proof-of-concept of biological processes underlying potential nuclear-mitochondrial interplay during muscle hypertrophy. These findings warrant future studies to identify a novel target for mitochondrial medicine to treat muscle atrophy. / Kinesiology

Hormônios como a testosterona, a desidroepiandrosterona (DHEA), o cortisol e o hormônio do crescimento (GH) e fatores de crescimento, como o fator de crescimento semelhante à insulina (IGF-1), são agudamente liberados no sangue logo após sessões de exercício de força (EF). Esses hormônios e fatores de crescimento estão relacionados com a modulação de processos fisiológicos na célula muscular esquelética. Mais recentemente, pesquisadores evidenciaram a presença de enzimas esteroidogênicas, responsáveis por metabolizar o colesterol em diferentes hormônios esteroides, no interior da célula muscular. Isso possibilitaria às células musculares regularem a concentração hormonal intramuscular. Essa modulação intramuscular pode ser capaz de afetar diferentes processos fisiológicos nessas células, como a atividade das células satélites (CS). Contudo, o papel da modulação da concentração hormonal sérica induzida pelo EF em regular as concentrações intracelular de hormônios nas células musculares, regulando a atividade das CS ainda não é bem conhecido em humanos. Para investigar esse fenômeno, indivíduos treinados em força foram submetidos a duas diferentes sessões de EF com o objetivo de modular diferentemente as respostas hormonais séricas entre elas. Uma sessão (HH) que elevaria expressivamente as concentrações agudas séricas da testosterona total e livre, do DHEA, do cortisol, do GH, e do IGF-1, enquanto outra sessão não induziria elevações expressivas desses hormônios (LH). Indivíduos treinados foram escolhidos por apresentarem menor impacto de sessões de exercício de força na modulação de processos fisiológicos nas células musculares por serem mais acostumados às mesmas. Isso favorece relacionar os processos da modulação hormonal sistêmica e local com a possível regulação da atividade das CSs. As sessões de EF foram efetivas em modular agudamente as concentrações séricas da testosterona total, DHEA, GH e cortisol. Contudo, apenas o Cortisol foi elevado mais para sessão HH comparado com a sessão LH. Consequentemente, apenas o cortisol teve sua concentração diferentemente alterada nas células musculares, estando mais aumentado também após a sessão HH. A ausência de elevação na célula muscular de hormônios androgênicos foi suportada pela ausência de mudança na expressão gênica das enzimas esteroidogênicas como a 5α redutase e a 17β Hidroxiesteróide Desidrogenase. Interessantemente, a expressão gênica da miostatina e da miogenina aumentaram aproximadamente nove e quatro vezes, respectivamente, 72 horas após as sessões de EF. Por fim, possivelmente afetados pelos níveis de cortisol elevado na célula muscular, que pode ter favorecido um expressivo aumento de expressão gênica da miostatina, a quantidade de CS e consequentemente de mionúcleos não sofreram nenhum efeito das sessões de EF. Essa ausência de modulação da quantidade de CS ocorreu mesmo com o aumento da expressão da miogenina que poderia ter favorecido um processo de diferenciação das CS. Assim, é possível sugerir que quando o hormônio é elevado agudamente no sangue de forma expressiva como o cortisol, o mesmo afetará sua concentração na célula muscular. Esse aumento da concentração hormonal no músculo pode regular a atividade das CS, já que não foi observada a esperada mudança na quantidade de CS nas células musculares após as sessões de EF / Hormones such as testosterone, dehydroepiandrosterone (DHEA), cortisol and growth hormone (GH), as well as growth factors such as insulin-like growth factor (IGF-1) are acutely released into the blood after resistance exercise (RE). These hormones are related to the regulation of physiological processes in skeletal muscle cells. Recently, researchers have shown the presence of steroidogenic enzymes, responsible for metabolizing cholesterol in different steroid hormones, inside the muscle cell. This metabolization would enable muscle cells to regulate intramuscular hormone concentration. This intramuscular modulation may affect different physiological processes in these cells, such as satellite cell activity (SC). However, the role of acute RT-induced changes in serum hormone concentrations in regulating intracellular hormonal concentrations in muscle cells and, consequently, activity of SC is yet unknown in humans. To investigate this phenomenon, resistance-trained individuals underwent two different RE sessions to differently modulate serum hormonal responses. One session (HH) should significantly elevate serum concentrations of total and free testosterone, DHEA, cortisol, GH, and IGF-1, while the other session should not induce expressive elevations in these hormones (LH). Trained individuals were chosen because muscle cells are less impacted by RE as these individuals are more accustomed to RE. This design allows relating systemic and local hormonal modulation with possible modulations on SC activity. RE sessions were effective in acutely modulating the serum concentration of total testosterone, DHEA, GH, and cortisol. However, only cortisol was significantly raised for HH compared to LH. Consequently, only cortisol had its concentration differently modulated in muscle cells, being higher also after the HH session. The lack of elevations in muscle cell androgenic hormones was supported by the absence of changes in the steroidogenic enzymes gene expression such as 5α reductase and 17β Hydroxysteroid Dehydrogenase. Interestingly, myostatin and myogenin gene expression increased approximately nine and four times, respectively, 72 hours after the RE sessions. Finally, high cortisol levels in the muscle cell may have favored an expressive increase in myostatin gene expression, did not induce the expected changes in SC activity. This lack of modulation in the amount of SC and myionuclear content occurred regardless of the increase in myogenin expression, which could have favored the SC differentiation. Therefore, it is possible to suggest that when systemic hormones are expressively elevated like cortisol, there is a parallel increase in hormonal concentration in the muscle cell. The increase in muscle cell hormone concentration may have regulated the SC activity based as we did not observe the expected changes in the SC content after the RE sessions

Strength training is widely used to increase performance in sports with high physical demands. The use of drugs such as anabolic steroids among athletes is a wellknown phenomenon, and the effects of these drugs on physical performance documented. The studies presented in this thesis focused on the mechanisms of muscle fiber hypertrophy in the vastus lateralis and the trapezius muscles of strength trained elite athletes. The main hypothesis was that the muscle adaptations to strength training and anabolic steroids are muscle specific. Biopsies were obtained from the trapezius and the vastus lateralis from three groups of elite power lifters. Nine used drugs, ten did not and seven had previously used drugs. Six sedentary males served as controls. The biopsies were frozen and cut in serial cross sections. Histological and immunohistochemical staining techniques were used to analyze muscle fiber morphology and pathology. Fiber type distribution, fiber area, myonuclei number and distribution, satellite cell number and proportion of split fibers were counted and compared for the two muscles within and between the groups. The main findings were that: a) Muscle fiber hypertrophy by strength training is further increased by anabolic steroids. b) The number of nuclei per muscle fiber is higher in power lifters using anabolic steroids compared to non-steroids using lifters. c) Among power lifters who have withdrawn from anabolic steroid usage and training for several years, the number of myonuclei, both subsarcolemmal and internal, remains high. d) In active power lifters, anabolic steroids have no further effect on the number of satellite cells per fiber. e) Power lifters have a high proportion of split fibers. High intensity resistance training increases muscle strength and banned substances such as testosterone and anabolic steroids can enhance the training effects. The studies on muscle cell morphology presented in this thesis reveals that anabolic steroids and testosterone increases muscle fiber size and adds more nuclei to the muscle cell. Based on the morphological appearance of muscle sections from doped and nondoped power lifters, we conclude that testosterone and anabolic steroids enhances the hypertrophic effects of training without adding new features. The addition of myonuclei by training and doping appears to be longer lasting in some muscles than in others. The high proportion of split fibers in power lifter is probably due to high mechanical stress. The findings and conclusions in this thesis raise questions regarding relevant suspension times for athletes caught with banned substances in the body.