Skeletal muscle is a plastic tissue capable of responding to environmental perturbations. Increased loading via resistance exercise (RE) activates muscle protein synthesis (MPS) and, to a lesser extent, muscle protein breakdown (MPB). The ingestion of protein further stimulates MPS and suppresses MPB, inducing a positive net protein balance and protein accretion – i.e., muscle hypertrophy. In contrast, muscle unloading reduces MPS, which is thought to be the key driver underpinning skeletal muscle atrophy. The degree of muscle hypertrophy and atrophy in response to loading and unloading varies significantly between individuals and provides an opportunity to investigate the molecular regulators of skeletal muscle remodelling. To that end, we developed a novel unilateral model in which one leg was subjected to RE to induce hypertrophy (Hyp) and the contralateral limb was immobilized to induce atrophy (At). In study 1, we characterized the morphological changes induced by our HypAt model and validated the use of ultrasonography to measure changes in muscle size in both limbs. We discovered that by assessing the differential change in muscle size between legs we reduced the coefficient of variation between subjects. This enabled a more in-depth means-based characterization of the molecular regulators of skeletal muscle remodelling. Indeed, we discovered significantly more genes regulated by muscle remodelling than similarly-sized studies. We also identified a transcriptional signature that scaled with lean mass gains in three independent cohorts and included RNA species that were only modulated at their untranslated regions. Finally, in study 3 we simultaneously measured MPS and MPB in response to short-term immobilization (4 days) and demonstrated for the first time that MPB is statistically unchanged by unloading. Taken together, these studies contribute significantly to our understanding of skeletal muscle remodelling under different loading states and provide a valuable hypothesis-generating resource for future research in the field. / Thesis / Doctor of Philosophy (PhD) / Adaptations of skeletal muscle to loading and unloading are variable between individuals. Herein, we employed a unilateral approach to better understand the drivers of this variability by assessing the influence of resistance training (RT) and disuse on muscle protein turnover and gene expression. First, we validated the use of ultrasound for measuring changes in muscle size in response to loading and unloading. We then identified thousands of genes regulated by loading status and discovered many that were correlated with lean mass gain – some of which would not have been detected without our model. We also demonstrated that RT-induced increases in muscle protein synthesis were not associated with changes in muscle size; however, reductions in muscle protein synthesis were associated with the degree of muscle atrophy observed in response to disuse. Together, these studies contribute significantly to our understanding of how skeletal muscle size is regulated by muscle loading and unloading.

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<p><strong>Introduction:</strong> Mitochondria are important organelles for skeletal muscle function. Mitochondria are susceptible to many forms of stress that alters their morphology, energy generation and reactive oxygen species (ROS) production, which collectively promote degeneration and dysfunction in skeletal muscle. These processes are implicated in many health disorders, particularly in the aging process itself. Exercise is well established to increase muscle mitochondrial content and thus may attenuate several aspects of mitochondrial deterioration. <strong>Methods:</strong> Both human and animal models of mitochondrial stress (aging, ROS) were utilized in order to determine their effects on mitochondrial and muscle function. Additionally, exercise training was used in order to assess its therapeutic potential in ameliorating defects in oxidative capacity, muscle atrophy and metabolic adaptation in skeletal muscle. <strong>Results:</strong> Aging resulted in reduced strength, aerobic capacity, larger intramyocelluar lipid droplets and fewer mitochondria in skeletal muscle. These changes were related to suppressed lipid metabolism, mitochondrial dynamics and organelle turnover. Habitual aerobic exercise partially attenuated the age-related loss of muscle strength and aerobic capacity, presumably due in part to improved mitochondrial function. Persistent mitochondrial oxidative stress prevented mitochondrial adaptations to exercise training in mice, a phenomenon that may explain why exercise cannot fully counteract the effects of aging in skeletal muscle. <strong>Conclusions and significance:</strong> This work furthers our knowledge of the mitochondrial consequences of aging and the therapeutic potential of aerobic exercise within skeletal muscle. These results can be applied to other differentiated tissues that are severely affected by aging (brain, heart) and the effects described here are likely relevant to other conditions that result in muscle atrophy and energetic insufficiency.</p> / Doctor of Philosophy (PhD)

Yes / Background: Understanding and countering the well-established negative health consequences of spaceflight remains a primary challenge preventing safe deep space exploration. Targeted/personalized therapeutics are at the forefront of space medicine strategies, and cross-species molecular signatures now define the 'typical' spaceflight response. However, a lack of direct genotype-phenotype associations currently limits the robustness and, therefore, the therapeutic utility of putative mechanisms underpinning pathological changes in flight. Methods: We employed the worm Caenorhabditis elegans as a validated model of space biology, combined with 'NemaFlex-S' microfluidic devices for assessing animal strength production as one of the most reproducible physiological responses to spaceflight. Wild-type and dys-1 (BZ33) strains (a Duchenne muscular dystrophy (DMD) model for comparing predisposed muscle weak animals) were cultured on the International Space Station in chemically defined media before loading second-generation gravid adults into NemaFlex-S devices to assess individual animal strength. These same cultures were then frozen on orbit before returning to Earth for next-generation sequencing transcriptomic analysis. Results: Neuromuscular strength was lower in flight versus ground controls (16.6% decline, p

A amiotrofia espinal (AEP) é a mais freqüente das doenças neuromusculares (DNM) responsáveis pela Síndrome da Criança Hipotônica (SCH), causada pela degeneração das células do corno anterior da medula espinhal. O comprometimento neurológico se estabelece de forma rápida e intensa nos primeiros meses de vida de tal maneira que, em pouco tempo, a única atividade motora voluntária persistente pode ser a motricidade ocular. O Ácido Valpróico é um medicamento de fácil aquisição e administração, demonstrou-se eficaz em evitar a deleção do exon 7, o que prolongaria ainda mais a sobrevivência dos neurônios. Com base nesta nova perspectiva terapêutica para esta drástica doença e por não existir nenhuma forma de avaliação motora validada e de fácil reprodutibilidade, foi desenvolvido um protocolo de avaliação física-funcional para acompanhamento deste grupo de pacientes e de outros com a SCH, com o objetivo de avaliar quantitativamente e qualitativamente a evolução de aquisição motora nos pacientes, selecionando provas funcionais e criando uma nova forma de avaliação utilizando o recurso da filmagem para posterior examinação, de fácil aplicação em consultório, que permitam uniformizar a interpretação dos resultados em diferentes pesquisas ou individualmente ao longo de um acompanhamento clínico. Cinco pacientes com AEP-I, com idades variando de 04 a 18 meses, sem a necessidade de auxílio respiratórios, e submetidos à intervenção medicamentosa com o Ácido Valpróico, foram avaliados em 05 visitas, totalizando um acompanhamento de 2 meses e meio. A análise das aquisições motoras obedeceu a um roteiro de movimentação utilizando a filmagem e posteriormente atribuindo uma pontuação. Os resultados demonstraram que todas as crianças participantes do estudo não apresentaram piora do quadro motor, o que seria esperado pela evolução natural da doença. Consideramos assim que: os pacientes submetidos à intervenção medicamentosa com o Ácido Valpróico apresentaram uma tendência à melhora das aquisições motoras, em relação ao descrito na evolução natural da doença e a utilização da filmagem como método de análise de aquisição motora se mostrou ser um recurso eficaz no acompanhamento da evolução motora dos pacientes. / The spinal muscle atrophy (SMA) is the most frequent neuromuscular disease responsible for Hypotonic Children Syndrome (HCS). SMA is caused by anterior horn cells degeneration in the narrow. The neurological impairment is quickly and strongly established in the first months of life, so, after few months, the only movement that the children can do is moving the eyes. The Valproic acid is a drug easy to buy and to manage, and seems to avoid exon 7 deletion, which could prolong the neurons life. By this new therapeutic knowledge, and for don\'t exist a validate and easy to reproduce way to evaluate the motor function, there were made a functional evaluation protocol to follow the group of patients with SMA and other patients with HCS, with the purpose to qualify and to quantify the motor gain in these patients, creating a new form to evaluate, using film to exam in the end. It\'s easy to apply in the office and can became the only way to evaluate the results in different trials. There were evaluate five patients with SMA type 1, ages from 04 to 18 months, without respiratory assistance use, underwent to Valproic acid intervention. They were evaluated in five visits, in a total 2 years and a half of following. The motor acquisitions were made from the film and scores. The results showed that no one off al children presented a motor loss, which would be expected for the natural evolution of SMA type 1. Consequently, the patients that underwent to valproic acid trial presented a tendency to gain motor abilities when compared to the natural evolution of the disease and the film as an analyze method seem to be an efficient way to follow the motor evolution of these patients.

A amiotrofia espinal (AEP) é a mais freqüente das doenças neuromusculares (DNM) responsáveis pela Síndrome da Criança Hipotônica (SCH), causada pela degeneração das células do corno anterior da medula espinhal. O comprometimento neurológico se estabelece de forma rápida e intensa nos primeiros meses de vida de tal maneira que, em pouco tempo, a única atividade motora voluntária persistente pode ser a motricidade ocular. O Ácido Valpróico é um medicamento de fácil aquisição e administração, demonstrou-se eficaz em evitar a deleção do exon 7, o que prolongaria ainda mais a sobrevivência dos neurônios. Com base nesta nova perspectiva terapêutica para esta drástica doença e por não existir nenhuma forma de avaliação motora validada e de fácil reprodutibilidade, foi desenvolvido um protocolo de avaliação física-funcional para acompanhamento deste grupo de pacientes e de outros com a SCH, com o objetivo de avaliar quantitativamente e qualitativamente a evolução de aquisição motora nos pacientes, selecionando provas funcionais e criando uma nova forma de avaliação utilizando o recurso da filmagem para posterior examinação, de fácil aplicação em consultório, que permitam uniformizar a interpretação dos resultados em diferentes pesquisas ou individualmente ao longo de um acompanhamento clínico. Cinco pacientes com AEP-I, com idades variando de 04 a 18 meses, sem a necessidade de auxílio respiratórios, e submetidos à intervenção medicamentosa com o Ácido Valpróico, foram avaliados em 05 visitas, totalizando um acompanhamento de 2 meses e meio. A análise das aquisições motoras obedeceu a um roteiro de movimentação utilizando a filmagem e posteriormente atribuindo uma pontuação. Os resultados demonstraram que todas as crianças participantes do estudo não apresentaram piora do quadro motor, o que seria esperado pela evolução natural da doença. Consideramos assim que: os pacientes submetidos à intervenção medicamentosa com o Ácido Valpróico apresentaram uma tendência à melhora das aquisições motoras, em relação ao descrito na evolução natural da doença e a utilização da filmagem como método de análise de aquisição motora se mostrou ser um recurso eficaz no acompanhamento da evolução motora dos pacientes. / The spinal muscle atrophy (SMA) is the most frequent neuromuscular disease responsible for Hypotonic Children Syndrome (HCS). SMA is caused by anterior horn cells degeneration in the narrow. The neurological impairment is quickly and strongly established in the first months of life, so, after few months, the only movement that the children can do is moving the eyes. The Valproic acid is a drug easy to buy and to manage, and seems to avoid exon 7 deletion, which could prolong the neurons life. By this new therapeutic knowledge, and for don\'t exist a validate and easy to reproduce way to evaluate the motor function, there were made a functional evaluation protocol to follow the group of patients with SMA and other patients with HCS, with the purpose to qualify and to quantify the motor gain in these patients, creating a new form to evaluate, using film to exam in the end. It\'s easy to apply in the office and can became the only way to evaluate the results in different trials. There were evaluate five patients with SMA type 1, ages from 04 to 18 months, without respiratory assistance use, underwent to Valproic acid intervention. They were evaluated in five visits, in a total 2 years and a half of following. The motor acquisitions were made from the film and scores. The results showed that no one off al children presented a motor loss, which would be expected for the natural evolution of SMA type 1. Consequently, the patients that underwent to valproic acid trial presented a tendency to gain motor abilities when compared to the natural evolution of the disease and the film as an analyze method seem to be an efficient way to follow the motor evolution of these patients.

Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models.

A miopatia esquelética em doenças sistêmicas é um importante preditor de mortalidade e prognóstico em diversas síndromes, incluindo a insuficiência cardíaca. Os danos músculo-esqueléticos em situações de comprometimento cardíaco são descritos pela literatura há décadas, entretanto, nenhum recurso farmacológico proposto até o momento mostrou-se eficiente em reverter esses prejuízos, ressaltando o papel do treinamento físico aeróbio. Apesar dos inegáveis benefícios desta terapia adjuvante no tratamento da insuficiência cardíaca, muito pouco se sabe sobre a intensidade de exercício capaz de otimizar os ganhos promovidos por esta intervenção. Dado isso, nesse estudo avaliamos a eficácia do treinamento físico aeróbio em alta intensidade na musculatura esquelética em ratos submetidos ao infarto do miocárdio, comparando-a com protocolo isocalórico realizado em intensidade moderada. Observamos que os animais infartados apresentaram alterações patológicas na musculatura esquelética, similarmente ao observado em pacientes com IC, como prejuízos em enzimas metabólicas fundamentais, atrofia muscular, perturbação da homeostase redox e ativação do complexo proteassomal 26S. Ambos os protocolos de treinamento físico aeróbio foram capazes de aprimorar substancialmente a capacidade funcional e potência aeróbia máxima nos animais infartados, prevenindo a queda da atividade máxima das enzimas hexoquinase e citrato sintase, restaurando a morfologia da musculatura esquelética e aumentando a distribuição de fibras musculares do tipo I, o que foi acompanhado por melhora do balanço redox e redução da atividade do complexo proteassomal 26S. Apesar do treinamento físico aeróbio em alta intensidade ter proporcionado resultados superiores ao protocolo de intensidade moderada em relação a capacidade funcional dos animais, as adaptações músculo-esqueléticas às diferentes intensidades de TFA apresentaram-se muito semelhantes / Impaired skeletal muscle performance in systemic diseases is shown to strongly predict mortality and long-term prognosis in a wide variety of syndromes, including heart failure. The clinical picture of skeletal muscle damage in cardiac situations has been described for decades. However, no pharmacological strategy proposed so far was shown to effectively prevent the onset of skeletal myopathy, reinforcing the role of aerobic exercise training in counteracting such phenomenon. Despite the well-known benefits of exercise training in sets of cardiac dysfunction, very little is known about the optimal exercise intensity to elicit maximal outcomes. Therefore, in the present study we compared the effects of high-intensity aerobic exercise training with those of an isocaloric moderate-intensity protocol on skeletal muscle adaptations in infarcted rats. Our data suggest that infarcted rats presented signs of skeletal myopathy resembling those observed in HF patients, such as metabolic enzymes impairment, skeletal muscle atrophy, disrupted redox balance and proteasomal overactivation. Here we show that both high- and moderate-intensity aerobic exercise training were able to substantially increase aerobic capacity in infarcted rats, preventing the decay of citrate synthase and hexokinase maximal activities, reestablishing normal skeletal muscle morphology to a healthy profile and increasing the number of type I muscle fibers. Such outcomes were accompanied by an improved redox balance and reduced proteasomal activity in skeletal muscle. Even though high-intensity aerobic interval training was superior to moderate-intensity in improving functional capacity, the observed adaptations in skeletal muscle were remarkably similar between the protocols. Therefore, our data allow us to conclude that high-intensity and moderate-intensity aerobic exercise training equally prevent skeletal myopathy induced by myocardial infarction in rats

Neste estudo analisamos o envolvimento de E3 ligases sob três aspectos da plasticidade muscular esquelética: a perda de massa decorrente do desuso, a manutenção de fibras tipo I e II e a regeneração do tecido muscular. O primeiro objetivo foi determinar se leucina atenuaria a perda de massa provocada pela atrofia. Nossos dados mostraram que este aminoácido evitou a perda de massa, sobretudo através da inibição da expressão de E3 ligases. A seguir, analisamos se a ausência de duas E3 ligases, MuRF1 e MuRF2, poderia alterar a proporção de miofibras tipo I e tipo II. Detectamos que na ausência destas E3 ligases, a identidade de fibras tipo II era perdida, além destas fibras estarem protegidas da atrofia sem MuRF1. Por fim, analisamos o papel de MuRF1 e MuRF2 durante a regeneração. Os resultados mostraram que estas E3 ligases em conjunto são cruciais para a fisiologia das células-satélites e portanto para a regeneração do tecido. Esta tese mostrou que determinadas E3 ligases exercem um papel crucial para a plasticidade muscular. / In the present thesis we analyzed the involvement of E3 ligases under three aspects of skeletal muscle plasticity: mass loss resulting from disuse, maintenance of fiber type I and II and regeneration of muscle tissue. Our first aim was to determine whether leucine, was able to attenuate the mass loss caused by wasting. Our results showed that this amino acid prevented the mass loss, mainly by inhibiting the expression of E3 ligases. The second aim was to determine whether the absence of two E3 ligases, MuRF1 and MuRF2, could alter the proportion of type I and type II fibers. We found that in the absence of these E3 ligases, the identity of type II fibers was lost, and these fibers were protected against atrophy in the absence of MuRF1. Lastly, we analyze the role of MuRF1 and MuRF2 in muscle tissue regeneration. The results showed that these E3 ligases together are crucial to satellite-cells physiology and consequently an adequate tissue regeneration. This thesis show that certain E3 ligases can play a crucial role in muscle plasticity.