Restless legs syndrome (RLS) and Obstructive sleep apnoea syndrome (OSAS) are two sleep disorders that affect daily life with symptoms such as sleepiness and fatigue. It was therefore hypothesised that the skeletal muscle could be affected as symptoms from skeletal muscle are common. The overall aim of the thesis was to investigate aerobic capacity and structure of skeletal muscle in patients with OSAS and RLS and an age matched control group to provide information regarding the underlying mechanisms. The specific aims were to investigate muscle fibre composition, capillary network, capillary proliferation and sings of local inflammation in musculus tibialis anterior of RLS and OSAS.OSAS and RLS patients had a significantly lower predicted VO2 max expressed in ml/min/kg compared with the control group and in the OSAS group apnoes-hyponea index (AHI) was inversely correlated to maximal oxygen uptake Fibre type composition and muscle fibre cross sectional area in the tibialis anterior muscle was equal in all groups with a predominant proportion of slow type I fibres and a smaller fibre area in slow type I fibres compared to fast type II fibres. The distribution of fast fibres (I/IIA, IIA) did not differ except for the group IIX and IIA/IIX where OSAS and RLS had a significantly higher percentage. OSAS patients had a significantly higher number of capillaries per fibre (CAF) for slow type I fibres and CAF per fibre area (CAFA) for fast type II fibres. CFPE- index (capillary to fibre perimeter exchange) and LC/PF-index (length of capillary/perimeter of fibre) were higher in both patient groups. Vascular endothelial growth factor (VEGF) and proliferating endothelial cells were analysed by double-immunofluorescence staining and were presented to a greater extent in the patient groups compared with the healthy controls. Based on normal amounts of T-cells and macrophages in the histological picture it was also demonstrated that local inflammation was not present in the tibialis anterior muscle of RLS and OSAS whish was also supported by the absence of expression of major histocompatibility complex class I molecules (MHC class I) on the surface of the tibialis anterior muscle fibres.In conclusion, the low predicted VO2 max together with higher percentage of type IIX and IIA/IIX muscle fibres indicates a low central capacity in the patient groups. The increased capillary network and the absence of inflammation indicate the occurrence of local hypoxia in tibialis anterior muscle in patients OSAS and RLS.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2019-03-29T16:56:55Z No. of bitstreams: 2 Camila_Kuhn2018.pdf: 2657327 bytes, checksum: 9eb518e6dcb5d576b52f1606ea47d2fa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2019-03-29T16:57:16Z (GMT). No. of bitstreams: 2 Camila_Kuhn2018.pdf: 2657327 bytes, checksum: 9eb518e6dcb5d576b52f1606ea47d2fa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-03 / Studies show that maternal obesity can affect fetal development, resulting in diseases in adult life, such as diabetes mellitus type 2, cardiovascular disease, obesity itslef. To reduce the effects of obesity and its comorbidities, bariatric surgeries stand out among the most effective interventions, with Roux-en-Y gastric bypass (RYGB) being the most frequently performed type of bariatric procedure. However, there are still few studies in the literature that evaluate the effects of obesity and bariatric surgeries on the morphology of skeletal muscle tissue in adult offspring. Thus, the objective of this study was to evaluate microscopic parameters of muscle fibers and neuromuscular junctions (NMJs) of the extensor digitorum longus muscle (EDL) in obese rats’ offspring submitted or not to RYGB surgery. Three-week Wistar rats were randomly divided into three groups: Control Group (CTL) which received a standard diet; 2) Cafeteria False Operated (CAF FO) and 3) Cafeteria RYGB (CAF RYGB), the latter two received a cafeteria diet before and after the surgical procedure until the weaning of the offspring. In the 18th of life, the surgical procedure and false operation were performed in the CAF RYGB and CAF FO groups, respectively. The mating of the animals occurred five weeks after the surgical procedure. The birth of the offspring was postnatal day 0, and weaning occurred at three weeks of age, and only the male offspring were separated for the experiment. The offspring of the first generation (F1) were named CTL-F1, OB-F1, CAF FO-F1 and CAF RYGB and received standard diet. At 17 weeks the animals were euthanized and the EDL muscle collected for analysis of fiber muscles and NMJs. When the CTL-F1 and OB-F1 groups were analyzed, the latter had an increase in body weight, retroperitoneal and periepididymal fats, and capillary/fiber ratio. Reduction in the number of nuclei, conjunctive and morphological changes in the parameters evaluated in the ultrastructure. The area and larger diameter of NMJs also showed reduction. The analysis between CAF RYGB-F1 and CAF FO-F1 groups showed reduction of body weight, ELD muscle weight, retroperitoneal and periepididimal fat, nasoanal length, fiber area and nuclei/fiber ratio in the CAF RYGB-F1 group. This group also presented increase in the number of fibers of type I and IIa and number of capillaries, as well as reduction in the area of the NMJs and morphological alterations in the ultrastructure. These results demonstrate that both obesity and bariatric surgery expose the offspring, through metabolic programming, to effects on the morphology of skeletal muscle tissue, being found greater aggravations in the muscular fiber of the offspring of mothers submitted to RYGB. / Estudos apontam que a obesidade materna pode afetar o desenvolvimento fetal, resultando em doenças na vida adulta, tais como diabetes mellitus tipo 2, doenças cardiovasculares e a própria obesidade. Para reduzir os efeitos da obesidade e as suas comorbidades, as cirurgias bariátricas destacam-se entre as intervenções mais eficazes, sendo a derivação gástrica em Y de Roux (DGYR) o tipo de procedimento bariátrico mais frequentemente realizado. No entanto, ainda são escassos na literatura estudos que avaliem os efeitos da obesidade e das cirurgias bariátricas na morfologia do tecido muscular esquelético da prole adulta. Diante disso, o objetivo deste estudo foi avaliar a morfologia e a morfometria das fibras musculares e as junções neuromusculares (JNMs) do músculo extensor longo dos dedos (ELD) da prole de ratas obesas submetidas ou não à cirurgia de DGYR. Para tanto, ratas Wistar de três semanas de vida foram separadas aleatoriamente em três grupos: 1) Controle (CTL), que recebeu dieta padrão; 2) Cafeteria Falso operado (CAF FO) e 3) Cafeteria DGYR (CAF DGYR); esses dois últimos receberam dieta de cafeteria antes e após o procedimento cirúrgico, até o desmame da prole. Na 18ª semana de vida, foi realizado o procedimento cirúrgico e a falsa operação nos grupos CAF DGYR e CAF FO, respectivamente. O cruzamento dos animais ocorreu cinco semanas após o procedimento cirúrgico. O nascimento dos animais foi considerado o dia zero pós-natal e o desmame se deu na terceira semana vida, quando somente os machos foram separados para o experimento. A prole da primeira geração (F1) foi nomeada em CTL-F1, OB-F1, CAF FO-F1 e CAF DGYR-F1 e todos os animais receberam dieta padrão. Na 17ª semana, os animais foram eutanasiados e o músculo ELD coletado para análise das fibras musculares e JNMs. Quando analisado os grupos CTL-F1 e OB-F1, esse último apresentou aumento do peso corpóreo, das gorduras retroperitoneal e periepididimal, e relação capilar/fibra. Além disso, houve a redução do número de núcleos, conjuntivo e alterações morfológica nos parâmetros avaliados na ultraestrutura. A área e diâmetro maior das JNMs também apresentaram redução. A análise entre os grupos CAF DGYR-F1 e CAF FO-F1 evidenciou redução do peso corporal, do peso do músculo ELD, da gordura retroperitoneal e periepididimal, don comprimento nasoanal, da área das fibras e relação núcleo/fibra no grupo CAF DGYR-F1. Esse grupo também apresentou aumento no número de fibras do tipo I e IIa e no número de capilares, assim como redução na área das JNMs e alterações morfológicas na ultraestrutura. Esses resultados demonstram que tanto a obesidade como a cirurgia bariátrica expõem a prole, por meio da programação metabólica, com efeitos na morfologia do tecido muscular esquelético, sendo encontrado maiores agravos na fibra muscular da prole de mães submetidas à DGYR.

Introdução. A insuficiência cardíaca (IC) é caracterizada por exacerbação da atividade nervosa simpática muscular (ANSM), baixa tolerância ao esforço e dispneia. Além disso, é característico nessa população o desequilíbrio entre o anabolismo e catabolismo, favorecendo dessa maneira uma acentuada perda da massa magra muscular, o que agrava ainda mais a qualidade de vida nos pacientes com IC. Dentre as alterações anabólicas observadas na IC avançada destaca-se a diminuição dos hormônios GH, IGF-1 e testosterona. A testosterona, um importante hormônio para as características masculinizantes e na manutenção da massa muscular, apresenta acentuada redução com o avançar da doença. Esta perda da massa magra, leva ao processo de caquexia muscular e consequente atrofia, com diminuição da força e da capacidade funcional do paciente com IC. A reposição de testosterona nesses pacientes tem sido estudada e se mostra uma importante terapêutica para melhorar a capacidade funcional e força muscular. Porém, não se conhece claramente o papel deste tratamento medicamentoso sobre o processo anabólico muscular, bem como na melhora da composição corporal. O exercício físico como tratamento não medicamentoso tem sido amplamente recomendado na IC por reduzir a ANSM, melhorar o fluxo sanguíneo periférico, aumentar a força muscular e melhorar a qualidade de vida. Entretanto, a combinação das estratégias do exercício físico associado à terapia de reposição de testosterona, não é conhecido em pacientes com IC. Métodos. 24 pacientes com IC foram randomizados em 3 grupos: Treinamento (TR, n=9), Testosterona (T, n=8) e Treino+Testosterona (TRT, n=7). A ANSM foi avaliada pela técnica de Microneurografia. O fluxo sanguíneo do antebraço foi avaliado pela pletismografia de oclusão venosa. A composição corporal foi avaliada pela densitometria (DEXA). A biópsia do músculo vasto-lateral foi feita para avaliarmos a área de secção transversa da fibra e a tipagem de fibras musculares. A qualidade de vida foi avaliada pelo questionário de Minnesota. O treinamento físico aeróbio em bicicleta foi realizado 3 vezes por semana, com 40 minutos de exercício por sessão, pelo período de 4 meses. A reposição de testosterona foi realizada pela administração intramuscular de undecilato de testosterona pelo período de 4 meses. Resultados. Após 4 meses de intervenção, observamos restauração dos níveis de testosterona em todos os grupos. A ANSM reduziu nos grupos TR e TRT. Não houve aumento do fluxo sanguíneo entre os grupos. O consumo de oxigênio aumentou em todos os grupos, porém apenas o grupo TRT aumentou a potência máxima ao exercício. A massa magra apresentou aumento significativo apenas no grupo TRT. Não observamos mudança no conteúdo mineral ósseo entre os grupos. Apenas o grupo TRT aumentou de maneira significativa a área de secção transversa das fibras tipo I (oxidativas). A qualidade de vida melhorou apenas nos grupos TR e TRT. Conclusões. O exercício físico associado à terapia de reposição de testosterona se mostrou mais eficaz em reduzir a ANSM, aumentar a capacidade funcional, a força muscular, a massa magra com um importante aumento das fibras do tipo I. Nossos resultados enfatizam a importância do exercício físico em pacientes com IC e traz uma nova perspectiva com a associação da testosterona para pacientes com hipogonadismo / Introduction. Heart failure (HF) is characterized by exacerbation of muscle sympathetic nerve activity (MSNA), exercise intolerance and dyspnea. Furthermore, is characteristic in this population the imbalance between anabolism and catabolism which lead to loss of skeletal muscle mass worsening quality of life in HF patients. Prior studies have demonstrated decrease in anabolic hormones such as GH, IGF-1 and testosterone. Testosterone, an important hormone for masculinization feature and maintenance of muscle mass, shows sharp decline in advanced HF. Loss muscle mass leads to cachexia and atrophy which decrease strength and functional capacity in HF patients. Testosterone replacement in these patients has been studied and shows an important therapeutic to enhance functional capacity and muscle strength. However it is not known the role of this medical treatment on muscle anabolic process as well as on body composition. Physical exercise as a non-medication treatment has been widely recommended to reduce MSNA, enhance peripheral blood flow, increase muscle strength and improve quality of life. However, the combination of the strategies of physical exercise associated with testosterone replacement therapy is not known in HF patients. Methods. 24 HF patients were randomized in 3 groups: Training (TR, n=9), Testosterone (T, n=8) and Training+Testosterone (TRT, n=7). MSNA was recorded by microneurography technic. Forearm blood flow was evaluated by venous occlusion plethysmography. Body composition was measured by densitometry (DEXA). Muscle biopsy was done in vastus lateralis to evaluate the cross-sectional area and type of fibers. Quality of life was assessed by Minnesota living with heart failure questionnaire. Aerobic exercise training on a bicycle was performed 3 times per week, with 40 minutes of exercise per session, for a period of 4 months. Testosterone replacement was performed by intramuscular administration of testosterone undecylate for a period of 4 months. Results. After 4 months testosterone levels were restored in all groups. MSNA decreased in TR and TRT groups. There was no increase in blood flow between groups. Oxygen consumption increased in all groups, but only the TRT group showed increase in maximum power to exercise. Lean body mass increased significantly only in the TRT group. We did not observe changes in bone mineral content between groups. Only TRT group significantly increased the cross-sectional area of type I fibers (oxidative). The quality of life improved only in TR and TRT groups. Conclusions. Exercise training associated with testosterone replacement therapy was more effective in reducing MSNA, increase functional capacity, muscle strength, lean mass with a significant increase in type I fibers. Our results emphasize the importance of physical exercise in patients with HF and bring a new perspective to association testosterone for patients with hypogonadism

Introdução. A insuficiência cardíaca (IC) é caracterizada por exacerbação da atividade nervosa simpática muscular (ANSM), baixa tolerância ao esforço e dispneia. Além disso, é característico nessa população o desequilíbrio entre o anabolismo e catabolismo, favorecendo dessa maneira uma acentuada perda da massa magra muscular, o que agrava ainda mais a qualidade de vida nos pacientes com IC. Dentre as alterações anabólicas observadas na IC avançada destaca-se a diminuição dos hormônios GH, IGF-1 e testosterona. A testosterona, um importante hormônio para as características masculinizantes e na manutenção da massa muscular, apresenta acentuada redução com o avançar da doença. Esta perda da massa magra, leva ao processo de caquexia muscular e consequente atrofia, com diminuição da força e da capacidade funcional do paciente com IC. A reposição de testosterona nesses pacientes tem sido estudada e se mostra uma importante terapêutica para melhorar a capacidade funcional e força muscular. Porém, não se conhece claramente o papel deste tratamento medicamentoso sobre o processo anabólico muscular, bem como na melhora da composição corporal. O exercício físico como tratamento não medicamentoso tem sido amplamente recomendado na IC por reduzir a ANSM, melhorar o fluxo sanguíneo periférico, aumentar a força muscular e melhorar a qualidade de vida. Entretanto, a combinação das estratégias do exercício físico associado à terapia de reposição de testosterona, não é conhecido em pacientes com IC. Métodos. 24 pacientes com IC foram randomizados em 3 grupos: Treinamento (TR, n=9), Testosterona (T, n=8) e Treino+Testosterona (TRT, n=7). A ANSM foi avaliada pela técnica de Microneurografia. O fluxo sanguíneo do antebraço foi avaliado pela pletismografia de oclusão venosa. A composição corporal foi avaliada pela densitometria (DEXA). A biópsia do músculo vasto-lateral foi feita para avaliarmos a área de secção transversa da fibra e a tipagem de fibras musculares. A qualidade de vida foi avaliada pelo questionário de Minnesota. O treinamento físico aeróbio em bicicleta foi realizado 3 vezes por semana, com 40 minutos de exercício por sessão, pelo período de 4 meses. A reposição de testosterona foi realizada pela administração intramuscular de undecilato de testosterona pelo período de 4 meses. Resultados. Após 4 meses de intervenção, observamos restauração dos níveis de testosterona em todos os grupos. A ANSM reduziu nos grupos TR e TRT. Não houve aumento do fluxo sanguíneo entre os grupos. O consumo de oxigênio aumentou em todos os grupos, porém apenas o grupo TRT aumentou a potência máxima ao exercício. A massa magra apresentou aumento significativo apenas no grupo TRT. Não observamos mudança no conteúdo mineral ósseo entre os grupos. Apenas o grupo TRT aumentou de maneira significativa a área de secção transversa das fibras tipo I (oxidativas). A qualidade de vida melhorou apenas nos grupos TR e TRT. Conclusões. O exercício físico associado à terapia de reposição de testosterona se mostrou mais eficaz em reduzir a ANSM, aumentar a capacidade funcional, a força muscular, a massa magra com um importante aumento das fibras do tipo I. Nossos resultados enfatizam a importância do exercício físico em pacientes com IC e traz uma nova perspectiva com a associação da testosterona para pacientes com hipogonadismo / Introduction. Heart failure (HF) is characterized by exacerbation of muscle sympathetic nerve activity (MSNA), exercise intolerance and dyspnea. Furthermore, is characteristic in this population the imbalance between anabolism and catabolism which lead to loss of skeletal muscle mass worsening quality of life in HF patients. Prior studies have demonstrated decrease in anabolic hormones such as GH, IGF-1 and testosterone. Testosterone, an important hormone for masculinization feature and maintenance of muscle mass, shows sharp decline in advanced HF. Loss muscle mass leads to cachexia and atrophy which decrease strength and functional capacity in HF patients. Testosterone replacement in these patients has been studied and shows an important therapeutic to enhance functional capacity and muscle strength. However it is not known the role of this medical treatment on muscle anabolic process as well as on body composition. Physical exercise as a non-medication treatment has been widely recommended to reduce MSNA, enhance peripheral blood flow, increase muscle strength and improve quality of life. However, the combination of the strategies of physical exercise associated with testosterone replacement therapy is not known in HF patients. Methods. 24 HF patients were randomized in 3 groups: Training (TR, n=9), Testosterone (T, n=8) and Training+Testosterone (TRT, n=7). MSNA was recorded by microneurography technic. Forearm blood flow was evaluated by venous occlusion plethysmography. Body composition was measured by densitometry (DEXA). Muscle biopsy was done in vastus lateralis to evaluate the cross-sectional area and type of fibers. Quality of life was assessed by Minnesota living with heart failure questionnaire. Aerobic exercise training on a bicycle was performed 3 times per week, with 40 minutes of exercise per session, for a period of 4 months. Testosterone replacement was performed by intramuscular administration of testosterone undecylate for a period of 4 months. Results. After 4 months testosterone levels were restored in all groups. MSNA decreased in TR and TRT groups. There was no increase in blood flow between groups. Oxygen consumption increased in all groups, but only the TRT group showed increase in maximum power to exercise. Lean body mass increased significantly only in the TRT group. We did not observe changes in bone mineral content between groups. Only TRT group significantly increased the cross-sectional area of type I fibers (oxidative). The quality of life improved only in TR and TRT groups. Conclusions. Exercise training associated with testosterone replacement therapy was more effective in reducing MSNA, increase functional capacity, muscle strength, lean mass with a significant increase in type I fibers. Our results emphasize the importance of physical exercise in patients with HF and bring a new perspective to association testosterone for patients with hypogonadism

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.