Protein arginine methyltransferase 1 (PRMT1), -4 (also known as coactivator-associated arginine methyltransferase 1; CARM1), and -5 catalyze the methylation of arginine residues on target proteins. In turn, these marked proteins mediate a variety of biological functions. By regulating molecules that are critical to the remodelling of skeletal muscle phenotype, PRMTs may influence skeletal muscle plasticity. Our study tests the hypothesis that the intracellular signals required for muscle adaptation to exercise will be associated with the induction of PRMT expression and activity. C57BL/6 mice were assigned to one of three experimental groups: sedentary (SED), acute bout of exercise (0PE), or acute exercise followed by 3 hours of recovery (3PE). The mice in the exercise groups performed a single bout of treadmill running at 15 m/min for 90 minutes. We observed that PRMT gene expression and global enzyme activity are muscle- specific, generally being higher in slow, oxidative muscle, as compared to faster, more glycolytic tissue. Despite the activation of canonical exercise-induced signalling involving AMPK and PGC-1α, PRMT expression and activity at the whole muscle level were unchanged. However, subcellular analysis revealed the exercise-evoked myonuclear translocation of PRMT1 prior to the nuclear translocation of PGC-1α, which colocalizes the proteins within the organelle after exercise. Acute physical activity also augmented the targeted methyltransferase activities of CARM1, PRMT1, and -5 in the myonuclear compartment, suggesting that PRMT-mediated histone arginine methylation is an integral part of the early signals that drive skeletal muscle plasticity. In summary, our data supports the emergence of PRMTs as important players in the regulation of skeletal muscle plasticity. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that can adapt to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important in the regulation of skeletal muscle remodeling. However, their role in exercise-induced skeletal muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the association between the intracellular signals required for muscle adaption and various metrics of PRMT biology. Our data demonstrate that PRMTs exhibit muscle-specific expression and function in mice. The movement of PRMT1 into myonuclei increased following exercise, while the specific methylation status of PRMT targets were also elevated. Overall, our data suggests that muscle-specific PRMT expression may be important for the determination and/or maintenance of different fiber type characteristics. Moreover, distinct PRMT cellular localization and methyltransferase activity may be key signals that contribute to skeletal muscle phenotypic plasticity.

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.

In the present study Fourier Transform Infrared Spectroscopy was used to examine the effects of streptozotocin-induced diabetes mellitus on the structural components of slow- and fast-twitch rat skeletal muscles, at molecular level. Diabetes mellitus is a chronic disorder of carbohydrate, fat and protein metabolism, which is characterized by hyperglycemia caused by a defective or deficient insulin secretory response. The effect of diabetes is seen on a variety of tissues leading to important secondary complications such as kidney failure, liver dysfunction, cardiac disorders, etc. Skeletal muscle is one of the major tissues determining carbohydrate and lipid metabolism in the body / therefore, is one of the target tissues of diabetes. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. The FTIR spectra revealed a considerable decrease in lipid and protein content of diabetic skeletal muscles, indicating an increased lipolysis and protein breakdown or decreased protein synthesis. Moreover changes in protein structure and conformation were observed. In diabetes, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. Diabetes caused a decrease in the content of nucleic acids, especially RNA, and hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated slow-twitch muscle was more severely affected from diabetes. Thus, FTIR spectroscopy appears to be a useful method to evaluate the effect of diabetes on skeletal muscle tissues at molecular level.

Orientador: Denise Vaz de Macedo / Tese (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T02:09:44Z (GMT). No. of bitstreams: 1 Stancanelli_Mirtes_M.pdf: 527727 bytes, checksum: 8ebada11e8ef5688039bd52cf92a6a14 (MD5) Previous issue date: 2006 / Resumo: Os objetivos desse trabalho foram: i) analisar as concentrações plasmáticas de uréia e creatina quinase (CK) de jogadores de futebol que consumiram caldo de cana imediatamente pós-treinos ou jogos durante todo o Campeonato Brasileiro ii) analisar o efeito da gavagem estomacal com caldo de cana em ratos submetidos a um protocolo de exercício exaustivo agudo na reposição dos estoques de glicogênio muscular e hepático. A sessão de exercício agudo consistiu em natação com carga de 10% do peso corporal até a exaustão após jejum de 30 horas. Imediatamente após o exercício, os animais suplementados receberam água ou caldo de cana por gavagem estomacal. Os dados com os jogadores mostraram uma concentração plasmática de uréia, aliada a uma concentração estável da CK plasmática ao longo de todo o campeonato, sugerindo uma possível ação ergogênica do caldo de cana. O exercício agudo diminuiu significativamente os estoques de glicogênio somente nos músculos gastrocnêmio branco e vermelho dos ratos. A suplementação com caldo de cana (0,7g/kg de peso corporal) aumentou significativamente os estoques de glicogênio desses músculos, sem alterar a concentração de glicogênio hepático. Os ratos suplementados também exibiram maior resistência muscular pós-exercício. Esses dados comprovam o efeito ergogênico do caldo de cana na reposição dos estoques de glicogênio muscular. Entre outras vantagens, o caldo de cana é um produto de fácil obtenção e principalmente de baixo custo, ideal para as condições socio-econômicas dos atletas nacionais / Abstract: The goal of this work were i) monitoring soccer players (n=29) submitted to sugar cane juice consumption immediately after training or games through plasma urea and CK analyses along the Brazilian Championship, and ii) investigate the effect of sugar cane juice ingestion in the restoration of muscular and liver glycogen stores after high intense exercise in rats. The rats swam with 10% body weight load until exhaustion after 30 hours fasting. Immediately after exercise the supplemented animals got water or sugar cane juice from stomach gavages. We have used 56 male Wistar rats weighting from 280 to 310 g, which were divided in 7 groups; control (C); fasting (F); fasting and exercise (FE); water supplemented (WS); and sugar cane juice supplemented (SCS). Liver, white and red gastrocnemius and soleus muscles samples were removed after 40 and 60 min to glycogen concentration determinations. The players exhibited a decrease in plasma urea together with stable levels of plasma CK activity along all the championship, suggesting a recovery action. The exhaustive exercise decreased significantly only the red and white gastrocnemius muscle glycogen stores. Sugar cane juice supplementation (0,7 g/kg body weight) increased significantly glycogen stores of these muscles, but practically didn¿t change liver glycogen concentration after 1 h. These data strongly suggest that sugar cane juice is efficient to maximize the rate of glycogen storage in the early hour¿s post- exercise / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.

Com o envelhecimento observa-se redução da massa muscular, aumento da resistência à insulina e queda nos níveis séricos de alguns hormônios, entre eles o DHEA. Ratos com 12-14 meses de idade apresentam regulação músculo-específica em vias da insulina e IGF 1 em músculos com populações distintas de tipos de fibras e como indicador de sarcopenia apresentam redução da proporção e tamanho de fibras tipo 2b. Dose única de DHEA não modula as vias da insulina e IGF 1 no músculo esquelético e reduz tamanho de fibras tipo 1 no EDL. / Reductions in skeletal muscle mass, increased insulin resistance, and decrements in several hormones serum levels, including DHEA, are features observed in aging. Twelve to fourteen months old rats present specific regulation in the insulin and the IGF-1 intracellular pathway in distinct skeletal muscle fiber composition and show reductions in size and proportion of muscle composed predominantly by fiber type 2. DHEA treatment had no effect in these intracellular pathways but induces reduction in the size of type 1 fiber in the EDL muscle.

Le vieillissement musculaire (sarcopénie) conduit inéluctablement à une perte d'autonomie, et à une moindre capacité à lutter contre les agressions métaboliques. Or, les mécanismes mis en jeu sont complexes et restent mal connus. Ainsi, au cours de cette thèse, une étude protéomique comparative a été développée afin d'identifier de nouveaux biomarqueurs potentiels de la sarcopénie chez la femme âgée post-ménopausée, et 73 protéines exprimées différentiellement dans le muscle âga ont été identifiées. En plus des altérations du muscle squelettique, l'âge est connu comme étant un facteur favorisant l'apparition du syndrome métabolique (SM), facteur de risque pour les maladies cardiovasculaires et le diabète de type II. Cependant, les effets du SM sur le muscle squelettique des personnes âgées sont peu décrits dans la littérature. Des marquages immunohistologiques ont été réalisés à partir de biopsies du muscle vastus lateralis provenant de personnes jeunes (25 ans) et âgées avec ou sans SM (75 ans), afin de décrire les altérations structurales et fonctionnelles du muscle squelettique liées à l'âge et au MS. Les résultats montrent une atrophie des fibres de type II ayant une déformation accrue lors du vieillissement. Chez les personnes âgées atteintes de SM, l'aire des fibres est augmentée par rapport aux personnes âgées contrôles, et une forte diminution de l'activité cytochrome c oxydase a été observée. De plus, le vieillissement et plus particulièrement le SM sont associés à une forte accumulation de lipides intramusculaires. Enfin, alors que peu de différences ont été observées chez les personnes âgées contrôles, le contenu en capillaire est fortement altéré chez les individus atteints de SM. Par la suite, une étude protéomique comparative a permis d'identifier 42 biomarqueurs potentiellement impliqués dans le vieillissement musculaire et/ou dans le syndrome métabolique. L'ensemble des résultats obtenus au cours de cette thèse devrait permettre d'améliorer notre compréhension des facteurs impliqués dans le développement de la sarcopénie, et pourrait permettre d'identifier à la fois de nouvelles voies de régulation et suggérer des cibles thérapeutiques potentielles. / Muscle aging (sarcopenia) contributes to both loss of autonomy and decreased capacity to prevent metabolic aggressions, but the mechanisms involved are complex and remain unclear. Therefore in this thesis, we have undertaken a top-down differential proteomic approach to reveal novel potential biomarkers of sarcopenia, and 73 differentially expressed proteins were identified. In addition to alterations of skeletal muscle, aging favors metabolic syndrome (MS), a risk factor for cardiovascular disease and type II diabetes. However, the effects of MS on skeletal muscle in old individuals have poorly been investigated. Immunohistochemical studies were performed with vastus lateralis muscle biopsies from young (25 years) and old (75 years) men with and without MS, to reveal the importance of age-dependent and MS-associated modifications on fiber-type characteristics. An atrophy of type-II fibers and altered fiber shape characterized muscle aging in lean healthy men. In contrast, increased cross sectional area of fibers, and reduced cytochrome c oxidase activity in all fiber types characterized MS, even in active elderly men. Moreover, aging and particularly MS were associated with accumulation of intramyocellular lipid droplets. Finally, while few differences were observed in lean healthy men, the capillary supply was strongly altered in old men with MS. Thereafter, a differential proteomic approach identified 42 potential biomarkers implicated in muscle aging and/or in metabolic syndrome. Overall the results obtained in this thesis may improve our understanding of the factors influencing sarcopenia, and may both identify new regulatory pathways and provide potential therapeutical targets.

Un análisis de los tipos de fibras y de su distribución fue realizado en muestras de músculos de la lengua (mm. geniogloso, estilogloso, hiogloso y lingual propio), del paladar y fauces (mm. palatofaríngeo, palatino, elevador y tensor del velo palatino) y de la faringe (mm. terigofaríngeo, estilofaríngeo caudal, hiofaríngeo, tirofaríngeo y cricofaríngeo) en la especie canina. Asimismo, se procesaron muestras del músculo semitendinoso, que fue utilizado como músculo control. La tipificación fibrilar se basó en técnicas histoquímicas (mATPasa y NADH-TR) e inmunohistoquímicas (aplicación de anticuerpos monoclonales específicos frente a las cadenas pesadas de miosina tipos I, IIa y IIx). En todos los músculos estudiados se evidenciaron fibras puras (tipos I, IIA y IIX) e híbridas (tipos IIAX, IIXA y IIC). Por otra parte, en el músculo tensor del velo palatino también fueron identificadas fibras tipo IIM. / An analysis of fiber types and its distribution in canine muscles of the tongue (genioglossus, hyoglossus, styloglossus and lingualis propius muscles), soft palate (palatinus, levator veli palatini, tensor veli palatini and palatopharyngeus muscles), and pharynx (pterygopharyngeus, stylopharyngeus caudalis, hyopharyngeus, thyropharyngeus and cricopharyngeus muscles) was carried out. Additional samples were obtained from the semitendinosus muscle to be used as a control. Muscle fiber types were defined by histochemical (mATPase and NADH-TR), and immunohistochemical (monoclonal antibodies against myosin heavy chain isoforms I, IIa and IIx) techniques. In all analyzed muscles pure I, IIA and IIX fibers and hybrid IIAX, IIXA and IIC fibers were observed. Furthermore, type IIM fibers were identified in tensor veli palatini muscle

In the present study Fourier Transform Infrared (FTIR) and Attenuated Total Reflectance FTIR (ATR-FTIR) Spectroscopy were used to examine the effects of simvastatin on structure, composition and function of macromolecules of three different rat skeletal muscles EDL (Extensor Digitorium Longus), DIA (Diaphragm) and SOL (Soleus) containing different amount of slow and fast twitch fibers, at molecular level. Simvastatin, a lipophilic statin, is widely used in the treatment of hypercholesterolemia and cardiovascular diseases due to its higher efficacy. However, long term usage of statins give rise to many adverse effects on different tissues and organs. Skeletal muscle accounts for around 45 % of the total body weight and has a high metabolic rate and blood flow. As a consequence, it is highly exposed to drugs within the circulation. Therefore, it is one of the target tissues of statins. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. EDL is a fast twitch muscle while SOL is slow twitch muscle. On the other hand, DIA shows intermediate properties between slow and fast twitch muscle. The results of ATR-FTIR and FTIR spectra revealed a considerable decrease in protein and lipid content of simvastatin treated skeletal muscles, indicating protein breakdown or decreased protein synthesis and increased lipolysis. Moreover changes in protein structure and conformation were observed. In simvastatin treatment, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. The drug treatment caused a decrease in the content of nucleic acids, especially RNA, and hydrogen and non-hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated EDL muscle was more severely affected from statin treatments while SOL was less affected from the drug treatments. Thus, FTIR and ATR-FTIR spectroscopy appear to be useful methods to evaluate the effects of statin on skeletal muscle tissues at molecular level.