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The skeletal muscle is a dynamic tissue that has the ability to change and modify itself to fit the level of required activity; a phenomenon called muscle plasticity. Most studies of muscle plasticity are carried out in situ, a condition for which it is difficult to study and discern between the intrinsic properties of skeletal muscle, the myokines released by muscle fibers and the neurotrophic factors released by neurons innervating skeletal muscles that play various roles in the mechanisms of muscle plasticity. Another approach is to study the morphological and contractile properties of single adult muscle fibers under culture conditions for which one can fully control the level of activity and exogenous factors affecting muscle plasticity. However, the survival of single muscle fiber in culture is very low as most fibers degenerated or supercontracted within 5-7 days. The first objective of this study was to optimize fiber survival in culture. The application of chronic stimulation and beta-adrenergic agonists are two major factors that prevent muscle atrophy and loss of force in denervated muscles in situ. So, objective two was to determine if chronically stimulated single fibers in culture also improve fiber survival and contractile characteristic under culture conditions. The third objective was the same for salbutamol, a beta 2-adrenergic agonist. In regard to the optimization of fiber survival, the Minimum Essential Medium (MEM) was a better medium than Dulbecco’s Modified Eagle Medium (DMEM), changing 50% of the culture medium every two days also improved fiber survival compared to changing the medium every day. Interestingly, inhibiting the proliferation of satellite cells with AraC largely improved fiber survival when fibers were kept under resting conditions, but not when they were chronically stimulated. Finally, under conditions in which proliferation of satellite cells was inhibited, the use of a collagen/laminin mixture as adhering substrate to improve fiber adhesion to glass coverslip gave rise to a better fiber survival than Matrigel that contains not only collagen and laminin but several growth factors. The results suggest i) that when satellite cells (or fibroblasts) are allowed to proliferate they appear to contribute to the degeneration of fibers under resting conditions and ii) that the release of myokines by skeletal muscle fibers (or cytokines by other cells) likely play a role in fiber survival. Contrary to the situation in situ, neither the chronic stimulation nor salbutamol improved fiber survival and contractile characteristics of muscle fibers in culture suggesting that some important factors in culture are missing to allow chronic stimulation and salbutamol to reduce muscle atrophy and loss of force.

PRMT biology during skeletal muscle disuse. / Protein arginine methyltransferase 1 (PRMT1), PRMT4 (also known as co-activator-associated arginine methyltransferase 1; CARM1), and PRMT5 are critical components of a diverse set of intracellular functions. Despite the limited number of studies in skeletal muscle, evidence strongly suggests that these enzymes are important players in the regulation of phenotypic plasticity. However, their role in disuse-induced muscle remodelling is unknown. Thus, we sought to determine whether denervation-induced muscle disuse alters PRMT expression and activity in skeletal muscle within the context of early signaling events that precede muscle atrophy. Mice were subjected to 6, 12, 24, 72, or 168 hours of unilateral hindlimb denervation. The contralateral limb served as an internal control. Muscle mass decreased by ~30% following 168 hours of disuse. Prior to atrophy, the expression of muscle RING finger 1 and muscle atrophy F-box were significantly elevated. The expression and activities of PRMT1, CARM1, and PRMT5 displayed differential responses to muscle disuse. Peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase expression and activation were altered as early as 6 hours after denervation, suggesting that adaptations in these molecules are among the earliest signals that precede atrophy. AMPK activation also predicted changes in PRMT expression and function following disuse. Our study indicates that PRMTs are important for the mechanisms that precede, and initiate muscle remodelling in response to neurogenic disuse. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that is capable of adapting to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important players in the regulation of skeletal muscle remodelling. However, their role in disuse-induced muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the role of PRMTs within the context of early, upstream signaling pathways that mediate disuse-evoked muscle remodelling. We found differential responses of the PRMTs to muscle denervation, suggesting a unique sensitivity to, or regulation by, potential upstream signaling pathways. AMP-activated protein kinase (AMPK) was among the molecules that experienced a rapid change in activity following disuse. These alterations in AMPK predicted many of the modifications in PRMT biology during inactivity, suggesting that PRMTs factor into the molecular mechanisms that precede neurogenic muscle atrophy. This study expands our understanding of the role of PRMTs in regulating skeletal muscle plasticity.

Introduction:  Aging is related to a gradual decline in skeletal muscle mass, which is associated with morphological modifications such as reduced muscle fiber cross-sectional area and satellite cell content. Data also suggest that a short-term strength training period can be an effective instrument to rejuvenate these morphological parameters and to restore muscle mass. Therefore, the aim of this study is to investigate the effects of one year progressive strength training on fiber type-specific morphological parameters (fiber type composition, fiber area, satellite cell content, myonuclear number and domain) in skeletal muscle of elderly men.   Methods: Thirteen healthy elderly men (age range, 66-77 years) were randomly assigned into training (T) (n=7) and control (C) (n=6) groups. 52 weeks of progressive strength training was performed. Before and after the training, muscles biopsies were collected from the middle part of the vastus lateralis by percutaneous needle biopsy technique. Muscle biopsies were examined for muscle fiber type composition, fiber type-specific hypertrophy and alterations in satellite cell content, myonuclear content and domain using immuno-histochemistry.   Results: At baseline, myonuclear content and mean fiber area was larger in type I fibers compared to type II fibers (p&lt;0.05). No statistically significant differences were found in fiber type composition, mean fiber area, satellite cell content and myonuclear domain between T and C groups at baseline. By the end of the training period, fiber area was increased by 59% (p&lt;0.05) in type I and 71% (p&lt;0.05) in type II. Satellite cell content, myonuclear content and myonuclear domain were increased after training in type I by 58% (p&lt;0.05), 33% (p&lt;0.05), and 20% (p&lt;0.05), respectively. Similar increases in satellite cell content (+65%; p &lt;0.05), myonuclear content (+36%; p &lt;0.05) and myonuclear domain (+25%; p&lt;0.05) were seen in type II fibers. Conclusion: The current study reported that long-term strength training is an excellent tool to prevent sarcopenia. It is demonstrated that skeletal muscle in elderly is capable to enhance satellite cell and myonuclear content, which contributed to muscle hypertrophy. / <p>presentation was made in august 2012 and thesis is approved and got result as well in november 2012</p><p>For an enhanced reading experience go to a later version: http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-31017.</p> / This study was a part of a larger research project studying adaptations to strength, endurance and combined training

O músculo esquelético é o tecido mais abundante do organismo, é importante em diversas habilidades básicas nas atividades de vida diária, como: movimento, postura corporal e respiração, além de outros processos fisiológicos importantes para manutenção do equilíbrio metabólico e defesa imunológica. Para se manter em adequado funcionamento, o músculo esquelético possui uma alta plasticidade, remodelando sua estrutura e função de acordo com as exigências do ambiente. Os microRNAs são pequenos RNAs não codificadores de proteínas que podem regular a expressão gênica a nível pós-transcricional. Nas últimas décadas o conhecimento dos microRNAs na biologia do músculo esquelético vem abrindo portas para novas abordagens que visam otimizar a boa saúde da musculatura esquelética. Neste trabalho, nosso principal objetivo foi identificar e caracterizar microRNAs com potencial de modulação da regeneração e massa muscular esquelética. Utilizamos uma análise in silico para identificar os microRNAs que tem como alvo predito genes associados a vias que regulam a regeneração e massa muscular, em seguida manipulamos in vitro (células C2C12) e in vivo (camundongos C57BL/6) a expressão desses microRNAs, analisando a expressão desses genes e as consequências nas células e no tecido muscular. Na primeira parte deste trabalho, analisamos a hipótese de que certos microRNAs poderiam regular a expressão de MuRF1 e MuRF2 (E3-ligase importantes para o processo de regeneração muscular). Identificamos os microRNAs miR-29c e miR-101a que têm como alvo predito MuRF1, e miR-133a e miR-133b que tem como alvo predito MuRF2. MuRF1 é induzido nos primeiros estágios do processo de regeneração muscular e seus miRs potencialmente reguladores são reprimidos durante esse processo. A superexpressão de miR-29c e miR-101a reduz a expressão de MuRF1 em células C2C12, enquanto que, em um ensaio de luciferase, validamos MuRF1 como alvo direto apenas do miR-29c. Além disso, a superexpressão de miR-29c durante a diferenciação de células C2C12 promove a miogênese, com aumento do diâmetro e índice de fusão de miotubos, enquanto que a superexpressão do miR-101a provocou uma redução no diâmetro dos miotubos. Na segunda parte deste trabalho, identificamos in silico o miR-29c como um potencial regulador da massa muscular esquelética, em seguida através de um método de entrega gênica por meio de eletroporação, superexpressamos o miR-29c no músculo de camundongos. A superexpressão do miR-29c, promoveu um aumento da massa e do número de sarcomeros em serie, com ganho de força e função no músculo e esse efeito foi acompanhado de um remodelamento tecidual com aumento do número de células satélite ativadas. Tomados juntos, nossos resultados revelam que o miR-29c tem um efeito hipertrófico com ganho de função. A superexpressão deste microRNA pode ser uma ferramententa útil para futuras abordagens terapêuticas que visem a manipulação da massa muscular esquelética. / The skeletal muscle is the body most abundant tissue. It plays an important role in daily life activities, such as movement, posture, and breathing. In addition, this tissue is crucial at physiological processes like metabolic equilibrium and immune defense. The substantial adaptability in response to environmental change marks skeletal muscle as a plastic organ. This plasticity could be coordinated by microRNAs, those are small non-protein-coding RNAs that regulate post-transcriptional gene expression. This knowledge has fostered new approaches that aim to optimize the skeletal muscle health. Thus, in this work, we intended to identify and characterize microRNAs that modulate the muscle mass and the regeneration process. An in silico analyses has allowed the identification of microRNAs who possibly bind genes from pathways of skeletal muscle mass control and regeneration. After, we manipulated the expression of those microRNAs on C2C12 cells and C57BL/6 mice. Finally, we measured the transcriptional levels of target genes and the impact of these alterations on the cells and on muscle tissue. In the first section of this work, we hypothesized that microRNAs could regulate MURF1 and MURF2 expression, both E3-ligases important for the regeneration process. In our analysis, MURF1 was a predictable target of miR-29c and miR-101a while for MURF2 were identified miR-133a and 133b. During the regeneration process, MURF1 is up-regulated and its predicted target microRNAs are downregulated. In this context the hyperexpression of both miR-29c and miR-101a in C2C12 cells induced MURF1 down-regulation. Furthermore, miR-29c promotes myogenesis with an increase in myotubes diameter and fusion index. In contrast, miR-101a expression reduces myotubes diameter with no change at the fusion index. Lastly, luciferase assay validated only miR-29c directly target MURF1 3`UTR. In the second section of this work, we identified miR-29c as a potential regulator of skeletal muscle mass. Then through gene delivery by electroporation, we induce miR-29c overexpression in mice skeletal muscle. This procedure promoted an increase in muscle mass as well as a gain in strength, endurance and sarcomere number, furthermore the number of activated satellite cells. Taken together our results found that miR-29c has a hypertrophic effect with gain in muscle function. Thus, the overexpression of this microRNA could be a useful tool for future therapeutics that manipulate muscle mass.

Le muscle strié squelettique est un tissu fascinant qui permet d’assurer les fonctions essentielles à notre existence : se mouvoir, maintenir sa posture, se nourrir, communiquer ou tout simplement respirer. De nombreuses situations, engendrant principalement une hypoactivité, peuvent provoquer un déconditionnement musculaire caractérisé par une perte de masse et de force ainsi qu’un développement d’infiltrations graisseuses (IMAT), altérant ainsi la fonction musculaire. Le développement d’IMAT est également observé lorsque les processus de régénération musculaire sont altérés. Les fibro-adipogenic progenitors (FAPs) représentent la population de cellules souches principalement impliquée dans le développement d’IMAT. L’interaction entre FAPs et cellules satellites/immunitaires semble être un trio indispensable pour une régénération optimale, sans développement d’IMAT. Au regard de la littérature scientifique, une modulation du niveau d’activité contractile permet de faire varier le niveau d’expression de nombreuses cytokines impliquées dans la modulation des FAPs et donc dans l’apparition d’IMAT. Nos travaux ont contribué à l’accroissement des connaissances scientifiques relatives à la thématique des infiltrations graisseuses et à leurs exacerbations dans des situations d’hypoactivité ou de régénération musculaire. Nous avons montré que 3 jours d’hypoactivité chez l’homme, induite par le modèle novateur de dry immersion, suffisent à augmenter le contenu musculaire en IMAT. Dans un contexte de régénération musculaire, induite par le modèle glycérol chez la souris, nous avons démontré une inhibition de l’apparition des IMAT en diminuant les contraintes mécaniques appliquées au muscle lésé. Nous avons également précisé le rôle de l’axe TNFα/TGF-β1, et donc celui des processus inflammatoires nécessaires dans l’apoptose des FAPs afin de limiter le développement des IMAT dans ce modèle. Ces trois études ouvrent de nombreuses perspectives, afin i) de préciser le rôle des IMAT dans la dysfonction musculaire, ii) de définir les mécanismes de régulation qui contrôlent le développement et l’accumulation d’IMAT. / Skeletal muscle is a fascinating tissue that ensures core functions: moving, maintaining postures, feeding, communicating or just breathing. Many situations, associated with hypoactivity, are able to involve muscle deconditioning defined by a loss of mass and strength, as well as fat infiltration development (IMAT), altogether impairing muscle function. IMAT development occurs also with disrupted regeneration processes. Fibro-adipogenic progenitors (FAPs) appear as the main stem cell population involved in IMAT development. The interaction between FAPs and satellite/immune cells seems to be a crucial trio for an efficient regeneration, without IMAT development. According to the literature, the degree of contractile activity is able to affect the expression levels of different cytokines involved in FAPs fate, and therefore in IMAT accumulation. Our work contributed to increase scientific knowledge on muscle fatty infiltrations and their exacerbations in hypoactivity or regeneration situations. We showed that 3 days of hypoactivity in human, induced by the innovative model of dry immersion, are sufficient to promote an increase in IMAT content. In a context of muscle regeneration, induced by the mouse glycerol model, we highlighted an almost complete inhibition of IMAT accumulation by decreasing mechanical constraints applied to the injured muscle. We also investigated the role of the TNFα/TGF-β1 axis, and therefore the potential role of the inflammatory stage in FAPs apoptosis and inhibition of IMAT development. Our work open up new prospects 1°) clarifying the role of IMAT in muscle dysfunction, and 2°) defining the underlying mechanisms controlling IMAT development and accumulation.

Crustacean muscle fibers are some of the largest cells in the animal kingdom, with fiber diameters in the giant acorn barnacle (Balanus nubilus) exceeding 3 mm. Sessile animals with extreme muscle sizes and that live in the hypoxia-inducing intertidal zone – like B. nubilus – represent ideal models for probing the effects of oxygen limitation on muscle cells. We investigated changes in metabolism and structure of B. nubilus muscle in response to: normoxic immersion, anoxic immersion, or air emersion, for acute (6h) or chronic (6h exposures twice daily for 2wks) time periods. Following exposure, we immediately measured hemolymph pO2, pCO2, pH, Na+, Cl-, K+, and Ca+ then excised tergal depressor (TD) and scutal adductor (SA) muscles to determine citrate synthase (CS) activity, lactate dehydrogenase (LDH) activity, and D-lactate levels. We also prepared a subset of SA and TD muscles from the chronic barnacles for histological analysis of fiber diameter (Feret’s), cross-sectional area (CSA), mitochondrial distribution and relative density, as well as nuclear distribution and myonuclear domain size. There was a significant decrease in hemolymph pO2 and pCO2 following acute and chronic anoxic immersion, whereas air emersion pO2 and pCO2 was comparable to normoxic levels. Fiber CSA and diameter did not change significantly in either tissue, while myonuclear domain size in SA muscle was significantly lower in the anoxic and emersion groups than the normoxic control. Neither CS, nor LDH activity, showed any significant treatment effect in either tissue, whereas both muscles had significantly higher D-lactate levels after air emersion following acute (though not chronic) exposure. Thus far, our findings indicate that B. nubilus experience a general reduction in aerobic metabolism under anoxia, emersion is only mildly oxygen-limiting, and that muscle plasticity is occurring during chronic emersion and anoxia.