A traumatic skeletal muscle injury that involves the loss of a substantial portion of tissue will not regenerate on its own. Little is understood about the ability of the muscle to recover function after such a defect injury, and few research models exist to further elucidate the repair and regeneration processes of defected skeletal muscle. In the current research, a model of muscle injury was developed in the lateral gastrocnemius (LGAS) of the rat. In this model, the muscle gradually remodels but functional recovery does not occur over 42 days. Repair of the defect with muscle-derived extracellular matrix (ECM), improves the morphology of the LGAS. Blood vessels and myofibers grow into the ECM implant in vivo, but functional recovery does not occur. Addition of bone marrow-derived mesenchymal stem cells (MSCs) to the implanted ECM in the LGAS increases the number of blood vessels and regenerating myofibers within the ECM. Following 42 days of recovery, the cell-seeded ECM implanted LGAS produces significantly higher isometric force than the non-repaired and non-cell seeded ECM muscles. These results suggest that the LGAS muscle defect is a suitable model for the study of traumatic skeletal muscle injury with tissue loss. Additionally, MSCs seeded on an implanted ECM lead to functional restoration of the defected LGAS. / text

Le muscle strié squelettique est un tissu richement vascularisé. Au delà de l'apport en oxygène et en nutriments, de nouvelles fonctions des vaisseaux ont été récemment identifiées, par le biais des interactions établies entre les cellules du vaisseau (cellules endothéliales) et les cellules du muscle, en particulier les cellules souches musculaires (cellules satellites). Celles-ci interagissent étroitement avec les cellules endothéliales pour leur expansion et leur différenciation, puis avec les cellules péri-endothéliales pour leur auto-renouvellement et leur retour à la quiescence. Les vaisseaux participent ainsi au contrôle de l’homéostasie du muscle squelettique. Grâce à ces interactions, les cellules vasculaires jouent donc un rôle central dans le remodelage tissulaire après un phénomène destructif, survenant par exemple au cours d’un trauma ou d’une myopathie. Pour étudier, les mécanismes de la plasticité vasculaire au cours du remodelage tissulaire, deux situations paradigmatiques de muscle en régénération chez l’enfant : la dermatomyosite juvénile (DMJ) et la dystrophie musculaire de Duchenne (DMD) ont été étudiées. Il existe, dans ces deux pathologies une souffrance musculaire associée à des cycles de nécrose/régénération. Elles se différencient par leur plasticité vasculaire et par leur évolution. En effet, la DMJ, la myopathie inflammatoire la plus fréquente de l’enfant est caractérisée par une vasculopathie avec perte en capillaires. L’évolution peut être favorable avec restitution ad integrum du muscle. La DMD est une myopathie génétique conduisant à une dégradation progressive de la force musculaire associée à une néovascularisation compensatrice. Le volet clinique/histologique incluant une analyse multiparamétrique des critères évolutifs cliniques et de réponse thérapeutique couplée à une réévaluation des données histologiques de la DMJ (analyse morphométrique des muscles DMJ) a permis de montrer qu’il existait des sous groupes phénotypiques homogènes de sévérité différente dans la DMJ. Le degré de sévérité clinique est relié à la gravité de la vasculopathie musculaire Par ailleurs, des marqueurs cliniques et histologiques simples permettant de repérer au diagnostic les patients nécessitant une escalade thérapeutique rapide (CMAS>34, atteinte gastrointestinale, fibrose endomysiale musculaire au diagnostic) ont été identifiés. Le volet cellulaire a permis l’identification in vitro des interactions cellulaires spécifiques et différentielles des myoblastes issues de patients DMD et DMJ sur les cellules endothéliales normales par l’analyse de leur rôle sur la prolifération, migration et différenciation des cellules vasculaires. Dans la DMD, les myoblastes entrainent une réponse angiogénique importante mais non efficace (néovascularisation anarchique). Dans la DMJ, les myoblastes participent efficacement à la reconstruction vasculaire notamment via la sécrétion de facteurs proangiogéniques. Ces résultats ont été renforcés par analyse transcriptomique effectuée à partir de cellules endothéliales et satellites isolées de muscles de patients confirmant le rôle central de la vasculopathie associée à un contexte inflammatoire spécifique lié à l’interféron dans la physiopathologie de la DMJ et montrant dans la DMD une dérégulation de l’homéostasie normale des interactions vaisseau-muscle avec mise en jeu d’un remodelage tissulaire non efficace. Ces données permettent d'identifier de nouvelles fonctions des cellules vasculaires dans le remodelage du muscle strié squelettique au cours des pathologies musculaires de l'enfant, et devraient ouvrir la voie à de nouvelles approches thérapeutiques. / Skeletal muscle is highly vascularised. Beyond oxygen and nutriment supply, new functions for vessels have been recently identified, through the interactions that vessel cells (endothelial cells) establish with muscle cells, particularly with muscle stem cells (satellite cells). These latter closely interact with endothelial cells for their expansion and their differentiation, then with periendothelial cells for their self-renewal and return to quiescence. During skeletal muscle regeneration endothelial cells reciprocally interact with myogenic cells by direct contact or by releasing soluble factors to promote both myogenesis and angiogenesis processes. Skeletal muscle regeneration typically occurs as a result of a trauma or disease, such as congenital or myopathies. To better understand the role of vessel plasticity in tissue remodeling, we took advantage of two muscular disorders that could be considered as paradigmatic situations of regenerating skeletal muscle in the child: Juvenile Dermatomyositis (JDM), the most frequent inflammatory myopathy and Duchenne Muscular Dystrophy (DMD), the most common type of muscular dystrophy. Although these two muscular disorders share, at the tissue level, similar mechanisms of necrosis-inflammation, they differ regarding the vessel domain. In JDM patients, microvascular changes consist in a destruction of endothelial cells assessed by focal capillary loss. This capillary bed destruction is transient. The tissue remodeling is efficient and muscle may progressively recover its function. By contrast, in DMD, despite an increase of vessels density in an attempt to improve the muscle perfusion, the muscle function progressively alters with age. We identified clinical and pathological markers of severity and predictive factors for poor clinical outcome in JDM by computing a comprehensive initial and follow-up clinical data set with deltoid muscle biopsy alterations controlled by age-based analysis of the deltoid muscle capillarization. We demonstrated that JDM can be divided into two distinctive clinical subgroups. The severe clinical presentation and outcome are linked to vasculopathy. Furthermore, a set of simple predictors (CMAS<34, gastrointestinal involvement, muscle endomysial fibrosis at disease onset) allow early recognition of patients needing rapid therapeutic escalation with more potent drugs. We studied in vitro the specific cell interactions between myogenic cells issued from JDM and DMD patients and normal endothelial cells to explore whether myogenic cells participate to the vessel remodeling observed in the two pathologies. We demonstrated that MPCs possessed angiogenic properties depending on the pathological environment. In DMD, MPCs promoted the development of establishment of an anarchic, although strong, EC stimulation, leading to the formation of weakly functional vessels. In JDM, MPCs enhanced the vessel reconstruction via the secretion of proangiogenic factors. This functional analysis was supported by the transcriptomic analysis consistent with a central vasculopathy in JDM including a strong and specific response to an inflammatory environment. On the contrary, DMD cells presented an unbalanced homeostasis with deregulation of several processes including muscle and vessel development with attempts to recover neuromuscular system by MPCs. To summarize, our data should allow the definition of new functions of vessel cells in skeletal muscle remodelling during muscle pathologies of the child that will open the way to explore new therapeutic options and to gain further insights in the pathogenesis of these diseases.

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 squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires. / The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells.

O músculo esquelético é um tecido que tem a capacidade de se regenerar após lesão, seja ela patológica ou induzida. Para tanto, células musculares progenitoras, presentes no músculo adulto, atuam fundindo-se entre si, ou com as fibras musculares danificadas, para formar novas fibras. A via da macroautofagia, implicada na degradação e reciclagem de proteínas e organelas danificadas via lisossomo, é essencial para a manutenção da massa muscular, mas já foi também implicada na diferenciação e funcionamento de células progenitoras do músculo. Além disso, essa via está desregulada em diversas doenças neuromusculares, o que destaca seu papel nesse tecido. Nesse estudo, a regulação da autofagia foi investigada em diferentes situações de formação e degradação do músculo. Para estudar o processo de diferenciação muscular in vitro utilizamos um modelo de células musculares imortalizadas normais, e de paciente com miopatia ligada ao X com autofagia excessiva (XMEA). A análise dos genes e proteínas p62, BNIP3, BECLIN1, VPS34, ATG12 e LC3, além de alvos de mTOR, mostrou um padrão similar de expressão em mioblastos indiferenciados e miotubos diferenciados a partir de células controle e nas derivadas de paciente XMEA. Estes resultados sugerem que a desregulação da via autofágica relacionada à doença provavelmente surge em estágios mais avançados, como se observa em doenças de acúmulo lisossomal. A investigação da diferenciação muscular nessas células mostrou um aumento na capacidade de fusão de mioblastos XMEA, que não foi relacionado a mudanças na expressão de genes envolvidos na miogênese. Isso indica que o defeito primário relacionado a XMEA, como a deficiência da ATPase vacuolar, pode interferir no processo de diferenciação muscular. Para estudar o músculo em condições patológicas, utilizamos modelos animais para distrofias musculares que possuem distintos graus de afecção do músculo, como o DMDmdx, modelo para distrofia muscular de Duchenne, o SJL/J, modelo para distrofia muscular de cinturas tipo 2B e o Largemyd, modelo para distrofia muscular congênita 1D. Observamos que não há alterações globais na expressão de genes e proteínas da autofagia. Adicionalmente, cada modelo murino teve alterações pontuais, destacando a ausência de correlação entre o grau de degeneração do músculo e as alterações observadas na via autofágica. Por outro lado, quando uma lesão muscular é induzida em músculo normal, houve uma diminuição da expressão de todos os genes estudados, Bnip3, Beclin1, Vps34, Atg12, Lc3 e Gabarapl1, com possível acúmulo das proteínas autofágicas p62 e Beclin1. Com a recuperação do músculo, após cinco dias da lesão, a maior parte dos genes estudados teve sua expressão normalizada. Tais resultados indicam que a lesão aguda se relaciona a uma resposta drástica e recuperação rápida na via da autofagia. Em conjunto, nossos resultados mostram que a via da autofagia é diferencialmente afetada a depender do estímulo dado ao músculo, seja ele de regeneração e formação de novas células musculares ou de degeneração. Dessa forma, este estudo pode ter implicações para o desenvolvimento de terapias que tenham como alvo a via autofágica, já que indica que o momento da intervenção terapêutica pode ser importante, assim como o estímulo que levou a alterações no tecido muscular / The skeletal muscle is a tissue that has the ability to regenerate upon lesion, whether it occurs pathologically or induced. Therefore, progenitor muscle cells, present in the adult muscle, act by fusing with each other or with damaged fibers in order to recover the tissue. The macroautophagy pathway, related to degradation and recycling of proteins and damaged organelles via lysosome, is essential for the maintenance of muscle mass, and it was also implicated in the differentiation and functioning of muscle progenitor cells. Besides that, this pathway is deregulated in several neuromuscular disorders, highlighting its important role in this tissue. In this study, the autophagic regulation was investigated in distinct contexts of muscle formation and degradation. To study the muscle differentiation process in vitro, we used a model of immortalized muscle cells from both a normal control and a patient with X-linked myopathy with excessive autophagy (XMEA). The genes and proteins p62, BNIP3, BECLIN1, VPS34, ATG12, LC3 and mTOR targets showed a similar pattern of expression in both undifferentiated myoblasts and differentiated myotubes, from both control cells and XMEA patient-derived cells. This fact suggests that autophagic deregulation might arise in later stages of the disease, in a pattern observed in disorders with protein accumulation. The investigation of muscle differentiation in the studied cells showed an enhancement of the myoblast fusion capacity in XMEA cells, which was not related to changes in the expression of myogenic genes. This observation indicates that the primary defect related to the XMEA pathology, as the deficiency of the vacuolar ATPase, might interfere in the process of muscle differentiation. In order to evaluate muscle in pathological conditions, we studied animal models for muscular dystrophies that have distinct patterns of muscle affection, such as the DMDmdx, model for the Duchenne muscular dystrophy, the SJL/J, model for the limb-girdle muscle dystrophy type 2B and the Largemyd, model for the congenital muscular dystrophy type 1D. We did not find any global alterations in the expression of autophagic genes and proteins. Additionally, each animal model had discrete changes, highlighting the absence of correlation between the pattern of muscle degeneration and alterations in the autophagy pathway. On the other hand, when a lesion is induced in normal muscle, there is a decrease in the expression of all studied genes, such as Bnip3, Beclin1, Vps34, Atg12, Lc3 and Gabarapl1, with a possible accumulation of the autophagic proteins p62 and Beclin1. With muscle recovery, five days after lesion, most of the studied genes had their expression returning to normal levels. These results indicate that the acute lesion is related to a drastic response and rapid recovery of the autophagic pathway. Together, our results show that autophagy is differentially affected depending on the stimulus given to the muscle, either of regeneration and formation of new muscle cells or degeneration. In that sense, this study may have implications for the development of therapies that target autophagy, since it indicates that the time point of therapeutic interventions may be important, as well as the stimulus that led to alterations in the skeletal muscle tissue

Le muscle squelettique s'adapte en réponse à diverses influences en modulant sa masse et ses propriétés contractiles et métaboliques. Il est ainsi rapporté que l'hypoxie sévère a un effet délétère sur la masse et les capacités oxydatives du muscle, et pourrait ralentir la maturation du phénotype contractile au cours du développement post-natal. Cependant, les mécanismes de contrôle de cette plasticité musculaire ne sont pas clairement identifiés. Le but de ce travail était de déterminer le rôle de l'hypoxie environnementale sur le contrôle de la masse et l'adaptation du phénotype du muscle en croissance (hypertrophie de surcharge du plantaris après ablation de ses muscles agonistes et régénération du soléaire après lésions étendues induites par la notexine). L'exposition hypoxique limite transitoirement l'hypertrophie induite par la surcharge fonctionnelle, tandis qu'elle accentue la fonte musculaire en réprimant la formation et la croissance des néo-fibres au cours des étapes précoces de la régénération. Ces résultats seraient en partie expliqués par la désactivation partielle de la principale voie de protéosynthèse, la voie mTOR, par un mécanisme indépendant d'Akt. Parmi les inhibiteurs endogènes de mTOR étudiés (REDD1, BNIP-3 et l'AMPK), nous montrons que l'activation prononcée de l'AMPK en hypoxie pourrait réprimer l'activité de mTOR au cours de la régénération, alors que le mécanisme responsable de l'inhibition de mTOR n'a pas pu être identifié dans le modèle de surcharge. Le système protéolytique ubiquitine/protéasome-dépendant, évalué à partir de l'expression des atrogènes MURF1 et MAFbx, pourrait également expliquer en partie l'altération de l'hypertrophie de surcharge en hypoxie. Nos résultats soulignent par ailleurs que l'activité des cellules satellites serait réprimée au cours des premiers jours de régénération musculaire, conduisant à réduire la formation et la croissance des myotubes. Malgré cette perturbation précoce de la croissance musculaire, l'exposition prolongée en hypoxie ne limite pas l'hypertrophie de surcharge et la récupération de la masse du muscle lésé. Ceci démontre que les signaux anaboliques induits dans ces deux situations de croissance musculaire l'emportent très largement sur les signaux cataboliques de l'hypoxie. L'analyse des propriétés métaboliques et contractiles met en évidence que l'hypoxie altère les capacités oxydatives du muscle en croissance, mais les mécanismes impliqués dans cette réponse adaptative restent à identifier. Par ailleurs, l'hypoxie ne constitue pas un stimulus métabolique suffisant pour altérer la transition du phénotype contractile du muscle en surcharge et la récupération complète du phénotype contractile du muscle lésé. Elle contribue uniquement à ralentir très modérément et transitoirement l'adaptation phénotypique du muscle en surcharge, et à modifier le profil contractile du muscle durant la phase de dégénérescence musculaire. / Skeletal muscle adapts to various influences, by modulating both its mass and contractile and metabolic properties. It was reported that severe hypoxia impairs muscle mass and oxidative capacities and could reduce the fast-to-slow fiber transition during post-natal development. However, mechanisms involved in muscle plasticity during hypoxia exposure are not clearly identified. This work aimed to determine the role played by ambient hypoxia on the control of muscle mass and muscle phenotype during muscle growth (functional overload-induced hypertrophy of plantaris after removal of its synergist muscles and regeneration of soleus after extensive injury induced by notexin injection). Hypoxia exposure transiently minimizes the overload-induced hypertrophy, while it enhances the muscle-mass loss by repressing the formation and growth of nascent fibers during the early steps of regeneration. These results could be partly due to an impairment of the mTOR signaling activation, the main pathway involved in protein synthesis, independently of Akt. Among the endogenous repressors of mTOR studied (REDD1, BNIP-3 and AMPK), we show that the marked activation of AMPK in hypoxia could repress mTOR activity during regeneration, whereas the mechanism involved in mTOR inhibition remains unknown in the overload model. The ubiquitin/proteasome-dependant system, assessed from expression of the two atrogenes MURF1 and MAFbx, could also partly explain the hypoxia-induced alteration of muscle hypertrophy. Nevertheless, our findings show that activity of satellite cells could be repressed during the first days of regeneration, leading to reduce formation and growth of myotubes. Although muscle growth is early impaired, prolonged hypoxia exposure does not limit the overload-induced hypertrophy and the muscle mass recovery of injured muscle. This demonstrates that anabolic signals induced in these models of drastic muscle growth widely prevail on hypoxia-induced catabolic signals. The analysis of metabolic and contractile properties shows that hypoxia alters oxidative capacities in growing muscle, but mechanisms involved in this adaptive response remain to be elucidated. Moreover, hypoxia is not a sufficient metabolic stimulus to impair the fast-to-slow fiber transition in overloaded muscle, and the complete recovery of the contractile phenotype in injured muscle. It only contributes to transiently and modestly slow down the fast-to-slow fiber shift in overloaded muscle, and to modify the contractile profile of muscle during the degeneration phase.

Le muscle strié squelettique est un tissu richement vascularisé. Au delà de l'apport en oxygène et en nutriments, de nouvelles fonctions des vaisseaux ont été récemment identifiées, par le biais des interactions établies entre les cellules du vaisseau (cellules endothéliales) et les cellules du muscle, en particulier les cellules souches musculaires (cellules satellites). Celles-ci interagissent étroitement avec les cellules endothéliales pour leur expansion et leur différenciation, puis avec les cellules péri-endothéliales pour leur auto-renouvellement et leur retour à la quiescence. Les vaisseaux participent ainsi au contrôle de l’homéostasie du muscle squelettique. Grâce à ces interactions, les cellules vasculaires jouent donc un rôle central dans le remodelage tissulaire après un phénomène destructif, survenant par exemple au cours d’un trauma ou d’une myopathie. Pour étudier, les mécanismes de la plasticité vasculaire au cours du remodelage tissulaire, deux situations paradigmatiques de muscle en régénération chez l’enfant : la dermatomyosite juvénile (DMJ) et la dystrophie musculaire de Duchenne (DMD) ont été étudiées. Il existe, dans ces deux pathologies une souffrance musculaire associée à des cycles de nécrose/régénération. Elles se différencient par leur plasticité vasculaire et par leur évolution. En effet, la DMJ, la myopathie inflammatoire la plus fréquente de l’enfant est caractérisée par une vasculopathie avec perte en capillaires. L’évolution peut être favorable avec restitution ad integrum du muscle. La DMD est une myopathie génétique conduisant à une dégradation progressive de la force musculaire associée à une néovascularisation compensatrice. Le volet clinique/histologique incluant une analyse multiparamétrique des critères évolutifs cliniques et de réponse thérapeutique couplée à une réévaluation des données histologiques de la DMJ (analyse morphométrique des muscles DMJ) a permis de montrer qu’il existait des sous groupes phénotypiques homogènes de sévérité différente dans la DMJ. Le degré de sévérité clinique est relié à la gravité de la vasculopathie musculaire Par ailleurs, des marqueurs cliniques et histologiques simples permettant de repérer au diagnostic les patients nécessitant une escalade thérapeutique rapide (CMAS>34, atteinte gastrointestinale, fibrose endomysiale musculaire au diagnostic) ont été identifiés. Le volet cellulaire a permis l’identification in vitro des interactions cellulaires spécifiques et différentielles des myoblastes issues de patients DMD et DMJ sur les cellules endothéliales normales par l’analyse de leur rôle sur la prolifération, migration et différenciation des cellules vasculaires. Dans la DMD, les myoblastes entrainent une réponse angiogénique importante mais non efficace (néovascularisation anarchique). Dans la DMJ, les myoblastes participent efficacement à la reconstruction vasculaire notamment via la sécrétion de facteurs proangiogéniques. Ces résultats ont été renforcés par analyse transcriptomique effectuée à partir de cellules endothéliales et satellites isolées de muscles de patients confirmant le rôle central de la vasculopathie associée à un contexte inflammatoire spécifique lié à l’interféron dans la physiopathologie de la DMJ et montrant dans la DMD une dérégulation de l’homéostasie normale des interactions vaisseau-muscle avec mise en jeu d’un remodelage tissulaire non efficace. Ces données permettent d'identifier de nouvelles fonctions des cellules vasculaires dans le remodelage du muscle strié squelettique au cours des pathologies musculaires de l'enfant, et devraient ouvrir la voie à de nouvelles approches thérapeutiques. / Skeletal muscle is highly vascularised. Beyond oxygen and nutriment supply, new functions for vessels have been recently identified, through the interactions that vessel cells (endothelial cells) establish with muscle cells, particularly with muscle stem cells (satellite cells). These latter closely interact with endothelial cells for their expansion and their differentiation, then with periendothelial cells for their self-renewal and return to quiescence. During skeletal muscle regeneration endothelial cells reciprocally interact with myogenic cells by direct contact or by releasing soluble factors to promote both myogenesis and angiogenesis processes. Skeletal muscle regeneration typically occurs as a result of a trauma or disease, such as congenital or myopathies. To better understand the role of vessel plasticity in tissue remodeling, we took advantage of two muscular disorders that could be considered as paradigmatic situations of regenerating skeletal muscle in the child: Juvenile Dermatomyositis (JDM), the most frequent inflammatory myopathy and Duchenne Muscular Dystrophy (DMD), the most common type of muscular dystrophy. Although these two muscular disorders share, at the tissue level, similar mechanisms of necrosis-inflammation, they differ regarding the vessel domain. In JDM patients, microvascular changes consist in a destruction of endothelial cells assessed by focal capillary loss. This capillary bed destruction is transient. The tissue remodeling is efficient and muscle may progressively recover its function. By contrast, in DMD, despite an increase of vessels density in an attempt to improve the muscle perfusion, the muscle function progressively alters with age. We identified clinical and pathological markers of severity and predictive factors for poor clinical outcome in JDM by computing a comprehensive initial and follow-up clinical data set with deltoid muscle biopsy alterations controlled by age-based analysis of the deltoid muscle capillarization. We demonstrated that JDM can be divided into two distinctive clinical subgroups. The severe clinical presentation and outcome are linked to vasculopathy. Furthermore, a set of simple predictors (CMAS<34, gastrointestinal involvement, muscle endomysial fibrosis at disease onset) allow early recognition of patients needing rapid therapeutic escalation with more potent drugs. We studied in vitro the specific cell interactions between myogenic cells issued from JDM and DMD patients and normal endothelial cells to explore whether myogenic cells participate to the vessel remodeling observed in the two pathologies. We demonstrated that MPCs possessed angiogenic properties depending on the pathological environment. In DMD, MPCs promoted the development of establishment of an anarchic, although strong, EC stimulation, leading to the formation of weakly functional vessels. In JDM, MPCs enhanced the vessel reconstruction via the secretion of proangiogenic factors. This functional analysis was supported by the transcriptomic analysis consistent with a central vasculopathy in JDM including a strong and specific response to an inflammatory environment. On the contrary, DMD cells presented an unbalanced homeostasis with deregulation of several processes including muscle and vessel development with attempts to recover neuromuscular system by MPCs. To summarize, our data should allow the definition of new functions of vessel cells in skeletal muscle remodelling during muscle pathologies of the child that will open the way to explore new therapeutic options and to gain further insights in the pathogenesis of these diseases.

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

Made available in DSpace on 2016-06-02T19:02:41Z (GMT). No. of bitstreams: 1 5282.pdf: 2759184 bytes, checksum: ddd5666e5ff979383a7a5f6cdf80a469 (MD5) Previous issue date: 2013-03-01 / Financiadora de Estudos e Projetos / Skeletal muscle injuries from sport activities are usual, but despite the inherent healing capacity of muscle tissue, this process can be impaired depending on the injury extension, what can lead to atrophy, fibroses, pain and functional recovery incomplete, increasing the risks of recurrent lesions. In addition, biophysical and biochemical resources have been studied in an attempt to enhance the muscle regeneration process, as the low level laser therapy (LLLT). Many studies have showed that this therapeutic modality stimulates positives responses during muscle healing process. Thus, two studies were performed with the aim of evaluating the effects of LLLT (InGaAlP 660 nm), with the fluences of 10 J/cm2 and 50 J/cm2, during Tibiales anterior muscle regeneration after cryolesion. Sixty three Wistar rats (3 month of age, ±300 g) were randomly divided into 3 groups: injured control group (CG) injured animals without any treatment; treated group, at 10 J/cm2 (G10) and treated group, at 50 J/cm2 (G50). Each group was divided into 3 different subgroups (n=7) and on days 7, 14 and 21 post-injury, were sacrificed. The laser irradiation was performed at a single point and started 48 hours after injury, 5 times per week (each 24 hours), followed by an interval of 48 hours. At the first study, the effects of LLLT were analyzed on the muscle regeneration related on molecular mechanisms involved at repair process. The results showed a downregulation of mRNA Cox-2 expression in all treated groups after 14 and 21 days. On day 7, both treated groups had a downregulation of mRNA Vegf expression, but had a increase after 14 and 21 days of treatment, meanly at 50 J/cm2. The mRNA MyoD expression was upregulated with the higher fluence, in all periods evaluated, and with the lower fluence the MyoD levels increased only after 21 days. The Myogenin expression was downregulated in both treated groups on day 7, and was upregulated with dose of 10 J/cm2 after 21 days. These responses suggest that LLLT can improve the skeletal muscle regeneration through the gene expression stimulation. At the second study, the effects of LLLT were evaluated during muscle regeneration, through histological and immunohistochemical aspects. The treated groups had an inflammatory process modulation after 7 days. On day 14, the treated animals, with both fluences, showed organized new muscle fibers and less granulation tissue. After 21 days of lesion, all groups had complete tissue repair. Moreover, the irradiated groups had less necrosis area at the first experimental period evaluated, and, also had more blood vases quantity after 14 and 21 days, at 50 J/cm2. The immunohistochemistry analysis showed Myogenin expression in all experimental groups after 7 and 14 days, and at day 21st; the immunoexpression was detectable in the control group and in the treated group with lower fluence. Superior MyoD expression appeared in both treated groups during all evaluated periods. Concluding, both fluencies of LLLT played positive effects on muscle repair. Such findings are fundamentals to elucidate the biological and molecular mechanisms involved in the skeletal muscle regeneration. / Lesões do sistema músculo esquelético decorrentes de atividades esportivas são comuns, porém apesar da capacidade regenerativa intrínseca do tecido muscular, este processo pode ser prejudicado de acordo com a extensão da lesão, causando atrofia, fibrose, dor e incompleta recuperação funcional, aumentando o risco de lesões recorrentes. Dentro desse contexto, recursos biofísicos e bioquímicos tem sido estudados na tentativa de acelerar o processo de regeneração muscular, como a terapia laser de baixa intensidade (LLLT). Vários estudo demonstram que esta modalidade terapêutica estimula respostas positivas durante o reparo do tecido muscular. Diante disso, foram desenvolvidos dois estudos com o objetivo de verificar os efeitos da LLLT (InGaAlP 660 nm), com as fluências de 10 J/cm2 e 50 J/cm2, durante a regeneração do músculo tibial anterior criolesionado. Sessenta e três ratos da linhagem Wistar (3 meses de idade, ±300 g) foram distribuídos randomicamente em 3 grupos: grupo controle lesado sem nenhum tratamento (GC), grupo lesado e tratado com 10 J/cm2 (G10) e grupo lesado e tratado com 50 J/cm2 (G50). Ainda, todos os grupos foram subdivididos em 3 subgrupos (n=7), onde os animais foram eutanasiados depois de 7, 14 e 21 dias da indução da lesão. A irradiação laser foi realizada em um único ponto e se iniciou 48 horas após a criolesão, 5 vezes por semana (a cada 24 horas), seguidos de um intervalo de 48 horas. No primeiro estudo, foram analisados os efeitos da LLLT na regeneração muscular em relação aos mecanismos moleculares envolvidos no processo de reparo. Os resultados demonstraram que ocorreu uma redução da expressão do RNAm do Cox-2 em todos os grupos irradiados após 14 e 21 dias. No sétimo dia, a expressão do RNAm do VEGF não aumentou nos dois grupos tratados, entretanto sua expressão aumentou depois de 14 e 21 dias de tratamento, principalmente com a fluência de 50 J/cm2. A expressão do RNAm da MyoD foi aumentada com a fluência maior, em todos os períodos analisados, e com a fluência menor apenas depois de 21 dias. A expressão da Miogenina foi reduzida em ambos os grupos tratados em 7 dias, e foram aumentadas com a fluência de 10 J/cm2 depois de 21 dias. Estes resultados sugerem que a LLLT pode melhorar a regeneração muscular através da estimulação da expressão gênica. No segundo estudo, foram avaliados os efeitos da LLLT durante a regeneração muscular, nos seus aspectos histológicos e imunohistoquímicos. Uma modulação do processo inflamatório em 7 dias foi visualizada nos grupos tratados. No 14o dia, os animais irradiados, com as duas fluências, apresentaram fibras musculares jovens organizadas e pouco tecido de granulação. Depois de 21 dias de lesão, todos os grupos apresentaram tecido muscular organizado similar ao músculo esquelético sem lesão. Além disso, uma menor área de necrose no primeiro período experimental foi observada nos grupo tratados e uma maior quantidade de vasos sanguíneos depois de 14 e 21 dias foi visualizada no grupo tratado com a fluência de 50 J/cm2. A imunohistoquímica demonstrou expressão da Miogenina em todos os grupos experimentais depois de 7 e 14 dias, e no 21o dia, no grupo controle e no grupo tratado com a menor fluência. Maior expressão da MyoD esteve presente nos grupos tratados em todos os períodos avaliados. Concluindo, as duas fluências da LLLT desempenharam efeitos positivos durante o reparo muscular. Tais resultados são fundamentais na elucidação dos mecanismos biológicos e moleculares envolvidos na regeneração muscular.

Made available in DSpace on 2016-08-17T18:39:30Z (GMT). No. of bitstreams: 1 2518.pdf: 1232652 bytes, checksum: e57f0134a52e7bb7bc66a121273dc146 (MD5) Previous issue date: 2009-03-13 / Financiadora de Estudos e Projetos / The elderly still go through physical changes, especially musculoskeletal disorders such as sarcopenia, changes in central and peripheral nervous system, blood hypoperfusion, regenerative changes contributing to atrophy and muscle weakness, undermining the activities of daily living (AVL). The regenerative process happens about the determination, proliferation and differentiation of satellite cells through activation of muscle-specific genetic program, which is regulated by specific transcription factors, known as myogenic regulatory factors (MRFs). But in the elderly because of changes in skeletal muscle-activation of MRFs are inefficient, hindering the process of regeneration. However, recent findings showed very promising results of low laser therapy (LLT) in muscle regeneration, but the effects of this therapy when associated with aging are still unknown. This project aims to evaluate the effects of (LLT), &#955; = 830nm, the tibial muscle of aged rats after cryolesioning. We used 56 male Wistar rats randomly divided into 4 groups (n = 7) of young rats from 3 months to 4 groups (n = 7) of aged rats, 10 months, divided into control groups (C), groups in which the right Tibialis anterior muscle (TA) was only irradiated (I), groups in which the AT was submitted to cryolesioning (CL) and groups where the TA muscle cryolesioning and was subjected to irradiation (LI). Treatment with the laser model of the DMC, Class 3B, energy of 0.87 J, was performed every 24 h for five consecutive days, with the first application 24 hours after induction of injury. On the sixth day after injury, with the animals anesthetized and the TA muscle was carefully dissected and removed, and then the animals were euthanaziated. We carried out histological analysis of the area of the lesion with toluidine blue, and counting of blood capillaries with hematoxylin-eosin. Through analysis by RT-PCR, it was possible to analyze the expression of MyoD and VEGF genes. The results showed that there was significant increase (p <0.05) of the expression of MyoD gene, VEGF gene and capillary blood count of more prominent in elderly victims and irradiated groups than in the young. Probably the LLT increased the maturation of satellite cells into myoblasts and miotubos, enhancing the regenerative process of aged rats irradiated. / Os idosos passam por continuas mudanças físicas, principalmente músculo-esqueléticas, como sarcopenia, alterações no sistema nervoso central e periférico, hipoperfusão sanguínea, alterações regenerativas contribuindo para atrofia e fraqueza muscular, prejudicando as atividades de vida diárias (AVDs). O processo regenerativo ocorre pela determinação, proliferação, diferenciação das células satélites através da ativação do programa genético músculo-específico, que é regulado por fatores de transcrição específicos, conhecidos como fatores regulatórios miogênicos (FRMs). Porém no idoso por causa das alterações músculo-esqueléticas a ativação dos FRMs são ineficientes, prejudicando o processo de regeneração. Entretanto, recentes achados mostraram resultados muito promissores da laser terapia de baixa intensidade (LTBI) na regeneração muscular, mas os efeitos desta terapia quando associado ao envelhecimento continuam desconhecidos. Este projeto tem por objetivo avaliar os efeitos da (LTBI), &#955;=830nm, no músculo tibial de ratos idosos após criolesão. Foram utilizados 56 ratos machos Wistar, divididos aleatoriamente em 4 grupos (n=7) de ratos jovens de 3 meses e 4 grupos (n=7) de ratos idosos de 10 meses; subdivididos em: grupos controle (C), grupos em que o músculo tibial anterior direito (TAD) foi apenas irradiado (I), grupos em que o TAD foi submetido à criolesão (CL) e grupos onde o TAD foi submetido à criolesão e a irradiação (LI). O tratamento com o laser modelo da DMC, classe 3B, energia de 0,87 J, foi realizado a cada 24 h, durante cinco dias consecutivos, com a primeira aplicação 24 horas após a indução da lesão. No sexto dia pós lesão, com os animais vivos e anestesiados, o músculo TAD foi cuidadosamente dissecado e retirado, e logo depois os animais foram eutanaziados. Realizou-se analises histológicas da área da lesão com Azul de Toluidina e contagem dos capilares sanguíneos com Hematoxilina-eosina. Por meio da análise por RT-PCR, foi possível analisar a expressão dos genes MyoD e VEGF. Os resultados mostraram que houve aumento significativo (p<0,05) da expressão gênica da MyoD, do VEGF e da contagem de capilares sanguíneos mais proeminentes nos grupos idosos lesados e irradiados do que no grupo jovem. Provavelmente a LTBI aumentou a maturação das células satélites em mioblastos e miotubos, melhorando o processo regenerativo dos ratos idosos irradiados.