The skeletal muscle has a remarkable regenerative capacity upon injury, due to the presence of the satellite cells, which remain quiescent in the tissue, but, when required, they are able to proliferate and form and/or repair myofibers. Moreover, satellite cells are important to muscle growth and maintenance. However, in many neuromuscular disorders, the amount, function, and proliferative capacity of these cells are impaired. Centronuclear myopathies (CNM) are a group of muscle diseases characterized by generalized muscle weakness and myofibers with central nuclei. The autosomal dominant form (AD-CNM) is caused by mutations in the DNM2 gene. Dynamin 2 protein is ubiquitously expressed and is involved in membrane remodeling, intracellular trafficking, and cytoskeleton dynamics. Therefore, the pathophysiological mechanisms are equally diverse e not completely understood, mainly the fact to be a muscle-specific disease. In the present Ph.D. thesis, we sought to investigate the satellite cells in the context of centronuclear myopathy. For this, we used the mouse model KI-Dnm2R465W, bearing the most frequent mutation found in human patients. Since in centronuclear myopathy there is no evident degenerative process ongoing, we induced muscle lesion by electrical shock, a protocol developed for this thesis, comparatively to cardiotoxin injection. We verified that the number of satellite cells in gastrocnemius muscle is reduced in the KI-Dnm2R465W mouse in relation to wild-type animals. As a result, the regenerative potential of the mutant mouse is decreased and the muscle is not able to fully recover. In addition, we investigated the functional consequences of two mutations, p.R465W and p.E650K, in immortalized myoblasts. We examined the myogenic potential in vitro, the migratory property, and the endocytosis capacity. We found that both mutations impact on the myogenic potential, but in different ways. We also show that both mutations impair the migratory capacity of myoblasts that justify, in parts, the alterations in their myogenic potential. Finally, we verified that the endocytosis capacity is affected in a mutation-dependent manner, which may also indirectly disturb the myogenic differentiation efficiency / O músculo esquelético possui grande capacidade regenerativa após sofrer lesões, por causa da presença das chamadas células-satélite, que permanecem no tecido em estado quiescente, mas que, na presença de uma lesão, são capazes de proliferar e formar e/ou reparar miofibras. As células-satélite são importantes para o crescimento e manutenção do músculo adulto. Porém, em diversas doenças neuromusculares, a quantidade, a função e a capacidade proliferativa destas células podem estar comprometidas. As miopatias centronucleares (CNM) são um grupo de doenças musculares caracterizadas por fraqueza muscular generalizada e o posicionamento dos núcleos na porção central da miofibra. A forma autossômica dominante (AD-CNM) é causada por mutações no gene DNM2. A proteína dinamina 2 é expressa ubiquamente e está envolvida no remodelamento de membranas, no tráfego intracelular e na dinâmica do citoesqueleto. Consequentemente, os mecanismos fisiopatológicos também são diversos e não completamente compreendidos, principalmente o fato de ser uma doença músculo-específica. Nesta tese de doutorado, buscamos investigar as células-satélite no contexto da miopatia centronuclear. Para isto, utilizamos o camundongo modelo KI-Dnm2R465W, portador da mutação mais frequente em pacientes humanos. Como na miopatia centronuclear não há um processo degenerativo em atividade, induzimos nos camundongos a lesão muscular por choque elétrico, em protocolo desenvolvido nesta tese, comparativamente a injeção de cardiotoxina. Verificamos que o número de células satélite no músculo gastrocnêmio do camundongo KI-Dnm2R465W é reduzido em relação aos animais selvagens. Em consequência disto, o potencial regenerativo do animal mutante é reduzido e o músculo não se recupera completamente. Investigamos também os efeitos funcionais de duas mutações, p.R465W e p.E650K, em mioblastos imortalizados. Examinamos o potencial miogênico in vitro, a propriedade migratória e a capacidade de endocitose. Verificamos que o potencial miogênico destas células é afetado pelas mutações, porém de maneiras distintas. Mostramos também que ambas as mutações impactam negativamente na capacidade migratória dos mioblastos, o que em parte justifica as alterações no potencial miogênico dos mesmos. Por fim, verificamos que a capacidade endocítica em mioblastos é alterada a depender da mutação, o que indiretamente também pode afetar a capacidade de diferenciação miogênica

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La myopathie centronucléaire autosomique dominante (MCN-AD) est une maladie congénitale rare liée à des mutations principalement retrouvées dans le gène dynamine-2. La majorité des patients atteints de MCN-AD présente une évolution lentement progressive, avec une perte de masse et de force musculaire. A ce jour, aucune thérapie n’est disponible pour la MCN-AD. Des interventions thérapeutiques visant à limiter la progression et la sévérité de l’atteinte musculaire ainsi qu’à améliorer la qualité de vie des patients, sont donc nécessaires. Nous faisons l’hypothèse qu’une hypertrophie induite par l’invalidation de la myostatine (mstn), régulateur négatif majeur de la masse musculaire, pourrait être bénéfique pour la souris modèle de cette pathologie (KI-Dnm2R465W/+), permettant notamment le maintien de la masse et de la force musculaire. Nous avons généré un modèle doublement muté résultant du croisement de souris KI-Dnm2R465W/+ myopathe avec des souris KO-mstn hypermusclées. Notre étude démontre que l'inactivation du gène mstn permet une amélioration de la masse et du volume musculaire, limite la perte de force et de motricité. Nos données suggèrent également que cette amélioration est majoritairement due à une diminution du niveau d’expression de certains acteurs impliqués dans le système catabolique ubiquitine-protéasome. De plus, nous montrons une accélèration de la diminution de la fréquence des anomalies histologiques caractéristiques de la myopathie chez les souris KI-Dnm2R465W/+. Nous proposons que ces anomalies pourraient être dues à une altération de la structure et/ou de la fonction mitochondriale. / Autosomal dominant centronuclear myopathy (AD-CNM) is a rare congenital muscle disease caused by mutations predominantly found in the dynamin 2 gene (DNM2). The clinical features generally reported are progressive muscle atrophy and weakness. To date, no treatment is available. The mouse model for AD-CNM harboring a mutation of the dynamin-2 gene (KI-Dnm2R465W/+) reproduces some of the human clinical features, notably muscle atrophy and weakness. Mstn, is a master negative regulator of skeletal muscle mass. We hypothesized that inactivation of mstn could limit muscle atrophy and weakness reported in the AD-CNM mouse model (KI-dnm2R465W/+). To test this hypothesis, we intercrossed KI-Dnm2R465W/+ mice with mice inactivated for mstn (KO-mstn) to generate a double mutated lineage (KIKO). The present study demonstrates that mstn gene inactivation allows for an improvement of muscle weight and volume, prevents muscle weakness and motor skill alterations. Our data also reveal that inactivation of mstn essentially downregulates some actors implicated in the catabolic ubiquitin-proteasome system. Furthermore, we show that inactivation of mstn decreases the frequency of of histological abnormalities characteristical in KI mice. We hypothesize that these abnormalities could be due to an alteration of mitochondrial function and network. The perspective to this work is to verify this hypothesis in the mouse model, which will contribute to a better understanding of the physiopathological mechanisms and can open new insight in the therapeutical approach to AD-CNM.

L’édition du génome utilisant CRISPR/Cas9 est récemment apparue comme une stratégie thérapeutique potentielle des maladies génétiques. Pour les mutations dominantes de type gain de fonction, la correction allèle-spécifique pourrait être l'approche la plus appropriée. Ici, nous avons testé l'inactivation ou la correction d'une mutation hétérozygote du gène de la dynamine 2 (DNM2) causant la forme autosomique dominante de la myopathie centronucléaire (CNM). Des ARN-guides tronqués ciblant spécifiquement l'allèle muté ont été testés sur des cellules de patients et des myoblastes d'un modèle murin. L'allèle muté a été ciblé avec succès et des clones ont été obtenus avec inactivation ou correction précise du génome. Les myoblastes Dnm2R465W/+ ont montré une altération de l'endocytose et de l'autophagie. L'inactivation ou la correction allèle-spécifique a normalisé ces phénotypes. L'allèle muté a également été ciblé avec succès dans les muscles de la souris Dnm2R465W/+. Ces résultats illustrent le potentiel de CRISPR/Cas9 à cibler et corriger de manière allèle-spécifique les mutations ponctuelles hétérozygotes de type de gain de fonction. / Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. Here we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene causing the autosomal dominant form of centronuclear myopathies (CNM). Truncated single guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with both precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. The mutated allele was also successfully targeted in Dnm2R465W/+ mouse muscles. These findings illustrate the potential of CRISPR/Cas9 to target and correct heterozygous point mutations leading to a gain-of-function effect in an allele-specific manner.