Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Orientador: Robson Francisco Carvalho / Resumo: Os exossomos constituem uma classe de pequenas vesículas extracelulares de aproximadamente 40-150 nm originadas pelo sistema endossomal. Essas vesículas são produzidas constitutivamente por praticamente todos os tipos celulares, e são responsáveis por transportar distintas classes de moléculas, em resposta a diferentes estímulos. O TNF-α é uma citocina pró-inflamatória que em condições tais como AIDS, septicemia, diabetes, insuficiência cardíaca, doença pulmonar obstrutiva crônica, diabetes e câncer, contribui para a atrofia de fibras musculares esqueléticas. Nesse sentido, o objetivo do presente estudo foi caracterizar os RNAs transportados por exossomos de miotubos C2C12 com atrofia induzida por TNF-α e avaliar a ação desses exossomos no fenótipo de mioblastos e miotubos C2C12. Exossomos liberados no meio de cultura de miotubos C2C12 tratados com TNF-α, e de seus respectivos controles, foram isolados por ultracentrifugação e analisados quanto ao seu conteúdo de RNA por RT-qPCR (microRNAs) e sequenciamento de nova geração (RNA total). Além disso, mioblastos e miotubos C2C12 foram tratados com esses mesmos exossomos para analisar a expressão de genes envolvidos com ciclo celular e a capacidade de migração/proliferação (mioblastos), bem como na atrofia celular (miotubos). Nossos resultados demonstraram que exossomos provenientes de miotubos são capazes de alterar os níveis de transcritos de genes envolvidos na proliferação, motilidade e diferenciação de mioblastos em miotubos.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Exosomes constitute a class of small extracellular vesicles of approximately 40-150 nm originated by the endosomal system. These vesicles are constitutively produced by virtually all cell types and are responsible for carrying distinct classes of molecules in response to different stimuli. TNF-α is a proinflammatory cytokine that in conditions such as AIDS, septicemia, diabetes, heart failure, chronic obstructive pulmonary disease, diabetes and cancer, contributes to atrophy of skeletal muscle fibers. In this sense, the objective of the present study was to evaluate the expression and action of RNAs transported by exosomes from C2C12 myotubes with TNF-α-induced atrophy in the phenotype of myoblasts and myotubes. Exosomes released in the culture medium from TNF-α treated C2C12 myotubes and from their respective controls were isolated by ultracentrifugation and analyzed for their RNA content by RT-qPCR (microRNAs) and new generation sequencing (total RNA). In addition, myoblasts and C2C12 myotubes were treated with these same exosomes to analyze the expression of genes involved in cell cycle and migration / proliferation capacity (myoblasts), as well as cell atrophy (myotubes). Our results demonstrated that exosomes from myotubes alter the expression genes involved in the proliferation, motility and differentiation of myoblasts into myotubes. We also verified that exosomes from TNFα-treated myotubes decreased migration / proliferation capacity of myoblasts. In addition, we iden... (Complete abstract click electronic access below) / Doutor

Le développement du muscle squelettique est un processus complexe très finement régulé, qui inclus des étapes de prolifération de cellules progénitrices appelées myoblastes et des étapes de différenciation pour former des myotubes multi nucléés. La glycosylation est la principale modification post-traductionnelle des protéines. Son rôle dans divers processus biologiques et pathologiques est largement documenté, mais les mécanismes intimes de son implication lors du processus myogénique restent mal élucidés. Nous avons pris comme modèle cellulaire la lignée myoblastique C2C12 car elle est capable de mimer in vitro les étapes de prolifération et de différenciation de la cellule musculaire. En utilisant différentes lectines, nous montrons un changement de la sialylation périphérique en α2-6 des glycoconjugués de surface de la cellule C2C12 durant la différenciation myoblastique. En complément, nous avons analysé les N-glycannes des glycoprotéines par spectrométrie de masse et mesuré les niveaux d’expression des gènes des α2-6 sialyl-transférases et neuraminidases. Tous les résultats obtenus confirment bien que la différenciation des cellules C2C12 est accompagnée d’une diminution du taux de sialylation des glycoconjugués. Pour mieux comprendre l’implication de la sialylation en α2-6 dans la myogenèse, nous avons réalisé une étude fonctionnelle sur des cellules C2C12 qui sous-expriment St6gal1 du fait de l’introduction d’un shRNA spécifique. Les clones obtenus présentent de plus forts index de fusion et génèrent un plus grand nombre de myotubes qui, de surcroit, sont de grande taille. Ce phénotype est probablement dû à un engagement accru des cellules de réserve en différenciation. En effet, les clones sous-exprimant St6gal1 contiennent une plus petite proportion de cellules Pax7+, c’est-à-dire de cellules de réserve maintenues dans un état de quiescence.Ainsi, nos résultats montrent l’importante implication de la sialylation périphérique en α2-6 au cours de la différenciation myogénique. / Skeletal muscle development is a complex process highly regulated and which includes proliferation then differentiation of progenitor cells or myoblasts into multi-nucleated myotubes. Glycosylation is the main post-translational modification of proteins. Its role in various biological and pathological processes is well documented, but the precise mechanisms of its involvement during myogenesis are still poorly understood.We have used the C2C12 myoblast as a model cell line since it is able to mimic in vitro the steps of muscle cell proliferation and differentiation. Using different lectins we showed a change in the peripheral α2-6 sialylation of the cell surface glycoconjugates, during C2C12 differentiation. Besides, we also analyzed by mass spectrometry the N-glycans carried by glycoproteins and measured the expression levels of α2-6 sialyl-transferases and neuraminidases genes. All the results confirm that C2C12 differentiation is accompanied by a decrease of glycoconjugates sialylation. To highlight the involvement of α2-6 sialylation in myogenesis, we performed a functional study of C2C12 cells knockdown for St6gal1 by a specific shRNA. The generated clones exhibit a higher fusion index and generate more elongated myotubes. This phenotype probably results from an increased commitment of reserve cell in differentiation. Indeed, the clones knockdown for St6gal1 contain a lower proportion of Pax7+ cells, i.e. of reserve cells maintained in a quiescent state. Thus, our results show the significant involvement of the peripheral α2-6 sialylation during myogenic differentiation.

La famille Tead de facteurs de transcription reconnaît l'élément MCAT trouvé dans le promoteur de gènes spécifiques au muscle. L'analyse génétique de leur fonction dans la différenciation musculaire a révélé difficile en raison de la redondance susceptible parmi les membres de la famille. Dans cette étude, nous avons utilisé le silencing siRNA médiation pour aborder le rôle des facteurs TEAD dans la différenciation des myoblastes primaire.Contrairement aux cellules C2C12 où Tead4 joue un rôle essentiel, son silence dans les myoblastes primaires a eu peu d'effet sur leur différenciation. Silence de facteurs individuels TEAD n'a eu aucun effet significatif sur la différenciation des myoblastes primaires, alorsque le silençage combinatoire a conduit à l'inhibition de leur différenciation indiquant laredondance parmi ces facteurs. Dans les cellules C2C12 aussi, combinatoire silençageTead eu des effets beaucoup plus puissants que de faire taire Tead4 seule indiquant une contribution des autres Teads dans ce processus. En intégrant Tead1 et les données Tead4ChIP-Seq avec les données d'ARN-Seq suivante combinatoire Tead1 / 4 silencieux, nous identifions ensembles distincts, mais qui se chevauchent de gènes Tead réglementés dansles deux cellules C2C12 myoblastes et primaires. Nous avons également intégré les / 4 données Tead1 ChIP-seq avec des ensembles de données publiques sur Myog et MYOD1ChIP-Seq et chromatine modifications à identifier une série d'éléments de régulation actifsliés par des facteurs TEAD seul ou avec Myog et MYOD1. Ces données disséquer les fonctions spécifiques et combinatoires de ces facteurs de transcription dans les réseaux derégulation de le differentiation musculaire. / The Tead family of transcription factors recognise the MCAT element found in thepromoters of muscle-specific genes. Genetic analysis of their function in muscledifferentiation has proved elusive likely due to redundancy amongst the family members.We previously used shRNA-mediated silencing to show that loss of Tead4 function resultedin abnormal differentiation characterised by the formation of shortened myotubes. ChIP-chipcoupled to RNA-seq data identified a set of potential target genes that are either activatedor repressed by Tead4 during differentiation. In this study, we have used siRNA-mediatedsilencing to address the role of the Tead factors in primary myoblast differentiation. Incontrast to C2C12 cells where Tead4 plays a critical role, its silencing in primary myoblastshad little effect on their differentiation. Silencing of individual Tead factors had no significanteffect on primary myoblast differentiation, whereas combinatorial silencing led to inhibitionof their differentiation indicating redundancy amongst these factors. In C2C12 cells also,combinatorial Tead silencing had much more potent effects than silencing of Tead4 aloneindicating a contribution of other Teads in this process. By integrating Tead1 and Tead4ChIP-seq data with RNA-seq data following combinatorial Tead1/4 silencing, we identifydistinct but overlapping sets of Tead regulated genes in both C2C12 cells and primarymyoblasts. We also integrated the Tead1/4 ChIP-seq data with public data sets on Myogand Myod1 ChIP-seq and chromatin modifications to identify a series of active regulatoryelements bound by Tead factors alone or together with Myog and Myod1. These datadissect the specific and combinatorial functions of these transcription factors in muscledifferentiation regulatory networks.

Muscle loss from lack of activity is a serious issue for immobilized patients on Earth and in human spaceflight, where the low gravity environment prevents normal muscle activity. Simulating muscle loss in cultured cells is an important step in understanding how this condition occurs. This work evaluates different means of simulating muscle loss and selects the one that most closely mimics the cellular responses seen in animals and humans. To simulate the microgravity environment of spaceflight, mouse skeletal muscle cells were grown in a rotary cell culture system (RCCS). Growing the cells within a natural gelled substrate was compared against growing them on the surface of small plastic beads. Changes after culture under simulated microgravity were characterized by assessing proteins and genes known to change during muscle loss. The structure of the cells was also evaluated by microscopy. The mouse skeletal muscle cells grown on plastic beads in the RCCS had significant changes in multiple key genes associated with muscle loss and demonstrated physical characteristics expected of mature tissue in live animals. This model is a valuable platform for exploring muscle loss mechanisms and testing new drugs.

Muscle and bone are two tightly connected systems on both an anatomic and functional level. Bone and muscle diseases like osteoporosis and sarcopenia have been found to show an association with each other. These two organs form a complex musculoskeletal system and have been found to secrete hormone-like factors called myokines and osteokines that can influence and affect each other. Indeed, the crosstalk between bone and muscle plays an important role during development and aging. For example, myostatin, also known as growth differentiation factor 8 (GDF-8), a cytokine secreted by muscle cells, is a negative regulator of muscle and bone mass. Over expression or loss of function mutations of myostatin in mice have led to muscle atrophy and hypertrophy respectively. Interleukin-6 (IL-6) is expressed abundantly in muscle and is released during exercise and muscle contraction. It has been shown to increase osteoclast (bone cells that break down bone) formation. In the bone, osteocytes make up the majority of all cells and are terminally differentiated osteoblasts. Osteocytes control the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteocytes are also known to express receptors for various hormones, including parathyroid hormone (PTH) receptor. As osteocytes comprise more than 90% of all bone cells in adult bone, we hypothesize that osteocytes might secrete factors capable of controlling muscle cells and that PTH might control the expression of these factors. To test this hypothesis, we used an osteocytic cell line Ocy454-12H as well as C2C12 cells, which are a well-accepted model of myocyte differentiation. To investigate the effects of osteocyte-derived factors on myocytes, C2C12 cells were treated with conditioned medium (CM) from osteocytes during specific times. We found that during C2C12 proliferation, when compared to the αMEM control, mRNA expression of MSS51 was decreased for both cells that were treated with CM of osteocytes treated with PTH (PTH CM, p=0.00570) and cells that were treated with CM of osteocytes treated with vehicle only (CM control, p=0.0178). During C2C12 differentiation, mRNA expression of myostatin was significantly (p=0.0387) decreased in cells that were treated with PTH CM compared to cells that were treated with CM control. Considering the importance of mitochondrial respiration in cells, we next analyzed oxygen consumption and metabolism in C2C12 myocytes treated with CM from Ocy454-12H using a Seahorse XF Cell Mito Stress Test. Metabolic analysis revealed that during proliferation, PTH CM led to higher basal respiration, ATP production, and coupling efficiency in C2C12 cells while lowering spare respiratory capacity. In differentiation, there was a trend in which CM control would cause a decrease across all parameters compared to the control group and the PTH CM group. Interestingly, PTH CM-treated C2C12 cells were shown to have a higher oxygen consumption rate (OCR) than the CM-control treated group and would have similar values to that of the control group (C2C12 not treated with CM). Taken together these results suggest that osteocytes might control muscle cells differentiation and metabolism via a PTH-mediated signaling pathway.

The purpose of this dissertation was to investigate the molecular events associated with the onset of skeletal muscle disuse in humans. Study I examined global gene expression changes in vastus lateralis muscle following 48h unloading (UL) and 24h reloading (RL) in humans. Results showed that functions related to protein degradation and oxidative stress were enriched following UL and that these global gene expression patterns were not readily reversed following RL, thus indicating that molecular events associated with short-term disuse may persist beyond the duration of the stimulus. In contrast to previous work in IM, collagen gene expression increased in this study, demonstrating that differences in molecular signaling may exist among disuse models in humans. Study II of this dissertation expanded on the findings of Study I to investigate global gene expression patterns related to the early stage of multiple disuse models. Microarray data collected 48h post-UL in Study I were analyzed within the context of data previously collected in our laboratory following 48h immobilization (IM) and spinal cord injury (SCI). Results showed that the disuse models shared a small subset of commonly differentiated genes. Furthermore, the similarities between IM, SCI, and UL extended beyond specific genes to include commonly enriched functions and pathways such as protein degradation and oxidative stress, suggesting that these molecular mechanisms are involved in the early stages of disuse, regardless of specific stimulus. In Study III, an in vitro model of skeletal muscle was used to test the exploratory hypothesis that induction of oxidative stress response gene heme oxygenase-1 (HMOX1) would lead to decreases in gene expression associated with proteolysis, namely ubiquitin E3 ligases atrogin1 and MuRF1, as well as increased XXT cleavage (a marker of metabolic enzyme activity). In this study, C2C12 myotubes were pre-treated with hemin (an inducer of HMOX1) and then treated with H2O2 to elicit oxidative stress. Results showed that hemin treatment resulted in increased HMOX1 expression and decreased E3 ligase expression. Furthermore, hemin-treated cells exhibited increased XTT cleavage compared to controls. HMOX1 may be a promising gene target to protect against oxidative stress that accompanies early stages of disuse.