The extracellular matrix (ECM) of the skeletal muscle provides the framework for the muscle structure and plays a key role in the repair and maintenance of myofibers through the resident fibroblasts and muscle satellite cells. However, excessive production of ECM components, notably collagen, leads to fibrosis which impedes muscle function, impairs the natural repair process, and leads to muscle weakness. Fibrosis is a hallmark of muscular dystrophies, including Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy is a terminal, x-linked disorder characterized by progressive muscle wasting as muscle fibers are replaced by fibrosis and fat. There are approximately 300,000 DMD patients worldwide, and the few disease modifying treatments are genotype specific, only helping a small percentage of the patient population. Myostatin is a member of the transforming growth factor beta (TGF-β) family of ligands, is a negative regulator of muscle mass, and may also contribute to the fibrotic environment in dystrophic muscle through myofibroblast proliferation and survival. Therefore, myostatin blockade could potentially ameliorate muscle weakness in DMD patients by increasing skeletal mass and function while also reducing the accumulation of fibrosis. A murine anti-myostatin antibody, mRK35, and its humanized analogue, domagrozumab, are specific and potent inhibitors of myostatin. mRK35 was tested in multiple mouse models, from healthy C57Bl/6 and C57Bl/10, mildly dystrophic C57Bl/10.mdx, and severely dystrophic D2.mdx mice, for changes in muscle mass, muscle function, and fibrotic content. Additionally, inflammatory, fibrotic, and myogenic gene expression changes were analyzed in the severely dystrophic animals treated with mRK35. Domagrozumab was tested in non-human primates (NHPs) for changes in skeletal muscle mass. Myostatin blockade with mRK35 resulted in muscle anabolic and functional improvements in healthy murine models and NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume. However, as mice age or as the dystrophic severity of the model increases, the anabolic effect of myostatin inhibition is diminished. The extensor digitorum longus (EDL) muscle escapes this trend and is the most responsive to myostatin inhibition across all mouse strains and disease severities. However, analysis of the fibrotic content in the triceps and diaphragms of D2.mdx mice treated with mRK35 for 8 weeks does not reveal any change in fibrotic content. Gene expression changes in the muscles within these mice appear to be tightly tied to their healthy or dystrophic state and myostatin inhibition has minimal effect. In sum, while specific myostatin inhibition with mRK35 increases muscle weight and function in mice, there is no conclusive evidence of reduced muscle fibrosis. / 2023-06-22T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21827号 / 農博第2340号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5199(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 准教授 豊原 治彦, 准教授 田川 正朋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p>Skeletal muscle exhibits a remarkable capacity for growth and regeneration in response to physiological stimuli. This extensive plasticity is, in part, due to a tissue-resident stem cell called the satellite cell. Satellite cells respond to myotrauma by upregulating a class of transcriptional networks which orchestrate myogenic specification. This process is controlled by four main transcription factors known as the myogenic regulatory factors: Myf5, MyoD, MRF4 and Myogenin. Satellite cells respond to molecular cues released from the muscle fiber or inflammatory cells in response to muscle damage. Although several regulators have been implicated in the control of the satellite cell response to exercise or damage, very few of these have been examined in humans. Insulin-like growth factor-1 (IGF-1) and Interleukin-6 (IL-6) have been demonstrated to enhance satellite cell proliferation in animal and cell culture models. IGF-1 has also been shown to induce myogenic differentiation, however little is known about IGF-1 and IL-6 in humans, in response to physiological levels of muscle damage. Myostatin has been identified as a negative regulator of muscle growth and an inhibitor of satellite cells in mice. To date no data exists regarding the relation of myostatin to the satellite cell response to exercise and in the context of aging. The work outlined in this thesis provides support for the proposed divergent effects of the IGF-1 splice variants on satellite cell function. IGF-1 appears to be preferentially spliced as IGF-1Ec during the proliferative phase of the myogenic program while IGF-1Ea and Eb appear as the predominant splice variants during the initiation of differentiation based on the expression of the MRFs. Furthermore, the localization of IGF-1 with Pax7 in muscle-cross sections in the post-exercise time-course lends support to the importance of IGF-1 in the myogenic response to myotrauma. This thesis also provides novel evidence to support the role of IL-6 in the regulation of satellite cell proliferation in response to acute muscle damage in humans. These data confirm that IL-6 imparts its action on the satellite cell via the JAK2/STAT3 pathway. In addition, for the first time, myostatin is demonstrated to be altered by acute exercise in both young and older adults and this effect is most notable in the satellite cell compartment. In addition, these data implicate myostatin as a contributing factor to age-related satellite cell dysfunction in response to exercise (or myotrauma).</p> / Doctor of Philosophy (PhD)

No description available.

Essentielle à l'équilibre énergétique de la cellule, la mitochondrie, véritable sentinelle, joue, un rôle majeur dans le destin de la cellule, en modulant les voies de signalisation de mort cellulaire mis en jeu dans l'atrophie musculaire. L'objectif de cette thèse est de proposer des cibles thérapeutiques centrées sur la mitochondrie dans deux modèles murins dont la physiopathologie est caractérisée par une dysfonction mitochondriale associée à une atrophie musculaire : le vieillissement et la dystrophie musculaire de Duchenne (DMD). Pour lutter contre la perte de masse musculaire liée à l'âge, la déficience en myostatine (mstn), associée à un phénotype hypermusculé, est une stratégie thérapeutique prometteuse. Mais, l'altération du métabolisme mitochondrial et oxydatif induite par cette déficience réduit les effets bénéfiques d'une telle stratégie. Nous avons donc testé l'intérêt de l'utilisation de la molécule pharmacologique AICAR, activateur connu de l'AMPK, afin de « booster » la fonction mitochondriale chez la souris âgée KO mstn. Les résultats montrent chez la souris KO mstn, une amélioration du temps d'endurance de course. Au niveau signalétique, le traitement induit des effets bénéfiques mais limités sur la fonction mitochondriale. Les mécanismes restent à préciser mais tendent vers l'hypothèse d'un effet bénéfique de l'AICAR sur le stress du réticulum endoplasmique (RE). Le dysfonctionnement mitochondrial a été également largement impliqué dans la physiopathologie de la DMD. Dans notre seconde étude, ce même traitement à l'AICAR chez le modèle murin de la DMD, la souris mdx atténue le phénotype dystrophique et améliore la fonction contractile du diaphragme. Nous montrons que ces effets bénéfiques sont associés à une induction de mécanisme de survie, l'autophagie, et une limitation des phénomènes d'apoptose induit par la mitochondrie, mettant en évidence une amélioration de l'intégrité mitochondriale par stimulation de leur renouvellement dans des fibres musculaires dystrophiques. Enfin, ce travail a mis en avant pour la première fois la présence à l'état basal de stress du RE chez la mdx, propsant une nouvelle cible thérapeutique. L'impact de ce stress dans la fibre musculaire normal et pathologique est très mal connu. Nos résultats montrent que le stress du RE modifie les liens entre le réticulum sarcoplasmique et la mitochondrie, perturbe l'homéostasie calcique et active les voies de mort cellulaire associées à une dysfonction contractile. Ces résultats ouvrent une perspective de stratégie thérapeutique dans les pathologies musculaire impliquant un stress du RE, comme la DMD. Ce travail de thèse a mis en avant l'importance de développer des thérapies pharmacologiques dans les pathologies musculaires, permettant d'améliorer la fonction à la fois métabolique et de sentinelle de la mitochondrie. / Fundamental for the energetic balance of the cell, mitochondria play a key role for modulation of cell death pathway related to muscular atrophy. Thus, the purpose of this PhD is to find therapeutic strategy focus on mitochondria in two different murine models where the physiopathology is characterized by a mitochondria dysfunction associated with muscle atrophy: Aging process and Duchenne Muscular Dystrophy (DMD).To prevent loss of muscle mass associated with aging, the lack of myostatin, inducing a hypermuscular phenotype, is a promising therapeutic strategy. However, loss of myostatin is associated with a strong reduction of mitochondrial and oxidative metabolism in skeletal muscle, and this strategy need to be potentiated. In this context, we explore if mitochondrial alteration in aged wild-type mice or in aged mstn KO mice are rescued by chronic AMPK-activating treatment, using the synthetic agonist AICAR, considered as “an mimetic of exercise”. Our results show an improvement of aerobic running performance in mstn KO mice. Concerning to signaling pathways, AICAR treatment induces beneficial but limited effects on mitochondrial metabolism. Mechanisms are still under investigation but our results suggest a reduction in ER stress. Moreover, mitochondria dysfunction has been widely implicated in DMD physiopathology. This same treatment of AICAR, in the murine model of DMD, improves the diaphragm histopathology as well as maximal force generating capacity. These beneficial effects were linked with autophagy activation and apoptosis limitation, without inducing muscle fiber atrophy, and promoting the elimination of defective mitochondria.Finally, the last part of this study highlight for the first time, an increase of ER stress at basal level, suggesting a new therapeutic target. Nevertheless, ER stress impact in skeletal muscle fibers is sparsely known. The preliminary results show that ER stress decrease the link between RE and mitochondria, which have an impact on calcium homeostasis and stimulate cell death pathway with a decrease of contractile function.This study highlights the importance to develop pharmacological therapies in muscular pathology, focus on metabolic and sentinel mitochondria function.

Les muscles squelettiques, responsables des mouvements volontaires tels que la locomotion ou le maintien de la posture, représentent environ 40% de la masse corporelle.Cette masse musculaire est maintenue par plusieurs voies de signalisation qui régulent entre autres l’équilibre entre la synthèse et la dégradation des protéines myofibrillaires. En ciblant la voie de signalisation Akt/mTOR, la myostatine est un régulateur négatif de la myogenèse. Elle inhibe la différentiation myogénique et le renouvellement cellulaire. Parmi les différents facteurs moléculaires extracellulaires qui régulent la myostatine, les protéines GASP (Growth and differentiation factor Associated Serum Protein) ont été décrites comme des antagonistes de son activité. L’Unité de Génétique Animale a développé plusieurs stratégies qui permettent d’appréhender les mécanismes moléculaires qui régissent le(s) rôle(s) des protéines GASP au cours du développement musculaire. Ainsi, la création de la lignée murine appelée surGasp-1-20 a permis de montrer que la surexpression de Gasp-1 entraîne un phénotype hypermusclé associé à une hypertrophie des myofibrilles. Une analyse de l’expression génique dans des myoblastes dérivés des cellules satellites montre une surexpression de la myostatine corrélant avec une absence d’hyperplasie chez les souris surGasp-1-20. Des études similaires actuellement en cours pour la protéine GASP-2 devraient permettre de préciser son rôle dans le contexte musculaire. Les protéines GASP sont également définies comme des inhibiteurs composés hétérotypiques caractérisés par plusieurs domaines inhibiteurs pouvant moduler l’activité de différentes protéases. Parmi ces différents domaines,le second domaine Kunitz de GASP-2 a été précédemment décrit comme pouvant inhiber la trypsine. Dans ce travail, nous avons pu montrer que les deux protéines entières conservent cette capacité d’inhibition. Nos résultats indiquent cependant que GASP-1 et GASP-2 présentent une différence de spécificité due à la composition du second domaine Kunitz et non à l’environnement moléculaire présent dans chacune des protéines. Enfin, nous proposons un modèle structural du second domaine Kunitz impliqué dans la dualité fonctionnelle dans l'inhibition anti-trypsine de GASP-1 et GASP-2. / Skeletal muscles, responsible for voluntary movements such as locomotion or posture maintenance, represent about 40% of body mass. This muscle mass is maintened by several signaling pathways that regulate , among other things, the balance between synthesis and degradation of myofibrillar proteins. By targeting the Akt/mTOR pathway, myostatin is anegative regulator of myogenesis. It inhibits myogenic differentiation and cell turnover. Among the various endogenous molecular factors that regulate myostatin, proteins GASP (Growth and differentiation factor Associated Serum Protein) have been described as antagonists of its activity. The Animal Genetics Unit has developed several strategies to understand themolecular mechanisms that govern the role (s) of GASP proteins during muscle development.Thus, the creation of the transgenic mouse line named surGasp-1-20 has shown that overexpression of Gasp-1 results in a hypermuscular phenotype associated with myofibril hypertrophy. An analysis of gene expression in myoblasts derived from satellite cells showed overexpression of myostatin correlating with an absence of hyperplasia in Gasp-1-20 mice.Similar studies currently underway for the protein GASP-2 should clarify its role in the muscular context. Proteins GASP are also defined as compound heterotypic inhibitors characterized by several inhibitory domains that can modulate the activity of different proteases. Among these different modules, the second Kunitz domain of GASP-2 was previously been described asable to inhibit trypsin. In this work, we have shown that the two whole proteins conserve this capacity of inhibition. However, our results indicate that GASP-1 and GASP-2 exhibit a difference in specificity due to the composition of the second Kunitz domain and not to the molecular environment present in each of the proteins. Finally, by modeling, we propose a structural model of the second Kunitz domain of GASP-1 and GASP-2 implicated in the antitrypsin inhibition specificity

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive genetic disease, caused by a frame-shift mutation in the dystrophin gene. Current gene therapies for DMD target dystrophin transcripts in existing skeletal and cardiac muscle, rather than adipose and fibrotic tissues. These approaches may be unable to repair muscle functionality in DMD patients who have already undergone extensive muscle damage and wasting. Thus, successful DMD therapies must consider the underlying genetic cause and pathology. Inhibition of the gene myostatin, a negative regulator of muscle growth, has been shown to ameliorate muscle loss. Here, the CRISPR-Cas9 gene-editing platform is proposed to restore dystrophin expression and inhibit myostatin as a novel gene therapy in DMD patient derived induced pluripotent stem cells. Successful CRISPR-Cas9 mediated gene editing would be determined using PCR amplification, western blot analysis, immunofluorescence staining, and off target sequence analysis in differentiated skeletal muscle cells.

Le muscle squelettique est un tissu hétérogène et dynamique jouant un rôle important dans la mobilité et le métabolisme d’un organisme. C’est un organe actif qui sécrète de nombreuses cytokines participant au « crosstalk » entre tous les tissus impliqués dans ce métabolisme. Parmi ces myokines, la myostatine agit à la fois comme un régulateur négatif du développement musculaire et un médiateur dans l’homéostasie du glucose. En effet, les souris déficientes pour le gène de la myostatine (Mstn-/-) présentent une augmentation de leur masse musculaire associée à une hyperplasie et une hypertrophie des myofibres. Elles présentent également une diminution de leur masse adipeuse. L’expression de la myostatine est finement régulée par des inhibiteurs comme la follistatine, FSTL3 ou les protéines GASP-1 et GASP-2. Si de nombreuses études ont déjà été réalisées sur les autres inhibiteurs, les protéines GASPs sont à l’heure actuelle encore peu étudiées. Le modèle murin surexprimant Gasp-1 (Tg(Gasp-1) généré dans le laboratoire présente un phénotype hypermusclé associé à une hypertrophie mais sans hyperplasie et ne présentent pas de diminution de leur masse adipeuse. Afin de mieux comprendre les conséquences fonctionnelles de la surexpression de Gasp-1, nous avons analysé des cellules musculaires dérivées de cellules satellites de souris Tg(Gasp-1). Cette étude a révélé une dérégulation de l’expression de plusieurs gènes dont une surexpression de la myostatine qui pourrait expliquer l’absence d’hyperplasie. Nous avons voulu également expliquer l’absence de variation de masse adipeuse dans les souris Tg(Gasp-1) en réalisant des analyses métaboliques sur des souris jeunes et âgées. Ces travaux ont révélé une dérégulation globale de l’homéostasie du glucose dans les souris Tg(Gasp-1) associé à une dérégulation du sécrétome musculaire. Enfin nous avons voulu appréhender le rôle de GASP-2 dans le contexte musculaire. / Skeletal muscle is a heterogeneous and dynamic tissue which plays an important role in mobility and metabolism of organisms. It is an active organ that secretes numerous cytokines involved in "crosstalk" between all tissues implicated in metabolism. Among these myokines, myostatin acts both as a negative regulator of muscle development and a mediator in glucose homeostasis. Indeed, mice deficient for the myostatin gene (Mstn-/-) have an increase of muscle mass associated with hyperplasia and hypertrophy of myofibers. Mstn-/- mice also exhibit a decrease of fat mass. Expression of myostatin is tightly regulated by inhibitors such follistatin, FSTL-3 or GASP-1 and GASP-2 proteins. While many studies have already been performed on the other inhibitors, GASPs proteins are still poorly studied. The mouse model overexpressing Gasp-1 (Tg (Gasp-1)) generated in our lab presents a hypermuscular phenotype associated with hypertrophy without hyperplasia and exhibit no decrease in fat mass. To better understand the functional consequences of Gasp-1 overexpression, we analyzed muscle cells derived from Tg(Gasp-1) satellite cells This study revealed a deregulation of the expression of several genes with an upregulation of myostatin which could explain the absence of hyperplasia in the Tg(Gasp-1) mice. We then want to explain the absence of fat mass changes by performing metabolic assays in young and aged mice. These studies have revealed an overall dysregulation of glucose homeostasis and deregulation of muscle secretome in Tg(Gasp-1) mice. Finally we wanted to capture the role of GASP-2 in a muscular context.

La myostatine est un membre de la superfamille du TGF-β (transforming growth factor-β) impliqué dans la régulation négative de la masse musculaire. En effet, l’absence de myostatine (MSTN) chez la souris est responsable d’un phénotype hypermusclé. Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduit à une augmentation de la masse musculaire chez d’autres espèces, y compris l’Homme. L’identification de la MSTN et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine. Il existe de nombreuses situations pathologiques qui conduisent à une fonte musculaire importante : c’est le cas pour des maladies génétiques telles que les dystrophies musculaires ou pour d’autres pathologies comme le cancer et le sida. Différentes approches anti-MSTN ont été développées au cours des dernières années, par exemple un anticorps anti-MSTN ou des ligands de la MSTN. L’objectif majeur de ce projet de recherche a consisté à identifier de nouveaux inhibiteurs de la MSTN, en particulier appartenant à la famille de protéines appelées SLRP (Small Leucine Rich Proteoglycans). Il a été mis en évidence que des membres de cette famille, notamment la décorine (DCN) ainsi que des fragments issus de la DCN dont le peptide 31-71, sont capables de se lier à la MSTN en présence de zinc. La DCN peut alors empêcher l’activité de la MSTN en s’opposant à la liaison de cette dernière à son récepteur. Dans ce contexte, nous avons étudié des séquences peptidiques plus restreintes de la DCN murine pouvant interagir efficacement avec la MSTN et des peptides dérivés d’autres SLRP pour leur aptitude à lier la MSTN. Afin de faciliter le criblage in vitro de ces composés, nous avons tout d’abord créé une lignée cellulaire HEK293T exprimant stablement une cassette inductible par la MSTN fusionnée au gène de la luciférase (pCAGA-Luc). Parmi les candidats testés, le peptide mDCN48-71 a été le plus intéressant de par sa forte activité anti MSTN in vitro comparée aux autres, avec un IC50 de 7 µM. Notons également que le peptide mDCN48-71 n’a pas inhibé d’autres membres de la superfamille du TGF-β : TGF-β2, activine A et GDF-11 – ce qui suggère une spécificité d’action du peptide. En outre, des études d’anisotropie de fluorescence ont permis de prouver l’interaction directe du peptide mDCN48 71 avec la MSTN et la dépendance au zinc de cette liaison. Pour finir, nous avons montré que des injections intramusculaires répétées de ce peptide chez le modèle murin dystrophique mdx, conduisent à une augmentation significative de la masse des muscles tibiaux antérieurs injectés de l’ordre de 21 % par rapport aux muscles contrôles. / Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily and a negative regulator of skeletal muscle growth. In 1997, lack of myostatin (MSTN) was related to increased muscle mass in mice. Since then, MSTN has been found in other species including humans. Inhibition of this protein offers opportunities in human medicine for many pathological conditions leading to a significant muscle loss: genetic disorders such as muscular dystrophy as well as other diseases like cancer and AIDS. Recently, several anti-MSTN approaches have been developed such as antibodies against MSTN or naturally occurring proteins that bind to and inactivate MSTN. The aim of this research was to identify novel inhibitors of MSTN, especially belonging to the SLRP (Small Leucine Rich Proteoglycans) family of proteins. Members of this family, including decorin (DCN) and fragments thereof (murine derived peptide mDCN31-71) can bind to MSTN in a zinc-dependent manner. In this context, smaller peptide sequences of mouse DCN and peptides from other SLRP have been studied for their ability to bind MSTN. First, we created a HEK293T stable cell line expressing the luciferase gene under control of a MSTN-inducible promoter (pCAGA-Luc) so as to screen these compounds in vitro. Here we report that the peptide mDCN48-71 shows the stronger activity anti MSTN in vitro among all the peptides tested (IC50 = 7 µM). Furthermore, other members of the TGF β superfamily: TGF β2, activin A and GDF-11 are not inhibited by the mDCN48-71 peptide - which suggests a specificity of its action. By performing fluorescence anisotropy studies, we proved the direct and zinc dependent interaction between peptide mDCN48-71 and MSTN. Finally, we showed that repeated intramuscular injections of this peptide in the dystrophic mdx mouse model led to a significant increase of the injected tibialis anterior muscle mass (21 %) compared to control muscles.

Orientador: Lucia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:30:05Z (GMT). No. of bitstreams: 1 Mantovani_CarolinaStefano_M.pdf: 7530088 bytes, checksum: 5c8cf5e001032d6962337caaccfcbcd8 (MD5) Previous issue date: 2015 / Resumo: A Miostatina (MSTN) é uma proteína que regula negativamente a formação de musculatura esquelética, e sua estrutura e função são conservadas em diversas espécies, incluindo humanos. O nocaute de seu gene causa hiperplasia e hipertrofia das fibras musculares, o que despertou grande interesse médico e agropecuário desde a descoberta deste gene. Trabalhos anteriores descrevem a função da proteína, mas ainda faltam dados sobre a regulação da sua expressão gênica. Recentemente, o promotor do gene da Mstn foi identificado e caracterizado pelo nosso grupo de pesquisa, a partir de uma abordagem filogenética. Entretanto, além do promotor, também existem outros elementos cis-reguladores que podem atuar como estimuladores ou silenciadores do processo de transcrição gênica. Em conjunto com o promotor gênico, esses elementos controlam a taxa de transcrição e conferem especificidade espacial e temporal para sua expressão. Sendo assim, a proposta deste trabalho foi identificar e caracterizar possíveis elementos cis-reguladores da Mstn a fim de ampliar o conhecimento sobre a sua regulação transcricional. Para tal, análises de genômica comparativa, semelhantes àquelas empregadas na identificação do promotor gênico da Mstn, foram realizadas para localizar regiões evolutivamente conservadas (ECRs) adjacentes ao lócus da Mstn de humano, camundongo e galinha, bem como para identificar potenciais sítios de ligação para fatores transcricionais conservados nessas regiões. Além disso, foi realizada uma análise da evolução desses possíveis elementos cis-reguladores, a fim de identificar polimorfismos que tenham o potencial de afetar a atividade transcricional da Mstn. Nossos resultados revelaram a existência de um arcabouço regulatório ancestral composto por sítios de ligação conservados em várias das ECRs analisadas, o qual tem sido mantido entre 310 e 430 milhões de anos. Além disto, foram identificados polimorfismos, bem como sítios de ligação grupo-específicos, os quais podem estar envolvidos na regulação diferencial da atividade da Mstn e, portanto, na geração de diferentes fenótipos musculares entre os animais. Dentre as oito ECRs estudadas, duas possuem maior potencial de estarem envolvidas na miogênese, uma vez que nelas foram identificados sítios de ligação para importantes fatores relacionados a este processo. Análises funcionais das ECRs 2 e 5/6 em células C2C12 em diferenciação confirmaram o potencial dessas ECRs de humano e camundongo de atuarem como elementos cis-reguladores, uma vez que mostraram que eles são capazes de modificar a expressão do gene repórter EGFP em comparação ao controle. As ECRs de galinha, por outro lado, não geraram resultados significativos, reforçando a hipótese de que as diferenças nos TFBS encontradas no grupo das aves geram alterações funcionais nas ECRs. O desenrolar de novos estudos a partir dos resultados obtidos neste trabalho permitirão estabelecer o papel específico das ECRs identificadas, bem como determinar a importância das variações de sequências destes elementos reguladores em diferentes animais. No longo prazo, nossas pesquisas poderão subsidiar o desenvolvimento de estratégias que possibilitem a modulação da atividade da Mstn em patologias humanas que afetam a musculatura esquelética / Abstract: The Myostatin protein (MSTN) is an important regulator of skeletal muscle deposition in vertebrates, and its structure and function are conserved in several species, including humans. The knockout of this gene causes hyperplasia and hypertrophy of muscle fibers, which has aroused great medical and agricultural interest since its discovery. Previous studies have described the function of this protein, but data on the regulation of Mstn gene expression are still scarce. Recently, the Mstn gene promoter has been identified and characterized by our research group from a phylogenetic approach. However, in addition to the promoter, there are also other cis-regulatory elements that can act as enhancers or silencers of gene transcription process. Along with the gene promoter, these elements control the transcription rate and provide spatial and temporal specificity for its expression. Thus, the purpose of this study was to identify and characterize potential Mstn cis-regulatory elements in order to increase knowledge of the transcriptional regulation of this gene. For this purpose, comparative genomic analysis similar to those employed in the identification of the Mstn promoter were performed to locate evolutionary conserved regions (ECRs) adjacent to the locus of the Mstn gene in human, mouse and chicken, as well as to identify potential transcription factors binding sites (TFBS) conserved in these regions. Furthermore, an evolutionary analysis of the putative cis-regulatory elements has been performed in order to identify polymorphisms that have the potential to affect the transcriptional activity of Mstn. Our results indicate the existence of an ancestral regulatory framework that comprises conserved TFBS in almost all of the ECRs analyzed, which has been maintained between 310 and 430 million years. Furthermore, polymorphisms were identified, as well as group-specific binding sites, which may be involved in the differential regulation of Mstn activity and, therefore, in the generation of different muscle phenotypes in animals. Among the eight ECRs studied, two of them have great potential to be involved in myogenesis, given that binding sites for important factors related to this processes were identified. Functional analyses of ECRs 2 and 5/6 in differentiating C2C12 cells confirmed the potential of the human and mouse ECRs as cis-regulatory elements, once they have shown that they are able to enhance the expression of the EGFP reporter gene, when compared to the control. The chicken ECRs, on the other hand, did not generate significant results, supporting the hypothesis that the differences found in the TFBS pattern in birds generate functional modifications in the ECRs. The development of new studies from the results obtained here may help to establish the specific role of the ECRs identified, as well as determine the importance of the variation in the sequence of these regulatory elements in different animals. In the long run, our research may lay the foundation to the development of strategies that allow the modulation of Mstn activity in human pathologies affecting skeletal muscle / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural