[ES] Los procesos de comunicación celular permiten a las células desarrollar una acción coordinada durante la embriogénesis y asimilar de forma coherente las señales recibidas a través del entorno. Algunas de las moléculas señalizadoras más usadas en la clínica y la investigación son las citoquinas. Sin embargo, existe una tendencia creciente en el uso de otro tipo de moléculas, como los iones metálicos. Algunos iones como el calcio y el zinc actúan como segundos mensajeros intracelulares. Otros como el litio son capaces de inactivar proteínas quinasa alterando rutas de señalización. En el desarrollo de esta tesis doctoral, se ha estudiado el efecto del zinc en células musculares de ratón, el papel del zinc en la auto-renovación de células madre embrionarias (CMEs), y el papel del litio en la diferenciación de CMEs. El estudio del efecto del zinc sobre los mioblastos demostró que el zinc es capaz de estimular la diferenciación de los mioblastos. El análisis del zinc intracelular, en los diferentes estadios de diferenciación de las células musculares, demostró que los miotubos eran capaces de albergar mayor cantidad de zinc en su interior. Los resultados mostraron que la adición de zinc extracelular estimula la fosforilación y activación de la proteína quinasa Akt. También se ha visto que el transportador de zinc, Zip7, es crítico en el proceso de diferenciación celular mediado por el zinc, además, su activación incrementa la fosforilación de Akt. La inhibición de Zip7 mediante ARN interferente redujo la fosforilación de Akt y consecuentemente origino unos niveles menores de diferenciación de los mioblastos expuestos a zinc extracelular. Nuestros resultados demuestran que altas concentraciones de zinc extracelular producen un incremento en la diferenciación de los mioblastos debido a la activación de Akt mediada por Zip7. Para el segundo estudio, se analizó el efecto del zinc sobre las CMEs. Como control de mantenimiento de la pluripotencia se usó medio suplementado con factor inhibidor de leucemia (LIF). Se ha observado que la adición externa de concentraciones de zinc superiores a 100 µM produce un incremento inmediato de la concentración de zinc intracelular activando Akt. Los resultados demuestran que las células tratadas con altas concentraciones de zinc mantienen su capacidad de auto-renovación. Para demostrar que el efecto del zinc en CMEs está asociado a la activación de Akt mediada por Zip7, se inhibió la fosforilación de Akt y se silenció Zip7. Ambos abordajes dieron como resultado un incremento en la diferenciación de las células tratadas con zinc. Por otro lado, CMEs cultivadas durante 30 días en presencia de zinc fueron capaces de retener su pluripotencia, mientras que el control sin zinc presentaba rasgos claros de diferenciación celular. Por último, la combinación de LIF con zinc produjo un incremento importante del efecto del LIF en cuanto al mantenimiento de la capacidad de auto-renovación celular. Por último, se ha estudiado el efecto del litio en la diferenciación de las CMEs. El litio es un inhibidor de la glucógeno sintasa quinasa 3ß (GSK3ß). En términos de CMEs, GSK3ß activa los mecanismos de diferenciación. Los resultados obtenidos indican que altas concentraciones de litio (10 mM) son capaces de fosforilar e inhibir la proteína GSK3ß. Sin embargo, en lugar de mantener la pluripotencia, las células madre se diferenciaron hacia el linaje del mesodermo tras 3 días de cultivo. Después de un total de 6 días, las células tratadas con 10 mM de litio presentaron características de endotelio hemogénico. La inhibición de GSK3ß dio como resultado la activación de la proteína ß-catenina, cuya actividad transcripcional es necesaria para la hematogénesis embrionaria. La capacidad de las células endoteliales con potencial hemogénico obtenidas de derivar en células madre hematopoyéticas fue confirmada tras su maduración durante 11 día / [CA] Els processos de comunicació cel·lular permeten a les cèl·lules desenvolupar una acció coordinada durant la embriogènesis y assimilar de forma coherent als senyals rebudes a través de l'entorn. Algunes de les molècules senyalitzadores més usades en la clínica i la investigació són les citocines. No obstant, hi ha una tendència creixent en l'ús d'un altre tipus de molècules, com els ions metàl·lics. Alguns ions com el calci i el zinc són capaços de dur a terme funcions de missatger secundari. Altres com el liti són capaços d'inactivar proteïnes quinasa alterant rutes de senyalització. Durant el desenvolupament d'aquest treball de tesi doctoral, s'ha estudiat l'efecte del zinc sobre mioblasts de ratolí, el paper del zinc en l'auto-renovació de les cèl·lules mare embrionàries (CMEs), i el paper del liti sobre la diferenciació de les CMEs. L'estudi de l'efecte del zinc sobre els mioblasts ha demostrat que el zinc és capaç d'incrementar la diferenciació dels mioblasts. L'anàlisi del zinc intracel·lular ha demostrat que els mioblasts diferenciats eren capaços d'albergar major quantitat de zinc intracel·lular. Els resultats han mostrat que suplementar les cèl·lules amb zinc extracel·lular produïx una major fosforilació i activació de la proteïna quinasa Akt. D'altra banda, s'ha observat que el transportador de zinc Zip7 es crític per a la diferenciació cel·lular mediada pel zinc. S'ha demostrat que l'activació d'aquest transportador mitjançant zinc extracel·lular és capaç d'incrementar la fosforilació d'Akt. La inhibició d'aquest transportador mitjançant ARN interferent ha donat com a resultat una menor fosforilació d'Akt i una menor diferenciació dels mioblasts exposats a zinc. Aquests resultats demostren que altes concentracions de zinc extracel·lular produeixen un incrementar la diferenciació dels mioblasts a causa de l'activació d'Akt per mitja de Zip7. Per al segon estudi, s'ha analitzat l'efecte del zinc sobre les CMEs. Com a control de manteniment de la pluripotència es va usar medi suplementat amb factor inhibidor de leucèmia (LIF). S'ha observat que les concentracions extracel·lulars de zinc a partir de 100 µM produïxen un increment immediat de la concentració intracel·lular, produint l'activació d'Akt per mitja de Zip7. Les CMEs tractades amb altes concentracions de zinc mantenen l'auto-renovació. Per demostrar que aquest efecte està associat a l'activació d'Akt mediada per Zip7, es va inhibir la fosforilació d'Akt i es va silenciar el transportador Zip7. Tots dos abordatges han donat com a resultat un increment en la diferenciació de les CMEs tractades amb zinc. D'altra banda, les CMEs van ser capaços de retenir la seva pluripotència després de ser cultivades durant 30 dies en presència de zinc, mentre que el control sense zinc presentava trets clars de diferenciació cel·lular. Finalment, la combinació de LIF amb zinc ha produit un increment sinèrgic de l'efecte del LIF. Finalment, també s'ha estudiat l'efecte del liti en la diferenciació de les CMEs. El liti és un inhibidor de la glicogen sintasa quinasa 3 beta (GSK3ß). En termes de CMEs, aquesta proteïna activa els mecanismes de diferenciació. Els resultats obtinguts indiquen que altes concentracions de liti (10 mM) tenen la capacitat de fosforilar i inhibir la proteïna GSK3ß. No obstant això, en lloc de mantenir la pluripotència, les CMEs es van diferenciar cap al llinatge del mesoderma després de 3 dies. Després d'un total de 6 dies, les cèl·lules tractades amb 10 mM de liti presentaven característiques d'endoteli hemogénic. La fosforilació de GSK3ß va donar com a resultat l'activació de la proteïna ß-catenina, l'activitat trasncripcional d'aquesta proteïna és necessària per a la hematogénesis embrionària. La capacitat de les cèl·lules endotelials amb potencial hemogénic obtingudes de derivar en cèl·lules mare hematopoètiques va ser confirmada després de la / [EN] The cell signalling process allows cells to develop a coordinated action during embryogenesis and assimilate coherently the signals received through the environment. Some of the most currently used signalling molecules in clinics and research are growth factors and cytokines. However, there is a growing trend in the use of other types of molecules, such as metal ions. Some ions such as calcium and zinc are able to carry out secondary messenger functions, transmitting signals in cascade. Others ions, such as lithium, are capable to inactivate protein kinases altering signalling pathways. During the development of this doctoral thesis, we investigated the effect of zinc on mouse muscle cells (myoblasts), the role of zinc in embryonic stem cells (ESCs) self-renewal, and the role of lithium in the differentiation of ESCs. In the first chapter, we showed that zinc is able to increase the differentiation of myoblasts. The analysis of intracellular zinc indicated that the differentiated myoblasts were capable to harbour higher concentration of intracellular zinc than undifferentiated ones. Addition of high concentration of extracellular zinc increased protein kinase Akt phosphorylation and activation. Akt activity is critical for myoblasts differentiation and has been well studied by other authors. Our results indicated that zinc transporter Zip7 was critical for zinc-mediated cell differentiation. It was prior demonstrated that the activation of this transporter by extracellular zinc increased the phosphorylation of Akt. The inhibition of Zip7 by interfering RNA resulted in a lower phosphorylation of Akt and reduced differentiation of the myoblasts exposed to extracellular zinc. These results demonstrated that high concentration of extracellular zinc enhances the differentiation of myoblasts through activation of Akt mediated by Zip7. In the second chapter, we have analysed the effect of zinc on ESCs. Leukaemia inhibitory factor (LIF) was used as pluripotency sustaining factor. We observed that extracellular supplementation of 100 ¿M zinc produced an immediate increase of the intracellular concentration, which resulted in the activation of Akt mediated by Zip7 transporter. ESCs treated with high concentrations of zinc maintained self-renewal. The role of Akt on ESCs self-renewal has been well established in the literature. To demonstrate that this effect is associated with the activation of Akt mediated by Zip7, we inhibited Akt phosphorylation and silenced the expression of Zip7. Both approaches resulted in an increase in the differentiation levels of the ESCs treated with zinc. We further demonstrated that ESCs treated with zinc during 30 days were able to retain their pluripotency, while the control condition cultured 30 days without zinc presented evident traits of spontaneous cellular differentiation. Finally, the combination of LIF with zinc produced a synergistic-like increase in the effect of LIF on ESCs self-renewal. Finally, we addressed the effect of lithium on the differentiation of ESCs. Lithium is an inhibitor of glycogen synthase kinase 3 beta (GSK3ß). In terms of ESCs, GSK3ß activates differentiation mechanisms. Our results indicated that high concentration of lithium (10 mM) was able to phosphorylate and strongly inhibit the activity of GSK3ß. However, instead of maintaining pluripotency, ESCs differentiated into the mesoderm lineage after 3 days of culture. After a total of 6 days, ESCs treated with 10 mM lithium showed haemogenic endothelium characteristics, expressing CD31, Sca-1 and CD31/Sca-1 positive cells. The phosphorylation of GSK3ß resulted in the activation of the ß-catenin protein, whose transcriptional activity is necessary for embryonic hematogenesis. The ability of endothelial cells with hemogenic potential obtained from lithium-treated ESCs to derive into hematopoietic stem cells was confirmed after maturation of these cells, resulting in rounded cell aggregates positive for Sox17. / Ministerio de Ciencia, Innovación y Universidades a través de la beca BES-2013-064052 y los proyectos MAT2012-38359-C03-01 y MAT2015-69315-C3-1-R. / Mnatsakanyan Movsesyan, H. (2019). ROLE OF IONS IN STEM CELLS SIGNALLING [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123063

Avaliou-se os efeitos dos ácidos linoleico e oleico na regeneração do músculo gastrocnêmio lacerado em ratos e em mioblastos, miotubos e fibroblastos em cultura. Houve regeneração incompleta e recuperação parcial da função contrátil do músculo lesionado. O ácido linoleico diminuiu a massa, a atividade contrátil, a área de secção tranversa das fibras, aumentou tecido fibroso no músculo lesionado e elevou a expressão de PCNA, colágeno e fibronectina nos fibroblastos. O ácido oleico aboliu as alterações na atividade contrátil e o aumento de tecido fibroso e elevou a expressão de MyoD em mioblastos e desmina em miotubos e reduziu de PCNA, colágeno e fibronectina em fibroblastos. O ácido linoleico comprometeu a regeneração enquanto que o oleico otimizou a capacidade regenerativa e a função contrátil do músculo lesionado. / The effects of linoleic and oleic acids on regeneration of lacerated gastrocnemius muscle in rats and on cultured myoblasts, myotubes and fibroblasts were examined. There was incomplete regeneration and partial recovery of the contractile function of the injured muscle. Linoleic acid reduced the mass, contratile activity and cross-sectional area of the fibers, raised the fibrous area and reduced the expression of PCNA, collagen and fibronectin in fibroblasts. Oleic acid abolished the changes in contratile activity and the increase in the fibrous area, raised the expressions of MyoD in myoblasts, desmin in myotubes and inhibited the expressions of PCNA, collagen and fibronectin in fibroblasts. Linoleic acid impaired regeneration whereas oleic acid optimized the regenerative capacity and contractile function of the injured muscle.

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.

Avaliou-se os efeitos dos ácidos linoleico e oleico na regeneração do músculo gastrocnêmio lacerado em ratos e em mioblastos, miotubos e fibroblastos em cultura. Houve regeneração incompleta e recuperação parcial da função contrátil do músculo lesionado. O ácido linoleico diminuiu a massa, a atividade contrátil, a área de secção tranversa das fibras, aumentou tecido fibroso no músculo lesionado e elevou a expressão de PCNA, colágeno e fibronectina nos fibroblastos. O ácido oleico aboliu as alterações na atividade contrátil e o aumento de tecido fibroso e elevou a expressão de MyoD em mioblastos e desmina em miotubos e reduziu de PCNA, colágeno e fibronectina em fibroblastos. O ácido linoleico comprometeu a regeneração enquanto que o oleico otimizou a capacidade regenerativa e a função contrátil do músculo lesionado. / The effects of linoleic and oleic acids on regeneration of lacerated gastrocnemius muscle in rats and on cultured myoblasts, myotubes and fibroblasts were examined. There was incomplete regeneration and partial recovery of the contractile function of the injured muscle. Linoleic acid reduced the mass, contratile activity and cross-sectional area of the fibers, raised the fibrous area and reduced the expression of PCNA, collagen and fibronectin in fibroblasts. Oleic acid abolished the changes in contratile activity and the increase in the fibrous area, raised the expressions of MyoD in myoblasts, desmin in myotubes and inhibited the expressions of PCNA, collagen and fibronectin in fibroblasts. Linoleic acid impaired regeneration whereas oleic acid optimized the regenerative capacity and contractile function of the injured muscle.

L’interleukine (IL)-17 et le TNFa sont deux cytokines pro-inflammatoires jouant un rôle important dans diverses maladies inflammatoires systémiques et auto-immunes affectant différents organes et tissus comme le foie et les muscles. Cependant, les rôles de l’IL-17 et du TNFa restent encore mal compris dans les muscles et le foie, qui est impliqué dans la réponse en phase aiguë. En utilisant des cultures de myoblastes, d’hépatocytes et de cellules stellaires hépatiques humaines, nous avons trouvé que l’IL-17 et le TNFa augmentent en synergie la sécrétion de la cytokine pro-inflammatoire IL-6 et de plusieurs chimiokines. Dans les myoblastes, l’IL-17 et le TNFa induisent un stress oxydatif et une dérégulation de calcium montrant ainsi que les processus pathologiques immuns et non-immuns interagissent. Dans les hépatocytes, en augmentant en synergie les niveaux de la CRP et des transaminases, l’IL-17 et le TNFa participent à l’inflammation systémique et aux dommages cellulaires. Etant donné que des infiltrats de cellules immunitaires sont retrouvés lors d’atteintes inflammatoires, les interactions cellulaires contribuent certainement à la chronicité de l’inflammation. Des cellules mononuclées du sang périphérique activées ou non ont ainsi été placées en co-cultures avec les myoblastes, les hépatocytes et les cellules stellaires. Par comparaison aux monocultures, les productions de l’IL-6 et de la chimiokine IL-8 étaient augmentées dans les co-cultures. L’IL-17 et le TNFa contribuaient partiellement à ces effets. Les effets systémiques de l’IL-17 et du TNFa en font donc des cibles thérapeutiques attrayantes pour le traitement des nombreuses maladies inflammatoires systémiques / Interleukin-17A (IL-17) and tumor necrosis factor-a (TNFa) are two pro-inflammatory cytokines playing an important role in various systemic inflammatory and autoimmune disorders affecting different organs and tissues including the liver and the muscles. However, the roles of IL-17 and TNFa are not fully understood in the muscles and also in liver, which is crucial in the acute phase response. By using cultures of human myoblasts, primary human hepatocytes, human HepaRG cells and LX-2 hepatic stellate cells, we found that IL-17 and TNFa increase in synergy the production of the pro-inflammatory cytokine IL-6 and chemokines (IL-8, CCL20, MCP-1). In myoblasts, the IL-17 and TNFa stimulation induces endoplasmic reticulum stress and calcium dysregulation showing that immune and non-immune pathogenic mechanisms interplay. In hepatocytes, IL-17 and TNFa mediate systemic inflammation and cell damage by increasing in synergy the CRP acute-phase protein and transaminase levels through the induction of IL-6. Since active liver and muscle disorders are characterized by inflammatory infiltrates of immune cells, the cell interactions play certainly an important role in the chronicity of the inflammation. Peripheral blood mononuclear cells activated or not were therefore co-cultured with myoblasts, hepatocytes and/or hepatic stellate cells to assess the inflammatory role of the cell-cell interactions. Co-cultures enhance the production of IL-6 and IL-8 compared to monocultures. IL-17 and TNFa contribute partially to these inductions. The systemic effects of IL-17 and/or TNFa make them attractive therapeutic targets for the treatment of various systemic inflammatory disorders

Les myopathies nécrosantes auto-immunes (MNAI) appartiennent au groupe des myopathies inflammatoires idiopathiques. Les MNAI sont des maladies musculaires sévères pouvant conduire à un déficit musculaire définitif et handicapant. Pour rétablir une force normale et prévenir le handicap, des traitements prolongés associant corticothérapie et immunosuppresseurs sont nécessaires. Les MNAI sont définies histologiquement par la prédominance de fibres musculaires en nécrose qui contraste avec l’absence ou la faible abondance d'inflammation musculaire. Les mécanismes impliqués dans la nécrose comme ceux impliqués dans les séquelles atrophiques sont en revanche inconnus. Les MNAI peuvent être associées à des auto-anticorps (aAc) soit anti-SRP, soit anti-HMGCR. Ces aAc ciblent des protéines cytoplasmiques ubiquitaires. Leurs titres sont corrélés avec l'activité de la maladie, suggérant ainsi le rôle pathogène de ces aAc. Dans ce travail de thèse, nous avons émis l’hypothèse que les aAc pouvaient être impliqués dans les lésions musculaires observées aux cours des MNAI anti-SRP+ et anti-HMGCR+. Durant cette étude, nous avons eu pour objectifs : (i) de caractériser et de quantifier la nécrose musculaire et l’inflammation associée, ainsi que les mécanismes physiopathologiques impliqués (rôle des aAc) ; (ii) d’analyser la régénération et l’atrophie musculaire et l’effet des aAc sur ces phénomènes. L'analyse histologique des biopsies musculaires des patients MNAI a montré que la proportion des fibres musculaires en nécrose était plus importante chez les patients anti-SRP+. Les taux sériques de créatine phosphokinase étaient corrélés avec la proportion de fibres nécrotiques, montrant ainsi que ce taux est un bon marqueur de l’activité de la maladie. De façon inattendue, une inflammation musculaire était régulièrement observée. En particulier, la densité lymphocytaire T était dans un quart des cas comparable à celle des autres myopathies inflammatoires. Cette densité cellulaire était de plus corrélée au pourcentage de fibres en nécrose. L’infiltrat inflammatoire était néanmoins composé principalement de macrophages. Des macrophages CD68+ iNOS+, impliqués dans des phénomènes de myophagocytoses dans un environnement Th-1 étaient régulièrement observés. Des macrophages avec un phénotype suggérant une activation alternative par l’immunité humorale ont aussi été observés. Dans ce sens, la présence de dépôts de complexe d’attaque membranaire à la surface des fibres musculaires, mais aussi des dépôts de C1q et d’IgG montraient l’activation de la voie classique du complément au cours des MNAI. De plus, la détection des protéines SRP et HMGCR sur certaines fibres musculaires immatures (in vitro et in vivo) suggérait le rôle pathogène des aAc. Concernant les mécanismes de régénération et les phénomènes d’atrophie musculaire, l’analyse des biopsies musculaires a révélé l’importance de l’irrégularité du diamètre des fibres avec une prédominance de petites fibres. Ces petites fibres correspondaient à la fois à des fibres en régénération et à des fibres en nécrose. In vitro, les Ac anti-SRP et anti-HMGCR induisent une atrophie musculaire en augmentant la transcription de MAFbx et Trim63. En outre, l'atrophie des fibres musculaires a été associée à un niveau élevé de cytokines inflammatoires comme TNF, IL-6 et ROS. Concernant l’étude de la régénération musculaire, in vitro, la présence des aAc réduisait la fusion des myoblastes. Ce défaut était associé à une diminution de la production de cytokines anti-inflammatoires IL-4 et IL-13. L’ajout d'IL-4 et/ou d’IL-13 permettait de corriger la fusion des myoblastes. Dans ce travail, les données recueillies convergent pour suggérer le rôle pathogène des aAc anti-SRP et anti-HMGCR dans les lésions musculaires nécrotiques mais aussi dans la régénération musculaire et l’atrophie. Ces données nouvelles soulignent l’importance de traitements ciblés au cours des MNAI. / Immune mediated necrotizing myopathy (IMNM) is recognized as a separate entity among inflammatory myopathies. IMNM is a severe disabling muscle disease requiring prolonged combination of corticosteroid and immunosuppressive drugs. IMNM is morphologically defined by predominant muscle fiber necrosis and no or little inflammation, and is associated with important variation of the size of the fiber. However pathogenic mechanisms involved in muscle necrosis and muscle atrophy are largely unknown. IMNM may be associated with either anti-SRP or anti-HMGCR auto-antibodies (aAbs). The titer of these aAbs, targeting ubiquitous cytoplasmic proteins, is correlated with the disease activity suggesting their pathogenic role. In this thesis, we described the morphology of skeletal muscle alterations occurring in both conditions of anti-SRP+ or anti-HMGCR+ patients, studied the role of the Abs in (i) the necrosis mechanisms and the associated inflammation; (ii) by analyzing the atrophy and the regeneration mechanisms. Muscle histological analysis of anti-SRP+ and anti-HMGCR+ patients showed a random distribution of necrotic fibers that was more pronounced in anti-SRP+ patients. Creatine Phosphokinase levels; myolysis indicator, and muscle regeneration were correlated with the proportion of necrotic fibers. Inflammation was regularly observed in IMNM muscle patients. Macrophages were the most abundant but T cells densities were in a quarter of cases in the same range as myositis controls. CD68+iNOS+ macrophages and a Th-1 immune environment were also observed and involved in ongoing myophagocytosis. Of note, macrophages with alternative activation were also detected. Humoral immunity with activation of the classical pathway of the complement cascade was observed in IMNM. Positive membrane staining for SRP and HMGCR proteins, on some muscle fibers, was detected both in vitro and in muscle biopsies of IMNM patients. An important proportion of small fibers corresponding to both atrophic and regenerating fibers was observed in anti-SRP+ and anti-HMGCR+ patients. In vitro, anti-SRP and anti-HMGCR aAbs induced muscle fibers atrophy and increased the transcription of MAFbx and Trim63. In addition, the muscle fiber atrophy was associated with high level of inflammatory cytokines such TNF, IL-6 and ROS. Muscle regeneration in vitro was also affected by impairing the myoblasts fusion in presence of anti-SRP and anti-HMGCR Abs. This default was associated with a decrease production of anti-inflammatory cytokines: IL-4 and IL-13. Of note, the addition of IL-4 and/or IL-13 totally rescued the fusion. Together those data suggest that these aAbs have a pathogenic effect on muscle. Anti-SRP and anti-HMGCR are involved in muscle atrophy and affect the regeneration. The role of these aAbs in muscle damages occurring in IMNM was highlighted and emphasizes the potential interest of targeted therapies.

Doctor of Philosophy / Department of Animal Sciences and Industry / Bradley J. Johnson / We used a total of fifty-nine gilts (BW=137.7 kg) from three different breeding groups were used to assess the effects of feeding L-carnitine during gestation on gilt growth characteristics, gilt and fetal blood metabolites, litter characteristics, and IGF axis components in fetal hepatic and skeletal muscle, maternal uterine and chorioallantois tissues, and porcine embryonic myoblasts collected from fetuses. Experimental treatments were arranged in a 2 × 3 factorial arrangement with main effects of L-carnitine and day of gestation. Gilts were fed a constant feed allowance of 1.75 kg/d and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length (40, 55, or 70). No differences (P > 0.16) were observed for BW or estimated protein or fat mass at any gestation length. Gilts fed L-carnitine tended to have greater (P = 0.10) backfat at d 40 and were numerically heavier at d 70 compared to control gilts. No differences (P > 0.77) were observed in circulating total and free carnitine at breeding, but concentrations increased (P < 0.01) as gestation length increased for the gilts fed L-carnitine compared to those fed the control diet. Fetuses from the gilts fed L-carnitine tended to be heavier (P = 0.06) and fetal circulating IGF-II lower (P = 0.09) at day 70 compared to the fetuses from the control gilts. Insulin-like growth factor-I (IGF-I) mRNA was lower (P = 0.05) in fetal hepatic tissue in fetuses collected from gilts fed supplemental L-carnitine. Insulin-like growth factor binding protein-3 (IGFBP-3; P = 0.05) and IGFBP-5 mRNA increased (P = 0.01) and IGF-I mRNA numerically increased (P = 0.16) in the endometrium of gilts supplemented with L-carnitine. At d 55 or 70 gestation, fetuses were removed for collection of porcine embryonic myoblasts (PEM) from the semitendinosus. There were no treatment differences (P > 0.10) for the expression of IGF-I, IGF-II or IGFBP-5 mRNA levels. However, PEM isolated from fetuses collected from gilts fed L-carnitine had lower (P = 0.08) IGFBP-3 mRNA levels compared to the controls. Myoblasts isolated from fetuses obtained from gilts fed L-carnitine had greater (P = 0.09; 8.8 %) 5.1H11 monoclonal antibody attachment after 72 h in culture. Although not significant (P = 0.20), the total number of PEM in the S phase of the cell cycle was 4.7 % greater in PEM collected from fetuses from gilts fed L-carnitine compared to the controls. This study shows L-carnitine supplementation to gestating gilts has beneficial effects on average fetal weight, due in part to changes in the expression of the IGF axis at the fetal-maternal interface in swine. These changes in the IGF axis play a fundamental role in porcine fetal growth and development due to enhanced proliferation and delayed differentiation of PEM.

No description available.

La fusion des myoblastes est une étape cruciale pour une bonne formation musculaire pendant l'embryogenèse et après une blessure à l'âge adulte. Le système génétique simpliste des mouches a été largement utilisé dans le passé pour identifier les acteurs essentiels impliqués dans la fusion des myoblastes. Chez la drosophile, la protéine d'échafaudage Antisocial (Ants)/Rols7 joue un rôle essentiel dans la fusion des myoblastes en connectant les protéines de surface d'adhésion cellulaire au cytosquelette. Même si la plupart des voies moléculaires régissant la fusion des myoblastes sont évolutives conservées entre les mammifères et les mouches, les contributions relatives de Tanc1 et Tanc2, les orthologues mammifères de Ants/Rols7, dans la fusion de myoblastes n'ont pas été établies. Le premier objectif de la thèse était d'évaluer les contributions potentielles de Tanc1 et Tanc2 dans la fusion de myoblastes en utilisant la lignée cellulaire de myoblastes murins C2C12 comme modèle de différenciation et de fusion de myoblastes. Nous avons constaté que l'expression de Tanc1 et Tanc2 n'est pas modulée lors de la différenciation C2C12, mais que les deux échafaudages sont enrichis au niveau du cortex lors de la prolifération des myoblastes. De plus, le knockdown de Tanc1 ou Tanc2 a altéré la fusion des myoblastes sans affecter la différenciation des myoblastes. Notamment, l'expression du défaut de fusion humain entièrement restauré Tanc1 ou Tanc2 observé dans les cellules C2C12 épuisées pour Tanc1 ou Tanc2 suggérant qu'un niveau seuil de leur expression est critique pour une fusion efficace des myoblastes. De plus, ni Tanc1 ni Tanc2 n'ont pu se substituer à Ants/Rols7 lors de la fusion des myoblastes chez la drosophile, ce qui suggère que différents acteurs pourraient être impliqués dans la régulation de la fusion des myoblastes chez les mammifères. Le deuxième objectif de la thèse était de caractériser davantage le rôle de Tanc1 et Tanc2 dans la fusion de myoblastes en utilisant des modèles murins de souris. À cette fin, des souris knock-out Tanc1 totales (Tanc1 KO) et des souris knock-out Tanc2 conditionnelles (Tanc2 cKO) ont été générées. Bien que les souris Tanc2 KO aient été précédemment signalées comme étant mortelles sur le plan embryonnaire, nous rapportons ici que ces souris sont viables contrairement à ce qui a été rapporté. L'expression de Tanc1 et Tanc2 a été détectée dans les somites ainsi que dans les fibres musculaires primaires. L'analyse du phénotype musculaire au stade embryonnaire a révélé une différenciation normale des somites et la formation de fibres musculaires chez les souris Tanc1 KO et Tanc2 cKO. De plus, lors de l'analyse au stade adulte, aucune différence dans la section transversale des fibres musculaires entre les souris de type sauvage et les souris mutantes n'a été détectée. Cela pourrait-il impliquer une redondance potentielle entre Tanc1 et Tanc2 dans la régulation de la myogenèse ? Pour répondre à cette question, des souris double knockout Tanc1 et Tanc2 sont actuellement en cours de génération. En conclusion, nous avons identifié dans cette étude un nouveau rôle pour les protéines d'échafaudage Tanc1 et Tanc2 dans la fusion de myoblastes chez les mammifères. L'identification de nouveaux acteurs essentiels dans la fusion des myoblastes nous rapproche de sa compréhension et de son ciblage thérapeutique à long terme. / Myoblast fusion is a crucial step for proper muscle formation during embryogenesis and after in injury during adulthood. The simplistic genetic system of flies has been extensively used in the past to identify essential players involved in myoblast fusion. In Drosophila, the scaffold protein Antisocial (Ants)/Rols7 plays an essential role in myoblast fusion by connecting the cell adhesion surface proteins to the cytoskeleton. Even though most molecular pathways governing myoblast fusion are evolutionary conserved between mammals and flies, the relative contributions of Tanc1 and Tanc2, the mammalian orthologs of Ants/Rols7, in myoblast fusion have not been established. The first aim of the thesis was to assess the potential contributions of Tanc1 and Tanc2 in myoblast fusion by using the murine myoblast C2C12 cell line as a model for myoblasts differentiation and fusion. We found that Tanc1 and Tanc2 expression is not modulated during C2C12 differentiation, but that both scaffolds are enriched at the cortex during myoblast proliferation. Furthermore, the knockdown of either Tanc1 or Tanc2 impaired myoblast fusion without affecting myoblast differentiation. Notably, the expression of human Tanc1 or Tanc2 fully restored fusion defect observed in C2C12 cells depleted for Tanc1 or Tanc2 suggesting that a threshold level of their expression is critical for efficient myoblast fusion. Furthermore, neither Tanc1 nor Tanc2 could substitute for Ants/Rols7 during Drosophila myoblast fusion suggesting that different players might be involved in regulating myoblast fusion in mammals. The second aim of the thesis was to further characterize the role of Tanc1 and Tanc2 in myoblast fusion by using murine mice models. For this purpose, total Tanc1 knockout mice (Tanc1 KO) and conditional Tanc2 knockout mice (Tanc2 cKO) were generated. Although Tanc2 KO mice were previously reported to be embryonically lethal, we report here that those mice are viable contrary to what has been reported. Tanc1 and Tanc2 expression was detected in the somites as well as in the primary muscle fibers. Analysis of the muscle phenotype at the embryonic stage revealed normal somites differentiation and muscle fiber formation in both Tanc1 KO and Tanc2 cKO mice. Furthermore, when analyzed at the adult stage, no difference in the cross-sectional area of the muscle fibers between wild-type mice and mutant mice was detected. Could this imply a potential redundancy between Tanc1 and Tanc2 in regulating myogenesis? To answer this question, Tanc1 and Tanc2 double knockout mice are currently being generated. In conclusion, we identified in this study a novel role for the scaffold proteins Tanc1 and Tanc2 in myoblast fusion in mammals. Identifying new and essential players in myoblast fusion brings us a step closer to understanding it and on the long run target it therapeutically.

Muscle development involves concerted action of a repertoire of mechanisms governing myoblast proliferation, migration, fusion and differentiation. Subsequently, there are cellular events administrating proper muscle function and maintenance of muscle integrity. Chapter 1 covers what is known about muscle development, building up of mass and maintenance in vertebrates and Drosophila, highlighting the myogenic programs and factors that play a role in them. The formation of vertebrate skeletal muscles can be recapitulated in Drosophila indirect flight muscles (IFMs), making IFMs an interesting model to dissect and understand the mechanisms of muscle development and maintenance. The cellular and developmental events that occur during IFM development have been discussed in detail along with their genetic control which encompasses both cell autonomous and cell non-autonomous mechanisms. The fly resources and tools used for experimentations have been described in Chapter 2. One of the hallmark events during muscle development is myoblast fusion. Myoblasts are kept in undifferentiated state until they fuse through a balanced action of anti-differentiation and pro-differentiation factors. The swarming myoblasts are in semi-differentiated state and just prior to fusion should exit cell cycle to achieve terminal differentiation. The mechanisms of cyclin/CDK complexes and their regulation via CKI (CDK inhibitor) are known in a cell. However, tissue specific factors exerting additional control on molecules that participate in cell cycle have been proposed but have not been shown in vivo. Chapter 3 uncovers a novel role played by the transcription factor, Erect wing (Ewg) in IFM development and patterning. Despite the fact that Ewg is known to express in fusing myoblasts and nuclei of developing IFMs and has long been used as a nuclear marker for IFMs, the mechanism(s) behind Ewg‟s function has remained enigmatic. Historical perspective of Ewg has been presented in Chapter 1. One set of IFMs; dorsal longitudinal muscles (DLMs) require larval templates for their formation and the other set; dorsal ventral muscles (DVMs) form de novo. Chapter 3 shows that Ewg is required in a spatio-temporal fashion to initiate myoblast fusion process. In the absence of Ewg, the number of fusion events in a given time is reduced. In addition de novo fusion is observed in the region of DLM development just like DVM and overall IFM development is delayed resulting in an aberrant adult IFM pattern. Genetic studies undertaken reveal a requirement for Ewg in exerting a temporal control on myoblast fusion. This is achieved by down-regulating Cyclin E levels, as a result of which the myoblasts exit cell cycle at G1/S stage. Through this study the proposal for DLM development and pattern has been put forth as follows: i) appropriate progression of DLM development commences on synchronous exit of myoblasts from cell cycle. This function is facilitated by Ewg expression in fusing myoblasts assisting symmetrical DLM formation in hemithoraces. ii) DLM pattern of six muscles in each hemithorax is dependent on template survival which requires fusion of enough myoblasts and further subsequent fusion events to support the splitting of three larval templates or presumptive DLM. The muscles that develop should preserve their structural integrity for efficient functional output. Muscles perform extensive activities warranting high energy requirements. IFMs are widely utilised for thorax movements that aid flight. IFMs are exclusively oxidative in nature with upto 40% mass contributed by large mitochondria themselves. Chapter 4 describes yet another novel finding for Ewg function in IFM maintenance. Vertebrate homolog of Ewg is nuclear respiratory factor 1 (NRF1) known for its role in mitochondrial biogenesis. This prompted an investigation on the role of Ewg, if any, in mitochondrial function and IFM maintenance. In this chapter, Ewg null adult IFMs are shown to undergo degeneration. Mitochondria in these muscles show rounder and smaller phenotype. Mitochondria morphology is traced throughout Drosophila pupal DLM development and extensive fusion is observed in last one-fourth of pupal phase. In Ewg null condition transcripts of Opa1-like required for inner mitochondrial membrane fusion is found to be absent, suggesting lack of mitochondrial fusion behind the smaller mitochondrial morphology. This emerged as an intriguing problem since Ewg expression follows until sarcomerogenesis (formation of sarcomeres) initiates at mid pupal stage. Developmentally extending Ewg‟s expression beyond mid pupal stage is not observed to increase Opa1-like levels pointing an indirect regulation by Ewg. However, Opa1-like knock-down beyond mid pupal stage is not observed to result in any muscle or flight defect. It is thus proposed that Ewg expression early during muscle development helps to up-regulate Opa1-like levels needed to support mitochondrial growth and fusion. In addition, this chapter provides additional data on requirement of Opa1-like for maintenance of mitochondrial as well as muscle integrity. This is the first ever report of tissue specific temporal regulation of Opa1-like by Ewg. Chapter 3 and Chapter 4 conclude spatially segregated functional requirements of Ewg which are also mechanistically distinct. Expression in fusing myoblasts channelizes fusing myoblasts to exit cell cycle and undergo timely fusion saving the larval template, subsequent fusion assists template splitting thus forming the appropriate adult DLM pattern. On the other hand expression until mid pupal stage up-regulates Opa1-like expression, facilitating mitochondrial fusion during the late pupal stage. This as a result helps maintain structural integrity of muscles in the adult. Vertebrate skeletal muscle contains multiple muscle fibres that provide appropriate mass and size to muscles. As DLM share similarity in development to that of the vertebrate skeletal muscle, DLM organisation is studied to get insights into the mechanisms which regulate the process. Chapter 5 discusses role of nuclear number and nuclear activity in determining the DLM organisation. In order to alter nuclear number, myoblast population is reduced to amounts lesser than that of the wild type and to alter nuclear activity, two nuclear encoded genes Opa1-like and Marf , involved in inner and outer mitochondrial membrane fusion respectively have been knocked down in IFMs. First, the DLM organisation is established by comparing it to the vertebrate skeletal muscle organisation. This organisation is affected on lowering the number of myoblasts destined to fuse and form IFMs, without affecting the differentiation. On the other hand, when nuclear encoded mitochondrial fusion genes are knocked down, not only DLM organisation but their differentiation is also affected. A proposal for achieving DLM organisation has been presented which should also apply to vertebrate skeletal muscle given their developmental similarity. In conclusion, the studies decipher a novel mechanism by which a transcription factor, Ewg exerts a temporal control on myoblast fusions directly influencing progression of DLM formation, and thereby, symmetry and pattern. Moreover, Ewg is also shown here to regulate mitochondrial fusion during later pupal stages helping muscles to attain greater function and maintain structural integrity. Discovery of such regulatory mechanisms controlling mitochondrial dynamics in vertebrates can open up new avenues to understand and design new therapeutic approaches to tackle mitochondrial diseases. Additionally, myoblast fusion and hence myonuclear number and their efficient functioning are shown to be important determinants of muscle organisation. This has further implications in understanding and using stem cell science in dystrophic or atrophic or ageing related muscle loss and therapy.