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

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

<p>S. 1-96: sammanfattning, s. 97-134: 5 uppsatser</p> / digitalisering@umu

Made available in DSpace on 2016-06-02T20:18:16Z (GMT). No. of bitstreams: 1 4115.pdf: 3025208 bytes, checksum: 10c4684cd678fb5d22f0efb47de00306 (MD5) Previous issue date: 2012-02-24 / Universidade Federal de Minas Gerais / The quadriceps muscle weakness is common in subjects with knee osteoarthritis (OA), leading to functional deficits. Some risk factors are causes, such as morphological changes (atrophy and reduced number of muscle fibers), changes in non-contractile proteins of muscle and changes in the level of muscle activation. However, these factors are not well understood, particularly with respect if they are already present in earlier stages of the disease. Therefore, the aim of this study was to verify if men with early degrees of knee OA have functional and morphological impairments and immunohistochemical changes of the quadriceps muscle, as well as investigate if this subjects had compromising of their quality of life . The men (40-65 years) who participated in this study were divided into two groups: Control Group (CG) with healthy subjects and Osteoarthritis Group (OAG) with individuals with knee OA grade I or II. A biopsy of the vastus lateralis (VL) was performed for morphological (through the ATPase reaction) and immunohistochemical analysis(analysis of expression and localization of the receptor for advanced glycation end products - RAGE, and analysis of the expression of collagen type I and III ) . An evaluation of knee extensor torque (KET), concentric and eccentric at 90°/s and 180°/s, was performed simultaneously with an evaluation of the electromyographic activity of the VL (RMS value). Moreover, the WOMAC questionnaire was used. For the intergroup analysis, we used the independent t-test and the Mann-Whitney U test. Spearman s correlation coefficient was used to detect the relationship between the three subscales of WOMAC questionnaire and the average knee extensor peak torque (&#945;<0.05). The collagen was analyzed by semiquantitative analysis. We found lower values for the GOA in eccentric knee extensor peak torque at 90 °/s (p = 0.01) and 180°/s (p = 0.04), and in electromyography activity (RMS) during eccentric contraction, in two angular velocities (p <0.01). Higher values were found for the GOA in the proportion (p = 0.03) and in the relative crosssectional area of type 2b fibers (p = 0.02). It was found a negative correlation between the concentric and eccentric knee extensor torque and the three subscales of the WOMAC questionnaire (p<0.05). In the analysis of RAGE no difference was found between the groups. In semiquantitative analysis of collagen was found increased expression of collagen type I and type III for the GOA. Thus, men with knee OA grades I or II present functional, morphological and immunohistochemical changes of the quadriceps muscle, indicating possible neuromuscular adaptations. Therefore, in the early stages of knee OA, exercises should be recommended in order to minimize or delay the functional deficits resulting from OA. / A fraqueza do músculo quadríceps é comum em sujeitos com osteoartrite (OA) de joelho, levando a déficits funcionais. Alguns fatores são apontados como causas, tais como alterações morfológicas, como atrofia e redução no número de fibras musculares, alterações nas proteínas não contráteis do músculo e alterações no nível de ativação muscular. No entanto, esses fatores ainda não estão bem esclarecidos, principalmente com relação se os mesmos já estão presentes em estágios mais precoces da doença. Diante disso, o objetivo dessa tese foi verificar se homens com graus iniciais de OA de joelho apresentam alterações funcionais, morfológicas e imunohistoquímicas do músculo quadríceps, bem como comprometimento de sua qualidade de vida. Participaram desse estudo homens, com idade entre 40 e 65 anos, divididos em dois grupos: Grupo Controle (GC) com indivíduos saudáveis e Grupo Osteoartrite (GOA) com indivíduos com OA de joelho graus I ou II. Foi realizada biópsia do músculo vasto lateral (VL) para análise morfológica (por meio da reação de ATPase) e imunohistoquímica (análise da expressão e localização dos receptores dos produtos finais da glicosilação RAGE, e análise da expressão dos colágenos tipo I e III). A avaliação do torque extensor do joelho, concêntrico e excêntrico, a 90º/s e 180º/s, foi realizada simultaneamente à avaliação da atividade eletromiográfica do músculo VL (valor de RMS). O questionário WOMAC foi aplicado para análise da qualidade de vida. Na análise intergrupo, foi utilizado o teste T de student para amostras independentes e o teste não-paramétrico U Mann-Whitney. Foi utilizado o coeficiente de correlação de Spearman para analisar a relação entre as três seções do questionário WOMAC e os picos de torque extensor do joelho (&#945;<0.05). Para os colágenos foi feita análise semiquantitativa. Foram encontrados menores valores para o GOA no pico de torque extensor excêntrico a 90º/s (p=0,01) e a 180º/s (p=0,04), e na atividade eletromiográfica (valor RMS), durante contração excêntrica, nas duas velocidades angulares (p<0,01). Maiores valores para o GOA foram encontrados na proporção (p=0,03) e área transversa relativa das fibras tipo 2b (p=0,02). Foi encontrada correlação negativa entre as três seções do questionário WOMAC e os valores de pico de torque para os indivíduos com OA de joelho (p>0,05). Na análise do RAGE não foi encontrada diferença entre os grupos. Na análise semiquantitativa foi encontrada maior expressão dos colágenos tipo I e tipo III para o GOA. Assim, homens com OA de joelho graus I ou II apresentam alterações funcionais, morfológicas e imunohistoquímica do músculo quadríceps, podendo essas alterações serem possíveis adaptações neuromusculares. Dessa forma, desde os graus iniciais de OA de joelho, devem se recomendados exercícios com o objetivo de minimizar ou retardar os déficits funcionais decorrentes da OA.

The overall objective of this thesis was to examine mitochondrial bioenergetics in muscle biopsies from humans with type 1 diabetes (T1D) to gain a deeper understanding of the cellular mechanism(s) underlying changes to skeletal muscle health reported in T1D, a phenotype we have referred to as ‘diabetic myopathy’. It was hypothesized that humans with T1D, compared to their matched counterparts without diabetes (control), would demonstrate significant deficiencies in muscle mitochondrial function and ultrastructure/content as determined by the gold-standard in vitro methodology: high-resolution respirometry and transmission electron microscopy, respectively. It was further hypothesized that sex differences would not exist in mitochondrial function with T1D, and mitochondrial deficiencies would be more dramatic at an earlier age with T1D. Adults with uncomplicated T1D and strictly matched controls (age, sex, BMI, self-reported physical activity levels) were recruited from surrounding university-dwelling communities. Site-specific deficiencies in mitochondrial respiration, H2O2 emission, and calcium retention capacity were found in young, physically active adults with T1D despite normal mitochondrial content. Further experiments revealed that muscle mitochondrial respiration in women and men differentially adapt to the T1D environment where men with T1D have lower complex II but higher complex I respiration compared to women with T1D, while women (irrespective of T1D) have lower ADP sensitivity. Women with T1D also demonstrated lower H2O2 emission compared to men with T1D. In contrast, despite a lower mitochondrial content in middle- to older-aged adults with T1D, mitochondrial respiration (normalized to content) was either normal or increased in adults with T1D compared to control, with observable differences between sexes. Overall, this research has demonstrated that despite being recreationally to physically active, adults with uncomplicated T1D with moderately well-managed glycemia demonstrate alterations in skeletal muscle mitochondrial function and ultrastructure, including differences between sexes. / Dissertation / Doctor of Science (PhD) / Type 1 diabetes (T1D) is a complex disease that still has no known cure. Current treatment focuses on managing blood sugar levels with exogenous insulin injections and frequent blood sugar checks. However, over time, people with T1D still develop serious complications that inevitably impact their quality of life and lifespan. A potential adjuvant therapy to prevent complications in T1D is improving the health of skeletal muscle through exercise given its role in stabilizing blood sugar/lipid levels and whole-body insulin sensitivity. However, this area continues to be severely understudied in the T1D population. Thus, this thesis examined skeletal muscle metabolic ‘health’ from adults with T1D who do not have major diabetes complications and manage their blood glucose moderately-well. Through a series of novel experiments, we found that young and middle- to older-aged adults with T1D have alterations in the metabolic engines of their muscles, and depending on biological sex, the alterations manifest as either heightened or degraded cellular function. These findings are the first to provide a comprehensive cellular investigation of the impact of T1D on the metabolic health of skeletal muscle in people with T1D and provide the foundation for future research examining skeletal muscle as an essential and early adjuvant therapy in this population.