Pas de résumé disponible / Use of stem cells in regenerative medicine has attracted great interest in the past decade. Muscle stem cells such as satellite cells were shown to regenerate skeletal muscle tissue after injury and to contribute to muscle growth. These properties have raised an enormous interest in using satellite cells for the therapy of skeletal muscle wasting disorders where the intrinsic stem cell population is unable to repair muscle tissue. However, better understanding of the mechanisms controlling satellite cell lineage progression and self-renewal is crucial to exploit the power of these cells in combating myopathic conditions. In the studies described here, the mechanisms regulating the in vivo behavior and maintenance of quiescence of Drosophila Adult Muscle Precursors (AMPs) that share several properties with the vertebrate satellite cells are analyzed. We show that undifferentiated embryonic AMPs display homing behavior and that their survival depends on the somatic muscles. We observe that AMPs establish direct contact with muscle fibers by sending thin filopodia and that this AMP-muscle interaction is crucial for AMPs spatial positioning. Larval muscles also play an important role in promoting the AMP cell proliferation. They achieve this by secreting Drosophila Insulin like peptide 6 (dIlp6) that activate the AMPs from their quiescent state and induce proliferation during the end of the second larval instar. We also demonstrate that Notch acts downstream of Insulin pathway and positively regulates proliferation of AMPs via dMyc. In the second part of the thesis manuscript we report that the affected formation ofadult muscles impacts on persisting abdominal larval templates. In this section role of the Notch signaling pathway in specification of the Adult founder cells is also demonstrated. Finally, we report generation of new tools for the cell type specific genome wide approaches that can be applied to identify global gene expression profiles in quiescent versus activated AMPs. Together these studies identified several new features of AMPs and enhance our understanding on the processes regulating stem cells homing, quiescence and reactivation.

Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.

Introduction: Resistance training improves muscle strength and induces myofiber hypertrophy in young males and females with blunted responses occurring in older adults. These adaptations are partially due to the function of muscle stem cells (MuSCs) and fibro-adipogenic progenitors (FAPs). It remains unknown whether middle-aged males and females respond similarly to resistance training with protein supplementation, specifically at the cellular level. Purpose: The purpose of this study is to investigate the potential sex-specific responses of middle-aged males and females to whole-body resistance training. Methods: Middle-aged adults (N=28), 40-64 years, participated in a 10-week progressive, whole-body resistance training intervention coupled with protein supplementation. Muscle biopsies were collected from the vastus lateralis and stained for fibre morphology, MuSCs, and FAPs. Results: Both sexes increased type II fibre cross-sectional area with training. Myonuclear content, myonuclear domain size, and MuSC content were not altered with training in either sex. Both males and females altered FAP content with training. Interestingly, the change in MuSCs and both FAPs were correlated in males but not females (both P<0.05). It was concluded that there were no sex-specific responses to resistance training in middle-aged males and females; however, MuSCs and FAPs appear to be correlated in males but not females.

Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant and progressive neuromuscular disorder caused by the CTG trinucleotide repeat expansion in the 3’ untranslated region of the DMPK gene. Clinical manifestations include extensive atrophy of skeletal muscle (SkM) concomitant with muscle weakness, that develops in a distal to proximal fashion. Central to muscle plasticity is the satellite cell (SC), a muscle specific stem cell that, upon activation, facilitates muscle repair and regeneration. To date, SCs have yet to be elucidated in DM1; therefore, the aim of the present study was to extensively characterize the PAX7+ SC population, along with other indices of muscle quality in SkM. DM1 patients (6 women, 5 men) performed stationary cycling 3 times per week for 12wks, with biopsies taken from the Vastus lateralis pre- (PRE) and post-endurance exercise intervention (POST). Age-matched, healthy controls (CTRL) were used for comparison of baseline measures. Type 1 and 2 myofiber-specific PAX7+ cells were significantly greater in DM1 patients (PRE), in comparison to CTRL (2.24- and 1.84-fold, respectively), with type 2 SC content further increasing following training (p<0.05). In addition, protein expression of myogenic regulatory factors PAX7 and myogenin were significantly higher in DM1 compared to CTRL, with no training effects observed. Both immunohistochemical and immunoblotting analysis showed that activated MYOD+/PAX7+ cells did not significantly differ in DM1 vs. CTRL. FISH- IF analysis of CUG repeats show that 30% of SCs in DM1 were positive for these inclusions. Muscle capillarization was significantly lower in type 2 fibers in DM1 vs CTRL, which was fully rescued with training (p<0.05). At baseline, DM1 muscle showed the presence of de novo and fat infiltrated fibres, as well as fibrosis, that were relatively non-existent in the CTRL. In vitro results show patient-derived myoblasts exhibit a proliferation defect. Furthermore, myoblasts showed impairments in both glycolysis and mitochondrial respiration, with the latter being completely normalized to CTRL in myotubes. Our novel findings display an increased, albeit non-functional, SC pool in DM1 SkM indicated by disturbances in the myogenic program and overall poor muscle quality. We show that both SCs and SkM remain responsive to exercise training, suggesting therapeutic potential. We also suggest that mitochondrial dysfunction may underpin these impairments in the myogenic program. / Thesis / Master of Science (MSc) / Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults worldwide affecting 1:8000 individuals, with certain areas in northeastern Quebec having a higher prevalence of 1:600 individuals. DM1 is caused by an autosomal dominant genetic mutation that leads to muscle weakness, respiratory insufficiency, cataracts and cardiac conduction block, ultimately resulting in poor quality of life and shortened lifespan. Preliminary evidence suggests that the maintenance of muscle health can greatly improve quality of life and life-span of these individuals, making an in-depth research focus on this therapeutic intervention extremely important. Optimal muscle health is maintained by the functionality of muscle stem cells, that aid in muscle repair and facilitate adaptations in muscle following exercise interventions. These cells are shown to be dys- or non-functional in various muscular dystrophies which coincide with the observation of poor muscle health. Therefore, the aim of this study was to examine the number and functionality of muscle stem cells, and physiological factors of muscle health in DM1. In addition, we also aimed to explore whether exercise has therapeutic potential to alleviate poor muscle quality in DM1. In general, we found that DM1 patients have a higher proportion of muscle stem cells; however, they are inherently dysfunctional but did respond to exercise. Consistent with the latter observation, we found poor muscle quality metrics in DM1 patients, with aerobic training leading to improvements in muscle health. Altogether, our results provide in-depth analysis that underscores muscle dysfunction observed in DM1 and the benefits of exercise interventions.

<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)

Skeletal muscle possesses a remarkable plasticity, able to repair, remodel and adapt to various stressors. A population of resident muscle stem cells, commonly referred to as satellite cells (SC), are largely responsible for skeletal muscle plasticity. The loss of muscle mass and plasticity typically observed in aging has been attributed to the deterioration of SC function. SC reside in a quiescent state, but following stimuli they become active, proliferate and eventually differentiate, fusing to existing muscle fibres. The progression of SC through this process, termed the myogenic program, is orchestrated by a complex network of transcription factors, termed myogenic regulatory factors. SC function is regulated by various growth factors and/or cytokines. The delivery of these signalling factors to SC is, in part, dependent on their proximity and exposure to local microvascular blood flow. The purpose of this thesis was to examine the relationship between skeletal muscle capillaries and muscle SC. We examined the effect of age on the spatial relationship between SC and muscle fiber capillaries, and observed that type II muscle fiber SC were located at a greater distance from the nearest capillary in older men as compared to their younger counterparts. We then examined the changes in SC activation status following a single bout of resistance exercise, prior to and following a 16wk progressive resistance training (RT) program. We observed that following RT, there was an enhanced SC activation in response to a single bout of resistance exercise. This enhanced response was accompanied by an increase in muscle capillarization following training. Furthermore, we investigated the impact of muscle fiber capillarization on the expansion and activation status of SC in acute response to muscle damaging exercise in healthy young men. We observed that muscle capillarization was positively related to SC pool activation and expansion. Taken together, we demonstrate that muscle capillarization may be related to the SC response following acute resistance exercise or exercise-induced injury, and may be implicated in adaptation to RT. Furthermore, the spatial relationship between muscle capillaries and SC is negatively altered by aging. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle health is, in part, maintained by a population of stem cells associated with individual muscle fibres. When muscle is damaged or stressed, these cells become activated, aid in muscle repair, and help drive adaptations to exercise. The central purpose of this thesis was to examine the relationship between muscle capillaries and muscle stem cells, and determine how that relationship impacts muscle stem cell function. We demonstrated that muscle stem cells and capillaries exist in close proximity to each other in skeletal muscle. We observed that a greater muscle capillarization is linked to improved muscle stem cell function during muscle repair. However, we also report that the distance between muscle capillaries and muscle stem cells becomes greater in aging, and may be a root cause of impaired muscle stem cell function in aging.

O tecido muscular tem uma alta capacidade de regeneração após lesão, que está diretamente ligada à presença de células satélites (SCs). Essas células são as principais células-tronco do músculo e também têm um papel fundamental no desenvolvimento muscular na embriogênese. Embora quiescente nos músculos adultos normais, as SCs podem ser ativadas por sinais específicos após lesão muscular. Em doenças caracterizadas por processo de degeneração crônica, como distrofias musculares, as SCs são constantemente ativadas, e esta condição pode levar à depleção do pool de SCs e consequente falha no processo regenerativo. Nós estudamos as SCs musculares nas linhagens distróficas murinas DMDmdx, Largemyd, DMDmdx/Largemyd, em comparação a camundongos normais, com o principal objetivo de avaliar o comportamento das SCs em músculos distróficos com diferentes graus de degeneração histopatológica. A expressão de genes e proteínas de fatores de transcrição relacionados a SCs foram estudadas no músculo, e os resultados foram comparados com as características histopatológicas de regeneração e degeneração e estado de proliferação de células musculares. Nossos resultados mostraram que o músculo distrófico mantém seu pool de células satélites, expressando PAX7, um importante fator muscular para autorrenovação do pool de SCs, em níveis semelhantes em todas as linhagens distróficas e controle normal. As células isoladas de músculo distrófico apresentaram uma maior proporção de células em proliferação, como observado pela análise dos marcadores de ciclo celular no músculo gastrocnêmio dissociado, com maior número de células na fase G2/M. A cascata de genes de regeneração é ativada no músculo distrófico, com altos níveis de expressão de fatores de regeneração muscular, como MYOD e Myogenin. O músculo distrófico mantém a capacidade de formar novas fibras, observada por um número significativo de fibras recém formadas, que expressam dMHC, em todas as linhagens analisadas. No entanto, essas novas fibras mostram características de maturação incompleta, como tamanho pequeno e pouca variação em seu calibre, que pode ser determinante para sua disfunção. A degeneração muscular é intensa apesar da regeneração, com infiltração significativa de tecido conjuntivo em camundongos distróficos. Em conclusão, nossos achados sugerem que os músculos distróficos, independentemente do grau de degeneração, mantêm o pool de células satélites com capacidade proliferativa e estão prontos para responder aos estímulos regenerativos. Por outro lado, a maturação dessas novas fibras é incompleta e não previne a degeneração do músculo / Muscle tissue has a high regeneration capacity after injury, which is directly linked to satellite cells (SCs). These cells are the main stem cells of the muscle and also have a key role in muscle development in embryogenesis. Although quiescent in normal adult muscles, SCs can be activated by specific signals upon muscle injury. In diseases characterized by chronic degeneration process, such as muscular dystrophies, the SCs are constantly activated, leading to depletion of the SC pool and consequent failure of the regenerative process. We studied muscle SCs in the mouse dystrophic strains DMDmdx, Largemyd, DMDmdx/Largemyd, comparing to wild-type mice, with the main objective to evaluate SCs behavior in dystrophic muscles with different degrees of histopathological degeneration. Gene and protein expression of transcription factors related to SCs were studied in the muscle, and the results were compared to regenerating and degenerating histopathologic pattern and proliferative state of muscle cells. Our results showed that the dystrophic muscle retains its satellite cells pool, expressing PAX7, an important muscle factor for self-renewal of the SCs pool, at similar levels in all dystrophic strains and wild-type. Dystrophic muscle single cells presented a higher proportion of proliferating cells, as observed by the analysis of cell cycle markers in dissociated gastrocnemius muscle, with a greater number of cells in the G2/M phase. The cascade of regeneration genes is activated in the dystrophic muscle, with high levels of expression of muscle regenerating factors, such as MYOD and Myogenin. Dystrophic muscle retains the ability to form new fibers, as observed by a significant number of new fibers expressing dMHC in all dystrophic strains. However, these new fibers show incomplete maturation characteristics, such as small size and no variation in fiber caliber, which could be determinant for its dysfunction. Muscle degeneration is intense in spite of regeneration, with significant more connective tissue infiltration in dystrophic mice than wild-typemice. In conclusion, our findings suggest that dystrophic muscles, independently of the degree of degeneration, retain the pool of satellite cells with proliferating capacity and ready to respond to regenerating stimuli. On the other hand, the maturation of these new fibers is incomplete and do not prevent the degeneration of the muscle

Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

O tecido muscular tem uma alta capacidade de regeneração após lesão, que está diretamente ligada à presença de células satélites (SCs). Essas células são as principais células-tronco do músculo e também têm um papel fundamental no desenvolvimento muscular na embriogênese. Embora quiescente nos músculos adultos normais, as SCs podem ser ativadas por sinais específicos após lesão muscular. Em doenças caracterizadas por processo de degeneração crônica, como distrofias musculares, as SCs são constantemente ativadas, e esta condição pode levar à depleção do pool de SCs e consequente falha no processo regenerativo. Nós estudamos as SCs musculares nas linhagens distróficas murinas DMDmdx, Largemyd, DMDmdx/Largemyd, em comparação a camundongos normais, com o principal objetivo de avaliar o comportamento das SCs em músculos distróficos com diferentes graus de degeneração histopatológica. A expressão de genes e proteínas de fatores de transcrição relacionados a SCs foram estudadas no músculo, e os resultados foram comparados com as características histopatológicas de regeneração e degeneração e estado de proliferação de células musculares. Nossos resultados mostraram que o músculo distrófico mantém seu pool de células satélites, expressando PAX7, um importante fator muscular para autorrenovação do pool de SCs, em níveis semelhantes em todas as linhagens distróficas e controle normal. As células isoladas de músculo distrófico apresentaram uma maior proporção de células em proliferação, como observado pela análise dos marcadores de ciclo celular no músculo gastrocnêmio dissociado, com maior número de células na fase G2/M. A cascata de genes de regeneração é ativada no músculo distrófico, com altos níveis de expressão de fatores de regeneração muscular, como MYOD e Myogenin. O músculo distrófico mantém a capacidade de formar novas fibras, observada por um número significativo de fibras recém formadas, que expressam dMHC, em todas as linhagens analisadas. No entanto, essas novas fibras mostram características de maturação incompleta, como tamanho pequeno e pouca variação em seu calibre, que pode ser determinante para sua disfunção. A degeneração muscular é intensa apesar da regeneração, com infiltração significativa de tecido conjuntivo em camundongos distróficos. Em conclusão, nossos achados sugerem que os músculos distróficos, independentemente do grau de degeneração, mantêm o pool de células satélites com capacidade proliferativa e estão prontos para responder aos estímulos regenerativos. Por outro lado, a maturação dessas novas fibras é incompleta e não previne a degeneração do músculo / Muscle tissue has a high regeneration capacity after injury, which is directly linked to satellite cells (SCs). These cells are the main stem cells of the muscle and also have a key role in muscle development in embryogenesis. Although quiescent in normal adult muscles, SCs can be activated by specific signals upon muscle injury. In diseases characterized by chronic degeneration process, such as muscular dystrophies, the SCs are constantly activated, leading to depletion of the SC pool and consequent failure of the regenerative process. We studied muscle SCs in the mouse dystrophic strains DMDmdx, Largemyd, DMDmdx/Largemyd, comparing to wild-type mice, with the main objective to evaluate SCs behavior in dystrophic muscles with different degrees of histopathological degeneration. Gene and protein expression of transcription factors related to SCs were studied in the muscle, and the results were compared to regenerating and degenerating histopathologic pattern and proliferative state of muscle cells. Our results showed that the dystrophic muscle retains its satellite cells pool, expressing PAX7, an important muscle factor for self-renewal of the SCs pool, at similar levels in all dystrophic strains and wild-type. Dystrophic muscle single cells presented a higher proportion of proliferating cells, as observed by the analysis of cell cycle markers in dissociated gastrocnemius muscle, with a greater number of cells in the G2/M phase. The cascade of regeneration genes is activated in the dystrophic muscle, with high levels of expression of muscle regenerating factors, such as MYOD and Myogenin. Dystrophic muscle retains the ability to form new fibers, as observed by a significant number of new fibers expressing dMHC in all dystrophic strains. However, these new fibers show incomplete maturation characteristics, such as small size and no variation in fiber caliber, which could be determinant for its dysfunction. Muscle degeneration is intense in spite of regeneration, with significant more connective tissue infiltration in dystrophic mice than wild-typemice. In conclusion, our findings suggest that dystrophic muscles, independently of the degree of degeneration, retain the pool of satellite cells with proliferating capacity and ready to respond to regenerating stimuli. On the other hand, the maturation of these new fibers is incomplete and do not prevent the degeneration of the muscle