Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent. / May 2009

Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent.

Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

Background: Rhabdomyosarcoma (RMS) is a soft tissue malignancy of the skeletal muscle that occurs primarily in pediatric populations. The prevailing treatment for RMS is a combination of chemoradiation therapy and surgery which has contributed to its 5-year survival rate of 75%. However, the combination of RMS and chemoradiation therapy can lead to impaired muscle growth and development which results in life-long skeletal muscle atrophy and weakness for RMS survivors. Skeletal muscle is a highly plastic tissue due, in part, to dynamic interactions between muscle-resident stem and progenitor cells (i.e., satellite cells (SCs) and fibro/adipogenic progenitors (FAPs)), which are necessary for muscle maintenance, growth, and adaptation to anabolic stimuli such as resistance exercise training. There is a clear gap in research investigating whether resistance endurance training (RET) stimulates muscle growth and preserves muscle function after juvenile chemoradiation therapy. Purpose: To determine the extent to which RET ameliorates the skeletal muscle defects in a preclinical model of RMS survivorship. Hypothesis: RET will improve physical performance, muscle cross-sectional area (CSA), and stem/progenitor cell populations compared to sedentary mice following RMS and chemoradiation therapy. Methods: RMS (M3-9-M cells) was injected into a single hindlimb of juvenile (4 week) C57Bl/6 mice that underwent systemic chemotherapy followed by targeted, fractionated radiation therapy to the RMS-injected limb. Following therapy, mice underwent RET (RET; n=10) or remained sedentary (SED; n=10) for 8 weeks. Body composition and performance tests were completed pre- and post-therapy and throughout the exercise intervention. Fibre typing, cross-sectional area, myonuclear characteristics and trichrome staining were evaluated following muscle harvest and progenitor cell populations were assessed using flow cytometry. Results: RET led to increased endurance performance (p<0.0001) as well as a reduction in body fat percentage (p=0.0004). RET rescued atrophy induced by RMS+therapy as evidenced by a significant increase in gastrocnemius/soleus to body weight ratio for the RET group compared to the SED group (p=0.0303), despite the decrease in muscle weight observed in the treated limb compared to the control limb (p=0.015). A distinct increase in intramuscular fibrosis was noted in the treated limb compared to the control limb in both groups (p=0.0283). Furthermore, RET resulted in larger myofibre cross-sectional area (p<0.05) and a shift from Type IIX to IIA fibres (p<0.05). We also noted a greater Type IIA myonuclear domain in the RET group compared to the SED group (p=0.0015) and an overall decrease in myonuclear domain (the cytoplasmic volume controlled by each myonucleus) for the treated limb compared to the control limb (p<0.05). Interestingly, we noticed overall cell death and an increase in immune cells in the RMS treated limb, while exercise resulted in increased endothelial and mesenchymal stromal cells. Significance: These preclinical findings will provide the rationale for further investigation of the mechanisms responsible for the beneficial effects of RET as well as optimizing the RET protocol in this juvenile cancer survivorship model.

Le muscle squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires. / The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells.