Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

Once escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray–based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [3H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth

Mécanismes moléculaires impliqués dans la fusion des cellules précurseurs myogéniques humains

Les cellules musculaires striées squelettiques, sont des cellules multinucléées, formées par la prolifération, l'agrégation etla fusion de cellules précurseurs myogéniques (myogenic precursor cells, mpc) mononucléées. La caractérisation des mécanismes moléculaires impliqués dans le processus de fusion des cellules myogéniques humaines apparaît nécessaire à l'amélioration des biothérapies des maladies musculaires. Nous avons choisi d'évaluer in vitro l'implication de deux systèmes dans la fusion des mpc humaines l'ADAM12 et son partenaire intégrine alpha9betal et le récepteur purinergique P2X7. Nous avons montré l'expression du P2X7 par les mpc humaines et une partie de ses fonctions. Le récepteur P2X7 semble intervenir dan les mécanismes de régulation de la mort cellulaire plutôt que dans la fusion des mpc. Nous avons montré que les mpc humaines expriment constitutivement l'ADAM12 et l'intégrine alpha9betal in vitro et que les deux molécules interagissent entre elles aux zones de contact intercellulaire. L'inhibition du système ADAM12/ alpha9betal montre une diminution significative de l'index de fusion global. Cet effet n'est pas du à un détachement des mpc de leur support, et intervient principalement dans l'élongation des myotubes. Nous avons démontré in vivo chez l'homme une forte expression des 2 isoformes membranaire et secrétée d'ADAM 12 lors du processus de nécrose/régénération myocytaire; les cellules exprimant ADAM12 étant d'origine myogénique, hématopoïétique et interstitielle potentiellement myogénique. Le contrôle de la fusion des mpc avec les myocytes existants s'inscrit dans le développement de thérapies cellulaires efficaces des maladies musculaires.Cell therapy using transplantation of exogenous myogenic precursor cell (mpc) aimed at fusing with mature muscle fibres may help treating devastating muscle diseases. Characterization of molecular systems involved in mpc is a major goal to improve cell therapy. Fusion does not depend on the engagement of a single molecule, but rather on the coordinated recruitment of several molecules mediating ceIl aggregation, close cell-to-cell contact and, finally, the actual fusion event. Among the different human fusion systems, molecules from the ADAM family and the purinoceptor P2X7 could play a direct role in the cell fusion process itself. We showed in human mpc cultures, expression and partial functionality of P2X7. It seems to be implicated in cell death regulation during differentiation process more than in mpc fusion. We showed that human mpc constitutively express ADAMI2 and its integrin partner alpha9betal. Both molecuîes interact atcell-ceII contact areas. Inhibition strategies induce a significant decrease of fusion index. This effect is not due to ceIl de-adhesion from their support, and interaction of ADAM12 and alpha9betal s mainly operative in nascent myotube growth. We showed, in vivo, that in human the necrosislregeneration process is associated with high expression of both isoforms of ADAM1 2 (membrane-bound and secreted). Cells expressing ADAM12 are from myogenic, haematopoietic and interstitial origin. We believe that there is great interest in understanding mpc fusion, because of both ifs central role in muscle development and adult muscle repair, and the relevance of influencing muscle cell fusion for designing future therapeutic strategies.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Dual and beneficial roles of macrophages during skeletal muscle regeneration.

International audienceMacrophages are necessary for skeletal muscle regeneration after injury. Muscle recruits inflammatory monocytes/macrophages that switch toward an anti-inflammatory profile upon phagocytosis of debris. In vitro, proinflammatory macrophages stimulate myoblast proliferation, whereas anti-inflammatory macrophages stimulate their differentiation. Thus, macrophages are involved in both phases of skeletal muscle regeneration: first, inflammation and cleansing of necrosis, and then myogenic differentiation and tissue repair

ADAM12 and α(9)β(1) Integrin Are Instrumental in Human Myogenic Cell Differentiation

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding α(9)β(1) integrin. We report that normal human mpc express both ADAM12 and α(9)β(1) integrin during their differentiation. Expression of α(9) parallels that of ADAM12 and culminates at time of fusion. α(9) and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/α(9)β(1) integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to α(9)β(1), inhibited overall mpc fusion by 47–48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/α(4)β(1), which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-α(9)β(1) antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12–α(9)β(1) interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and α(9)β(1) blockade inhibited more importantly formation of large (≥5 nuclei) myotubes than that of small (2–4 nuclei) myotubes. We conclude that both ADAM12 and α(9)β(1) integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth

Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

International audiencence escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray-based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase O system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [ 3 H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth

Distinct interferon signatures stratify inflammatory and dysimmune myopathies

International audienceWhat is already known about this subject? ► among inflammatory/dysimmune myopathies (iDMs), dermatomyositis (DM) is the only associated with type i-interferon signature. ► Most iDMs are associated with myofiber expression of major histocompatibility complex (MHc)-class i. MHc-i is induced by interferons suggesting a possible role for type ii-interferon in iDMs other than DM. What does this study add? ► in this study, we showed that myofiber MHc-ii expression is observed in inclusion body myositis (iBM) and antisynthetase myositis (aSM), but not in DM and necrotizing autoimmune myopathy (naM). ► in accordance with this finding, we showed that iBM and aSM are specifically associated with type-ii iFnγ signature, DM only with type-i iFn signature, and naM with neither type-i nor type-ii iFn signature. How might this impact on clinical practice? ► Distinct iFn signatures allow a more distinct segregation of iDMs and therefore a more accurate diagnosis. ► Deciphering iFn signatures in iDMs will also lead to develop new therapeutic approaches targeting iFns pathways. AbstrAct Objective the role of interferons (iFn) in the pathophysiology of primary inflammatory and dysimmune myopathies (iDM) is increasingly investigated, notably because specific neutralisation approaches may constitute promising therapeutic tracks. in present work we analysed the muscular expression of specific iFnα/β and iFnγ-stimulated genes in patients with various types of iDM. Methods 39 patients with iDM with inclusion body myositis (iBM, n=9), dermatomyositis (DM, n=10), necrotising autoimmune myopathies (naM, n=10) and antisynthetase myositis (aSM, n=10), and 10 controls were included. Quantification of expression levels of iFnγ, iSg15, an iFnα/β-inducible gene and of six iFnγ-inducible genes (gBP2, Hla-DOB, Hla-DPB, ciita, Hla-DrB and Hla-DMB) was performed on muscle biopsy samples. Results DM usually associated with strong type i iFnα/β signature, iBM and aSM with prominent type ii iFnγ signature and naM with neither type i nor type ii iFn signature. immunofluorescence study in aSM and iBM showed myofibre expression of major histocompatibility class 2 (MHc-2) and ciita, confirming the induction of the iFnγ pathway. Furthermore, MHc-2-positive myofibres were observed in close proximity to cD8+ t cells which produce high levels of iFnγ. Conclusion Distinct iFn signatures allow a more distinct segregation of iDMs and myofibre MHc-2 expression is a reliable biomarker of type ii iFn signature

: Role of Cripto in skeletal muscle regeneration

International audienceSkeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-β ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine

Duchenne muscular dystrophy trajectory in R-DMDdel52 preclinical rat model identifies COMP as biomarker of fibrosis

International audienceAbstractDuchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene and for which there is currently no cure. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R-DMDdel52) causing a complete lack of dystrophin protein. Here we show that R-DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R-DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around 1 year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein, a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R-DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.</jats:p