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Rhabdomyosarcoma (RMS) is the most frequent form of paediatric soft-tissue sarcoma and remains a medical challenge, holding in failure current therapeutic strategies. RMS shares histological features with cells of the muscle lineage and this cancer is thought to arise from malignant transformation of myogenic precursors. It has been proposed that RMS and myogenesis could represent the “Jekyll and Hyde” of skeletal muscle. The underlying idea is that some early steps of myogenic differentiation are blocked in RMS, and that understanding how the normal process has gone awry could help to decipher the biological underpinnings of tumorigenesis and tumor escape.It is widely agreed that extracellular matrix (ECM) interferes in skeletal muscle regeneration and that defects in ECM components are clinically significant. The challenge is now to decipher actors and mechanisms responsible for the transmission of signals to muscle cells and the subsequent alterations that could be associated with RMS.Using an original transgenic mice model, we show here that ADAMTSL1 is involved in skeletal muscle regeneration. As previously reported for other members of its family, ADAMTSL1 is part of the TGF-β-ECM-sequestering complex and likely positively regulates TGF-β-pathway activity. Last, we observed that ADAMTSL1 expression behaves as a strong prognostic factor in the aggressive fusion-positive RMS and correlates with a neural-like phenotype of tumor cells, resulting from gain of SMAD2/3/4 targets.

Macrophages are key cells after tissue damage since they mediate both acute inflammatory phase and regenerative inflammation by shifting from pro-inflammatory to restorative cells. Glucocorticoids (GCs) are the most potent anti-inflammatory hormone in clinical use, still their actions on macrophages are not fully understood. We show that the metabolic sensor AMP-activated protein kinase (AMPK) is required for GCs to induce restorative macrophages. GC Dexamethasone activates AMPK in macrophages and GC receptor (GR) phosphorylation is decreased in AMPK-deficient macrophages. Loss of AMPK in macrophages abrogates the GC-induced acquisition of their repair phenotype and impairs GC-induced resolution of inflammation in vivo during post-injury muscle regeneration and acute lung injury. Mechanistically, two categories of genes are impacted by GC treatment in macrophages. Firstly, canonical cytokine regulation by GCs is not affected by AMPK loss. Secondly, AMPK-dependent GC-induced genes required for the phenotypic transition of macrophages are co-regulated by the transcription factor FOXO3, an AMPK substrate. Thus, beyond cytokine regulation, GR requires AMPK-FOXO3 for immunomodulatory actions in macrophages, linking their metabolic status to transcriptional control in regenerative inflammation., publishedVersion

International audience; Understanding the circuits that promote an efficient resolution of inflammation is crucial to deciphering the molecular and cellular processes required to promote tissue repair. Macrophages play a central role in the regulation of inflammation, resolution, and repair/regeneration. Using a model of skeletal muscle injury and repair, herein we identified annexin A1 (AnxA1) as the extracellular trigger of macrophage skewing toward a proreparative phenotype. Brought into the injured tissue initially by migrated neutrophils, and then overexpressed in infiltrating macrophages, AnxA1 activated FPR2/ALX receptors and the downstream AMPK signaling cascade, leading to macrophage skewing, dampening of inflammation, and regeneration of muscle fibers. Mice lacking AnxA1 in all cells or only in myeloid cells displayed a defect in this reparative process. In vitro experiments recapitulated these properties, with AMPK-null macrophages lacking AnxA1-mediated polarization. Collectively, these data identified the AnxA1/FPR2/AMPK axis as an important pathway in skeletal muscle injury regeneration.

International audience

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that was associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90) - the FAP(CD90+). We identified Platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPs(CD90+) showed a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity and production of extracellular matrix. FAP(CD90+) proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a sub-population of FAPs as a key event in the fibrosis development in T2DM muscle.

SummaryDuchenne Muscular Dystrophy is a genetic muscle disease characterized by chronic inflammation and fibrosis, which is mediated by a pro-fibrotic macrophage population expressing pro-inflammatory markers. The aim of this study was to characterize cellular events leading to the alteration of macrophage properties, and to modulate macrophage inflammatory status using the gaseous mediator H2S. We first analyzed the relationship between myofibers and macrophages in the mdx mouse model of Duchenne Muscular Dystrophy using coculture experiments. We showed that normal myofibers derived from mdx mice strongly skewed the polarization of resting macrophages towards a pro-inflammatory phenotype. Treatment of mdx mice with NaHS, an H2S donor, reduced the number of pro-inflammatory macrophages in skeletal muscle, which was associated with a decrease in the number of nuclei per fiber, a reduction of myofiber branching and a reduced fibrosis. These results identify an interplay between myofibers and macrophages where dystrophic myofibers contribute to the maintenance of a highly inflammatory environment that skews the macrophage status, which in turn favors myofiber damage, myofiber branching and fibrosis establishment. They also identify H2S donors as a potential therapeutic strategy to improve dystrophic muscle phenotype by modulating macrophage inflammatory status.

International audience; Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream ofGremlin 1(GREM1), which was associated with increasedGREM1expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

SummaryMacrophages are key immune cells which mediate both the acute inflammatory phase and the repair phase after tissue damage. Macrophages switch from pro-inflammatory to anti-inflammatory cells that sustain repair and return to tissue homeostasis. We show that the metabolic sensor, AMP-activated protein kinase (AMPK) is essential for glucocorticoid induction of an anti-inflammatory macrophage phenotype. While canonical gene regulation by glucocorticoids was not affected by loss of AMPK, we identified AMPK-dependent glucocorticoid-regulated genes in macrophages, related to efferocytosis. AMPK-deficient macrophages do not acquire phenotypic and functional anti-inflammatory features upon glucocorticoid exposure. We identified FOXO3 as an AMPK-dependent regulator of glucocorticoid activity in macrophages. Loss of AMPK in macrophages in vivo abrogates glucocorticoid anti-inflammatory actions during post-injury muscle regeneration and endotoxin induced acute lung injury. These data highlight that the glucocorticoid receptor is dependent on AMPK for its immunomodulatory actions in macrophages, linking their metabolic status to transcriptional control in resolving inflammation.

International audience; Macrophages are key players of immunity that display different functions according to their activation states. In a regenerative context, pro-inflammatory macrophages (Ly6Cpos) are involved in the mounting of the inflammatory response whereas anti-inflammatory macrophages (Ly6Cneg) dampen the inflammation and promote tissue repair. Reactive oxygen species (ROS) production is a hallmark of tissue injury and of subsequent inflammation as described in a bacterial challenge context. However, whether macrophages produce ROS following a sterile tissue injury is uncertain. In this study, we used complementary in vitro, ex vivo and in vivo experiments in mouse to show that macrophages do not release ROS following a sterile injury in skeletal muscle. Furthermore, expression profiles of genes involved in the response to oxidative stress in Ly6Cpos and Ly6Cneg macrophage subsets did not indicate any antioxidant response in this context. Finally, in vivo, pharmacological antioxidant supplementation with N-Acetyl-cysteine (NAC) following skeletal muscle injury did not alter macrophage phenotype during skeletal muscle regeneration. Overall, these results indicate that following a sterile injury, macrophage-derived ROS release is not involved in the regulation of the inflammatory response in the regenerating skeletal muscle.

The discovery of new non-canonical (i.e. non-innate immune) functions of macrophages has been a recurring theme over the past 20 years. Indeed, it has emerged that macrophages can influence the development, homeostasis, maintenance and regeneration of many tissues and organs, including skeletal muscle, cardiac muscle, the brain and the liver, in part by acting directly on tissue-resident stem cells. In addition, macrophages play crucial roles in diseases such as obesity-associated diabetes or cancers. Increased knowledge of their regulatory roles within each tissue will therefore help us to better understand the full extent of their functions and could highlight new mechanisms modulating disease pathogenesis. In this Review, we discuss recent studies that have elucidated the developmental origins of various macrophage populations and summarize our knowledge of the non-canonical functions of macrophages in development, regeneration and tissue repair.

Adult skeletal muscle is capable of complete regeneration after an acute injury. The main parameter studied to assess muscle regeneration efficacy is the cross-sectional area (CSA) of the myofibers as myofiber size correlates with muscle force. CSA analysis can be time-consuming and may trigger variability in the results when performed manually. This is why programs were developed to completely automate the analysis of the CSA, such as SMASH, MyoVision, or MuscleJ softwares. Although these softwares are efficient to measure CSA on normal or hypertrophic/atrophic muscle, they fail to efficiently measure CSA on regenerating muscles. We developed Open-CSAM, an ImageJ macro, to perform a high throughput semi-automated analysis of CSA on skeletal muscle from various experimental conditions. The macro allows the experimenter to adjust the analysis and correct the mistakes done by the automation, which is not possible with fully automated programs. We showed that Open-CSAM was more accurate to measure CSA in regenerating and dystrophic muscles as compared with SMASH, MyoVision, and MuscleJ softwares and that the inter-experimenter variability was negligible. We also showed that, to obtain a representative CSA measurement, it was necessary to analyze the whole muscle section and not randomly selected pictures, a process that was easily and accurately be performed using Open-CSAM. To conclude, we show here an easy and experimenter-controlled tool to measure CSA in muscles from any experimental condition, including regenerating muscle. Electronic supplementary material The online version of this article (10.1186/s13395-018-0186-6) contains supplementary material, which is available to authorized users.

International audience; Body composition has gained increasing attention in oncology in recent years due to fact that sarcopenia has been revealed to be a strong prognostic indicator for survival across multiple stages and cancer types and a predictive factor for toxicity and surgery complications. Accumulating evidence over the last decade has unraveled the "pharmacology" of sarcopenia. Lean body mass may be more relevant to define drug dosing than the "classical" body surface area or flat-fixed dosing in patients with cancer. Since sarcopenia has a major impact on patient survival and quality of life, therapeutic interventions aiming at reducing muscle loss have been developed and are being prospectively evaluated in randomized controlled trials. It is now acknowledged that this supportive care dimension of oncological management is essential to ensure the success of any anticancer treatment. The field of sarcopenia and body composition in cancer is developing quickly, with (i) the newly identified concept of sarcopenic obesity defined as a specific pathophysiological entity, (ii) unsolved issues regarding the best evaluation modalities and cutoff for definition of sarcopenia on imaging, (iii) first results from clinical trials evaluating physical activity, and (iv) emerging body-composition-tailored drug administration schemes. In this context, we propose a comprehensive review providing a panoramic approach of the clinical, pharmacological and therapeutic implications of sarcopenia and body composition in oncology.

International audience; Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-b family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

SummaryUnderstanding the circuits that promote an efficient resolution of inflammation is crucial to deciphering the molecular and cellular processes required to promote tissue repair. Macrophages play a central role in the regulation of inflammation, resolution and repair/regeneration. Using a model of skeletal muscle injury and repair, herein we identify Annexin A1 (AnxA1) as the extracellular trigger of macrophage skewing towards a pro-reparative phenotype. Brought into the injured tissue initially by migrated neutrophils, and then over-expressed in infiltrating macrophages, AnxA1 activates FPR2/ALX receptors and the downstream AMPK signalling cascade leading to macrophage skewing, dampening of inflammation and regeneration of muscle fibres. Mice lacking AnxA1 in all cells or in myeloid cells only display a defect in this reparative process.In vitroexperiments recapitulated these properties, with AMPK null macrophages lacking AnxA1-mediated polarization. Collectively, these data identify the AnxA1/FPR2/AMPK axis as a novel pathway in skeletal muscle injury regeneration.

In this chapter, we describe the methods to isolate and culture muscle stem cells (MuSCs) from murine skeletal muscle in order to decipher the intrinsic effect of AMP-activated kinase activity on MuSC fate. Culture of MuSCs is a powerful model to recapitulate every step of stem cell behavior observed in vivo: activation, proliferation, differentiation, fusion and also self-renewal. We provide the detailed procedures to isolate pure MuSCs by a flow cytometry-based method using the selection of a combination of specific markers and to characterize MuSC fate (quiescence, activation, and differentiation) in response to AMPK activity modulation by assessing of the expression of stem cell (e.g., Pax7) and myogenic marker (e.g., MyoD).

In this chapter, we describe the methods to isolate and culture muscle stem cells (MuSCs) from murine skeletal muscle in order to decipher the intrinsic effect of AMP-activated kinase activity on MuSC fate. Culture of MuSCs is a powerful model to recapitulate every step of stem cell behavior observed in vivo: activation, proliferation, differentiation, fusion and also self-renewal. We provide the detailed procedures to isolate pure MuSCs by a flow cytometry-based method using the selection of a combination of specific markers and to characterize MuSC fate (quiescence, activation, and differentiation) in response to AMPK activity modulation by assessing of the expression of stem cell (e.g., Pax7) and myogenic marker (e.g., MyoD).

Summary In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling., Graphical Abstract, Highlights • Endothelial cells (ECs) promote myogenesis • Myogenic progenitor cells (MPCs) stimulate angiogenesis as they differentiate • EC- and MPC-derived Apelin, Oncostatin M, and Periostin promote myo-angiogenesis • Restorative macrophages stimulate myo-angiogenesis via Oncostatin M secretion, In this study, Chazaud et al. demonstrate that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted to couple myogenesis and angiogenesis during skeletal muscle regeneration. EC- and MPC-derived Apelin, Oncostatin M, and Periostin controlled myogenesis/angiogenesis coupling and were required for myogenesis and vessel formation. They show that, via the production of Oncostatin M, restorative macrophages promoted myogenesis/angiogenesis coupling.

Skeletal muscle is highly irrigated by blood vessels. Beyond oxygen and nutrient supply, new vessel functions have been identified. This review presents vessel microanatomy and functions at tissue, cellular, and molecular levels. Mechanisms of vessel plasticity are described during skeletal muscle development and acute regeneration, and in physiological and pathological contexts.

17 Pages, 5 figures, Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate., This work was funded by grants from a European Commission integrated project (LSHM‐CT‐2004‐005272), the Framework Programme FP7 Endostem (under grant agreement 241440), the Association Française contre les Myopathies, Ligue Nationale Contre le Cancer, and the Société Française de Myologie. We thank D.G. Hardie (Dundee University, Scotland, UK) for providing antibodies against AMPKα1, R.A. Depinho (University of Texas, Austin, TX) for providing the LKB1fl/fl mice, Pierre Rocheteau for providing antibody against TOM22 (Institut Pasteur, Paris, France), T. Andrieu and S. Dussurgey from the AniRA‐Cytometry platform (UMS 3444—SFR Gerland Biosciences, Lyon, France) for their expertise in cell sorting, and Frederic Bouillaud for his expertise and help in bioenergetics and metabolism (Institut Cochin, Paris, France).

Skeletal muscle is a highly plastic tissue, which is able to regenerate after an injury. Effective and complete regeneration requires interactions between myogenic precursor cells and several cell types such as macrophages . Bone marrow derived macrophages in mouse and monocyte-derived macrophages in human are useful tools to obtain macrophage populations that may be specifically activated/polarized in vitro (e.g., pro-inflammatory, anti-inflammatory, and alternatively activated macrophages ). In vitro , human or murine primary myogenic cells recapitulate the adult myogenesis program through proliferation, myogenic differentiation, and fusion. Macrophages being highly secreting cells, they act on various biological processes including adult myogenesis . Here, we present protocols to analyze in vitro the effect of macrophage-secreted factors on muscle cell proliferation or differentiation in both mouse and human.

Skeletal muscle is a highly plastic tissue, which is able to regenerate after an injury. Effective and complete regeneration requires interactions between myogenic precursor cells and several cell types such as macrophages . Bone marrow derived macrophages in mouse and monocyte-derived macrophages in human are useful tools to obtain macrophage populations that may be specifically activated/polarized in vitro (e.g., pro-inflammatory, anti-inflammatory, and alternatively activated macrophages ). In vitro , human or murine primary myogenic cells recapitulate the adult myogenesis program through proliferation, myogenic differentiation, and fusion. Macrophages being highly secreting cells, they act on various biological processes including adult myogenesis . Here, we present protocols to analyze in vitro the effect of macrophage-secreted factors on muscle cell proliferation or differentiation in both mouse and human.

There are several open questions regarding the origin, development, and differentiation of subpopulations of monocytes, macrophages (MFs), and dendritic cells. It is a particularly intriguing question how circulating monocyte subsets develop and contribute to the generation of steady-state and inflammatory tissue MF pools and which transcriptional mechanisms contribute to these processes. In this study, we took advantage of a genetic model in which LyC6− circulating monocyte development is severely diminished due to the lack of the nuclear receptor, NUR77. We show that, in a mouse model of skeletal muscle injury and regeneration, the accumulation of leukocytes and the generation of LyC6+ and LyC6− MF pools are intact in the absence of circulating LyC6− blood monocytes. These data suggest that NUR77, which is required for LyC6− blood monocyte development, is expressed but not critically required for LyC6+ to LyC6− tissue MF specification. Moreover, these observations support a model according to which tissue macrophage subtype specification is distinct from that of circulating monocytes. Lastly, our data show that in the used sterile inflammation model tissue LyC6− MFs are derived from LyC6+ cells.

Marielle Saclier and Houda Yacoub-Youssef : Both should be considered as first author; International audience; Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.

Introduction: Duchenne Muscular Dystrophy (DMD) is characterized by the lack of dystrophin that leads to severe myofiber degeneration. We have shown that endomysial fibrosis is correlated with age at ambulation loss in DMD patients. However, the dystrophin-deficient mdx mouse does not have fibrotic lesions in adult limb muscles. Here, we describe a model of chronic mechanical muscle injury that triggers chronic lesions in mdx hindlimb muscle. Methods: Micromechanical injuries were performed daily in tibialis anterior muscles for 2 weeks. Results: Endomysial fibrosis appeared beginning 1 week post-injury, remained stable for 3 months and was associated with loss of specific maximal force. Fibrosis was associated with an increased expression of factors involved in fibrogenesis including α-smooth muscle actin, connective tissue growth factor, and lysyl oxidase, which colocalized with collagen deposits. Conclusions: This induced fibrotic dystrophic model may be useful to study mechanisms of fibrosis in dystrophinopathies and to evaluate antifibrotic treatments. Muscle Nerve 45: 803–814, 2012

Muscle injury triggers inflammation in which infiltrating mononuclear phagocytes are crucial for tissue regeneration. The interaction of the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axis that guides phagocyte infiltration is incompletely understood. Here, we show that CX3CR1 deficiency promotes muscle repair and rescues Ccl2−/− mice from impaired muscle regeneration as a result of altered macrophage function, not infiltration. Transcriptomic analysis of muscle mononuclear phagocytes reveals that Apolipoprotein E (ApoE) is upregulated in mice with efficient regeneration. ApoE treatment enhances phagocytosis by mononuclear phagocytes in vitro, and restores phagocytic activity and muscle regeneration in Ccl2−/− mice. Because CX3CR1 deficiency may compensate for defective CCL2-dependant monocyte recruitment by modulating ApoE-dependent macrophage phagocytic activity, targeting CX3CR1 expressed by macrophages might be a powerful therapeutic approach to improve muscle regeneration., Chemokine-driven infiltration of inflammatory macrophages is central to the muscle regenerative response to injury. Here the authors show that the function of infiltrating macrophages is also important as notexin-induced muscle injury in mice is rescued by CX3CR1 knockout owing to enhanced ApoE production.

Macrophage gene expression determines phagocyte responses and effector functions. Macrophage plasticity has been mainly addressed in in vitro models that do not account for the environmental complexity observed in vivo. In this study, we show that microarray gene expression profiling revealed a highly dynamic landscape of transcriptomic changes of Ly6CposCX3CR1lo and Ly6CnegCX3CR1hi macrophage populations during skeletal muscle regeneration after a sterile damage. Systematic gene expression analysis revealed that the time elapsed, much more than Ly6C status, was correlated with the largest differential gene expression, indicating that the time course of inflammation was the predominant driving force of macrophage gene expression. Moreover, Ly6Cpos/Ly6Cneg subsets could not have been aligned to canonical M1/M2 profiles. Instead, a combination of analyses suggested the existence of four main features of muscle-derived macrophages specifying important steps of regeneration: 1) infiltrating Ly6Cpos macrophages expressed acute-phase proteins and exhibited an inflammatory profile independent of IFN-γ, making them damage-associated macrophages; 2) metabolic changes of macrophages, characterized by a decreased glycolysis and an increased tricarboxylic acid cycle/oxidative pathway, preceded the switch to and sustained their anti-inflammatory profile; 3) Ly6Cneg macrophages, originating from skewed Ly6Cpos cells, actively proliferated; and 4) later on, restorative Ly6Cneg macrophages were characterized by a novel profile, indicative of secretion of molecules involved in intercellular communications, notably matrix-related molecules. These results show the highly dynamic nature of the macrophage response at the molecular level after an acute tissue injury and subsequent repair, and associate a specific signature of macrophages to predictive specialized functions of macrophages at each step of tissue injury/repair.

In skeletal muscle, satellite cells, that are responsible of muscle repair, are localized close to capillaries. Although angiogenesis is known for a long time to be crucial for muscle repair and satellite cell survival, cellular interplays between vessel cells and satellite/myogenic cells have been poorly explored. We analyzed the interrelationships between myogenic cells, endothelial cells, and periendothelial cells that includes smooth muscle cells and endomysial fibroblasts. We found that endothelial cells strongly stimulate myogenic cell growth and, inversely, myogenic cells increase angiogenesis. VEGF plays an essential role in this bidirectional interaction. On the contrary, periendothelial cells promote the return to quiescence of a subset of muscle precursor cells that ensures self-renewal of adult muscle stem cells. We have shown that Angiopoietin-1/Tie-2 signaling controls the entry into quiescence. We propose that during muscle regeneration, i.e., while vessels are not stabilized, endothelial cells and myogenic cells interact with each other to promote both myogenesis and angiogenesis, that have been shown to be concomitant processes in several models. On the other hand, once homeostasis of muscle is reached, the proximity of satellite cells and periendothelial cells allows the responsiveness of satellite cells, that bear Tie-2 receptor, to the secretion of Angiopoietin-1 by periendothelial cells, that, in the same time, stabilize vessels by promoting quiescence of endothelial cells.

We investigated the strength of the association between oxidative stress, hypoxia inducible factor 1 (HIF-1 alpha) and acute hypoxic ventilatory response (AHVR) after hypoxic training in elite runners. Six elite runners were submitted to 18-day of "living high-training low" (LHTL) and six performed the same training in normoxia. AHVR was measured during an acute hypoxic test before and after training. Plasma levels of protein oxidation (AOPP), malondialdehydes and (HIF-1 alpha) mRNA in the leukocytes were measured before and after the acute hypoxic test. LHTL increased AHVR and amplified the responses of HIF-1 alpha mRNA and AOPP (Delta(AOPP)) to the acute hypoxic test. Furthermore, between PRE and POST, the changes in Delta(AOPP) were correlated with the changes in AHVR (r=0.69, P=0.01). The ventilatory acclimatization to hypoxia occurring in athletes after LHTL seems to be modulated by oxidative stress. Furthermore, LHTL induced a higher sensitivity of HIF-1 alpha mRNA to acute hypoxia in elite athletes.

The aim of this study was to determine the response of blood markers to acute hypoxia in high-level endurance athletes before training based on "living high-training low" model. Thirty endurance athletes performed a hypoxic cycling test and spent 3 h at rest in a simulated altitude of 3,000 m. At the end of the hypoxic cycling test, the quantity of the natural antisense transcript of HIF-1alpha mRNA (aHIF) transcript increased significantly (+37%, P = 0.024). After 3-h exposure, at a simulated altitude of 3,000 m, the amount of HIF-1alpha mRNA increased significantly (+57%, P = 0.012). Moreover, a large inter-subject range was observed in response to the hypoxic cycling test and to the prolonged hypoxic exposure: -133%/+79% and -82%/+653% for HIF-1alpha mRNA, 69%/+324% and -76%/+229% for aHIF. This study shows a large inter-variability of blood markers in elite athletes in response to acute hypoxic exposure corroborating previous observations made in other populations.