MEK-inhibitors decrease Nfix in muscular dystrophy but induce unexpected calcifications, partially rescued with Cyanidin diet.

Muscular dystrophies (MDs) are incurable genetic myopathies characterized by progressive degeneration of skeletal muscles. Dystrophic mice lacking the transcription factor Nfix display morphological and functional improvements of the disease. Recently, we demonstrated that MAPK signaling pathway positively regulates Nfix in muscle development and that Cyanidin, a natural antioxidant molecule, strongly ameliorates the pathology. To explore a synergistic approach aimed at treating MDs, we administered Trametinib, a clinically approved MEK inhibitor, alone or combined with Cyanidin to adult Sgca null mice. We observed that chronic treatment with Trametinib and Cyanidin reduced Nfix in myogenic cells but, unexpectedly, caused ectopic calcifications exclusively in dystrophic muscles. The combined treatment with Cyanidin resulted in histological improvements by preventing Trametinib-induced calcifications in Diaphragm and Soleus. Collectively, this first pilot study revealed that Nfix is modulated by the MAPK pathway in MDs, and that Cyanidin partly rescued the unexpected ectopic calcifications caused by MEK inhibition.

A chromosome-level reference genome and pangenome for barn swallow population genomics.

Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.

Selective ablation of Nfix in macrophages attenuates muscular dystrophy by inhibiting fibro-adipogenic progenitor-dependent fibrosis.

Muscular dystrophies are genetic diseases characterized by chronic inflammation and fibrosis. Macrophages are immune cells that sustain muscle regeneration upon acute injury but seem deleterious in the context of chronic muscle injury such as in muscular dystrophies. Here, we observed that the number of macrophages expressing the transcription factor Nfix increases in two distinct mouse models of muscular dystrophies. We showed that the deletion of Nfix in macrophages in dystrophic mice delays the establishment of fibrosis and muscle wasting, and increases grasp force. Macrophages lacking Nfix expressed more TNFα and less TGFß1, thus promoting apoptosis of fibro-adipogenic progenitors. Moreover, pharmacological treatment of dystrophic mice with a ROCK inhibitor accelerated fibrosis through the increase of Nfix expression by macrophages. Thus, we have identified Nfix as a macrophage profibrotic factor in muscular dystrophies, whose inhibition could be a therapeutic route to reduce severity of the dystrophic disease. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Macrophages in Skeletal Muscle Dystrophies, An Entangled Partner.

While skeletal muscle remodeling happens throughout life, diseases that result in its dysfunction are accountable for many deaths. Indeed, skeletal muscle is exceptionally capable to respond to stimuli modifying its homeostasis, such as in atrophy, hypertrophy, regeneration and repair. In particular conditions such as genetic diseases (muscular dystrophies), skeletal muscle's capacity to remodel is strongly affected and undergoes continuous cycles of chronic damage. This induces scarring, fatty infiltration, as well as loss of contractibility and of the ability to generate force. In this context, inflammation, primarily mediated by macrophages, plays a central pathogenic role. Macrophages contribute as the primary regulators of inflammation during skeletal muscle regeneration, affecting tissue-resident cells such as myogenic cells and endothelial cells, but also fibro-adipogenic progenitors, which are the main source of the fibro fatty scar. During skeletal muscle regeneration their function is tightly orchestrated, while in dystrophies their fate is strongly disturbed, resulting in chronic inflammation. In this review, we will discuss the latest findings on the role of macrophages in skeletal muscle diseases, and how they are regulated.

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.

The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.

Interplay between myofibers and pro-inflammatory macrophages controls muscle damage in mdx mice.

Duchenne muscular dystrophy is a genetic muscle disease characterized by chronic inflammation and fibrosis mediated by a pro-fibrotic macrophage population expressing pro-inflammatory markers. Our aim was to characterize cellular events leading to the alteration of macrophage properties and to modulate macrophage inflammatory status using the gaseous mediator hydrogen sulfide (H2S). Using co-culture experiments, we first showed that 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 decreased number of nuclei per fiber, as well as reduced myofiber branching and fibrosis. Finally, we established the metabolic sensor AMP-activated protein kinase (AMPK) as a critical NaHS target in muscle macrophages. These results identify an interplay between myofibers and macrophages where dystrophic myofibers contribute to the maintenance of a highly inflammatory environment sustaining a pro-inflammatory macrophage status, which in turn favors myofiber damage, myofiber branching and establishment of fibrosis. Our results also highlight the use of H2S donors as a potential therapeutic strategy to improve the dystrophic muscle phenotype by dampening chronic inflammation. This article has an associated First Person interview with the first author of the paper.

Rebalancing expression of HMGB1 redox isoforms to counteract muscular dystrophy.

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.

Histological Analysis of Tibialis Anterior Muscle of DMDmdx4Cv Mice from 1 to 24 Months.

BACKGROUND: The mdx-C57/B6 mouse model does not show the clinical signs of Duchenne muscular dystrophy (DMD), although muscles exhibit hallmarks of permanent regeneration and alterations in muscle function. The DMDmdx4Cv strain exhibits very few revertant dystrophin positive myofibers, making that model suitable for studies on gene and cell therapies. OBJECTIVE: The study appraises the histological evolution of the Tibialis Anterior muscle of WT and DMD mdx4Cv mutant from 1 to 24 months. METHODS: Histological analysis included a series of immunostainings of muscle sections for assessing tissue features (fibrosis, lipid deposition, necrosis) and cellular characteristics (size of myofibers, number and distribution of myonuclei, number of satellite cells, vessels, macrophages). RESULTS: None of the investigated cell types (satellite cells, endothelial cells, macrophages) showed variations in their density within the tissue in both WT and DMD mdx4Cv muscle. However, analyzing their number per myofiber showed that in DMD mdx4Cv, myofiber capillarization was increased from 1 to 6 months as compared with WT muscle, then dropped from 12 months. Macrophage number did not vary in WT muscle and peaked at 6 months in DMD mdx4Cv muscle. The number of satellite cells per myofiber did not vary in WT muscle while it remained high in DMD mdx4Cv muscle, starting to decrease from 12 months and being significantly lower at 24 months of age. Myofiber size was not different in DMD mdx4Cv from WT except at 24 months, when it strongly decreased in DMD mdx4Cv muscle. Necrosis and lipid deposition were rare in DMD mdx4Cv muscle. Fibrosis did not increase with age in DMD mdx4Cv muscle and was higher than in WT at 6 and 12 months of age. CONCLUSIONS: As a whole, the results show a strong decrease of the myofiber size at 24 months, and an increased capillarization until 6 months of age in DMD mdx4Cv as compared with the WT. Thus, DMD mdx4Cv mice poorly recapitulates histological DMD features, and its use should take into account the age of the animals according to the purpose of the investigation.

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration.

Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.

Nutritional intervention with cyanidin hinders the progression of muscular dystrophy.

Muscular Dystrophies are severe genetic diseases due to mutations in structural genes, characterized by progressive muscle wasting that compromises patients' mobility and respiratory functions. Literature underlined oxidative stress and inflammation as key drivers of these pathologies. Interestingly among different myofiber classes, type I fibers display a milder dystrophic phenotype showing increased oxidative metabolism. This work shows the benefits of a cyanidin-enriched diet, that promotes muscle fiber-type switch and reduced inflammation in dystrophic alpha-sarcoglyan (Sgca) null mice having, as a net outcome, morphological and functional rescue. Notably, this benefit is achieved also when the diet is administered in dystrophic animals when the signs of the disease are seriously evident. Our work provides compelling evidence that a cyanidin-rich diet strongly delays the progression of muscular dystrophies, paving the way for a combinatorial approach where nutritional-based reduction of muscle inflammation and oxidative stress facilitate the successful perspectives of definitive treatments.

AMPK Activation Regulates LTBP4-Dependent TGF-ß1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy.

Chronic inflammation and fibrosis characterize Duchenne muscular dystrophy (DMD). We show that pro-inflammatory macrophages are associated with fibrosis in mouse and human DMD muscle. DMD-derived Ly6Cpos macrophages exhibit a profibrotic activity by sustaining fibroblast production of collagen I. This is mediated by the high production of latent-TGF-ß1 due to the higher expression of LTBP4, for which polymorphisms are associated with the progression of fibrosis in DMD patients. Skewing macrophage phenotype via AMPK activation decreases ltbp4 expression by Ly6Cpos macrophages, blunts the production of latent-TGF-ß1, and eventually reduces fibrosis and improves DMD muscle force. Moreover, fibro-adipogenic progenitors are the main providers of TGF-ß-activating enzymes in mouse and human DMD, leading to collagen production by fibroblasts. In vivo pharmacological inhibition of TGF-ß-activating enzymes improves the dystrophic phenotype. Thus, an AMPK-LTBP4 axis in inflammatory macrophages controls the production of TGF-ß1, which is further activated by and acts on fibroblastic cells, leading to fibrosis in DMD.

High mobility group box 1 orchestrates tissue regeneration via CXCR4.

Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.

Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration.

Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.

Effects of Macrophage Conditioned-Medium on Murine and Human Muscle Cells: Analysis of Proliferation, Differentiation, and Fusion.

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.

CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production.

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.

Monocyte/macrophage interactions with myogenic precursor cells during skeletal muscle regeneration.

Adult skeletal muscle has the remarkable property of regenerating after damage, owing to satellite cells and myogenic precursor cells becoming committed to adult myogenesis to rebuild the muscle. This process is accompanied by the continuing presence of macrophages, from the phagocytosis of damaged myofibres to the full re-formation of new myofibres. In recent years, there has been huge progress in our understanding of the roles of macrophages during skeletal muscle regeneration, notably concerning their effects on myogenic precursor cells. Here, we review the most recent knowledge acquired on monocyte entry into damaged muscle, the various macrophage subpopulations, and their respective roles during the sequential phases of muscle repair. We also discuss the role of macrophages after exercise-induced muscle damage, notably in humans.

Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

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.