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Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

Skeletal muscle has a high regenerative ability and maintains homeostasis by rapidly regenerating from frequent damage caused by intense exercise or trauma. In sports, skeletal muscle damage occurs frequently due to intense exercise, so practical methods to promote skeletal muscle regeneration are required. Recent studies have shown that it may be possible to promote skeletal muscle regeneration through new pathways, such as promoting autophagy and improving mitochondrial function. Spermidine is a type of polyamine, and oral intake of spermidine promotes autophagy and improves mitochondrial function without inhibiting mTOR. Therefore, we evaluate the effects of spermidine intake on skeletal muscle regeneration after injury using a mouse model of cardiotoxin‐induced muscle injury. Our results showed no significant change in skeletal muscle wet weight with spermidine intake at all time points. In addition, although spermidine intake significantly increased the mean fiber cross‐sectional area 14 days after injury, these effects were not observed at other time points. In addition, we analyzed stem cells, autophagy, mTOR signaling, inflammation, and mitochondria, but no significant effects of spermidine intake were observed at almost all time points and protein expression levels. Therefore, spermidine intake does not affect skeletal muscle regeneration after chemical injury, and if there is any, it is very limited.

Adult skeletal muscle stem cells, also known satellite cells (SCs), are a highly heterogeneous population and reside between the basal lamina and the muscle fiber sarcolemma. Myofibers function as an immediate niche to support SC self-renewal and activation during muscle growth and regeneration. Herein, we demonstrate that microRNA 378 (miR-378) regulates glycolytic metabolism in skeletal muscle fibers, as evidenced by analysis of myofiber-specific miR-378 transgenic mice (TG). Subsequently, we evaluate SC function and muscle regeneration using miR-378 TG mice. We demonstrate that miR-378 TG mice significantly attenuate muscle regeneration because of the delayed activation and differentiation of SCs. Furthermore, we show that the miR-378-mediated metabolic switch enriches Pax7 Hi SCs, accounting for impaired muscle regeneration in miR-378 TG mice. Mechanistically, our data suggest that miR-378 targets the Akt1/FoxO1 pathway, which contributes the enrichment of Pax7 Hi SCs in miR-378 TG mice. Together, our findings indicate that miR-378 is a target that links fiber metabolism to muscle stem cell heterogeneity and provide a genetic model to approve the metabolic niche role of myofibers in regulating muscle stem cell behavior and function.

Background Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. Methods To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. Results At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. Conclusion Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy.

Skeletal muscle regeneration is a highly orchestrated process that depends on multiple immune-system cell types, notably macrophages (MFs) and Foxp3⁺CD4⁺ regulatory T (Treg) cells. This study addressed how Treg cells rein in MFs during regeneration of murine muscle after acute injury with cardiotoxin. We first delineated and characterized two subsets of MFs according to their expression of major histocompatibility complex class II (MHCII) molecules, i.e., their ability to present antigens. Then, we assessed the impact of Treg cells on these MF subsets by punctually depleting Foxp3⁺ cells during the regenerative process. Treg cells controlled both the accumulation and phenotype of the two types of MFs. Their absence after injury promoted IFN-γ production, primarily by NK and effector T cells, which ultimately resulted in MF dysregulation and increased inflammation and fibrosis, pointing to compromised muscle repair. Thus, we uncovered an IFN-γ–centered regulatory layer by which Treg cells keep MFs in check and dampen inflammation during regeneration of skeletal muscle.

Key Points Changes in the stages of myogenesis during muscle regeneration following injury coincide with changes in the phenotype and activation state of leukocytes that invade the damaged, regenerating tissue. Macrophages dominate the inflammatory infiltrate in regenerating muscle, and they are biased towards an M1 phenotype during the early, proliferative stages of muscle regeneration and towards an M2 phenotype during the differentiation and growth phase of regeneration. Signalling initiated by tumour necrosis factor (TNF), interferon-γ (IFNγ), interleukin-10 (IL-10) and insulin-like growth factor 1 (IGF1) has key roles in controlling the normal inflammatory response and myogenic response to muscle damage that is required to achieve muscle regeneration. Disruptions of normal regulatory interactions between myeloid cells and muscle with regulatory T (T reg ) cells, CD8 + T cells and fibro-adipogenic progenitor (FAP) cells can prevent successful muscle regeneration following acute injury. Chronic muscle disease and muscle ageing disrupt the normal function of myeloid cells, FAP cells and T reg cells, which can lead to impaired muscle regeneration and increased muscle fibrosis. Manipulations of myeloid cell phenotypes can improve muscle regeneration and growth following muscle trauma. Following muscle injury, changes in the stages of muscle growth coincide with changes in the phenotype and activation status of leukocytes that enter the site of muscle damage. As described in this Review, complex and coordinated crosstalk between immune cells and muscle cells determines the success or failure of muscle regeneration. Diseases of muscle that are caused by pathological interactions between muscle and the immune system are devastating, but rare. However, muscle injuries that involve trauma and regeneration are fairly common, and inflammation is a clear feature of the regenerative process. Investigations of the inflammatory response to muscle injury have now revealed that the apparently nonspecific inflammatory response to trauma is actually a complex and coordinated interaction between muscle and the immune system that determines the success or failure of tissue regeneration.

Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we used mass spectrometry–based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed temporal regulation of glycerophospholipids and production of pro-inflammatory lipid mediators (for example, leukotrienes and prostaglandins) and specialized pro-resolving lipid mediators (for example, resolvins and lipoxins) that were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6C hi and Ly6C lo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6C lo macrophages. RNA sequencing of macrophages stimulated with resolvin D2 showed similarities to transcriptional changes found during the temporal transition from Ly6C hi macrophage to Ly6C lo macrophage. In vivo, resolvin D2 increased Ly6C lo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof of concept for their exploitable effector roles in muscle regeneration. Muscle damage elicits a sterile immune response that facilitates complete regeneration. Nagy and colleagues map the mediator lipidome during the transition from inflammation to resolution in skeletal muscle injury.

Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4–conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.

UV‐cured collagen‐based hydrogels hold promise in skeletal muscle regeneration due to their soft elastic properties and porous architecture. However, the complex triple helix conformation of collagen and environmental conditions, i.e., molecular oxygen, pose risks to reaction controllability, wet‐state integrity, and reproducibility. To address this challenge, a photoclick hydrogel platform is presented through an oxygen‐insensitive thiol‐ene reaction between 2‐iminothiolane (2IT)‐functionalized type I collagen and multiarm, nonhomopolymerizable norbornene‐terminated polyethylene glycol (PEG). UV‐induced network formation is demonstrated by oscillatory time sweeps on the reacting thiol‐ene mixture, so that significantly increased storage moduli are measured and adjusted depending on the photoinitiator concentration. Variations in PEG functionality (4‐arm and 8‐arm) and PEG content generate hydrogels with skeletal muscle native stiffness ( E c  = 1.3 ± 0.2‒11.5 ± 0.9 kPa), diffusion‐controlled swelling behavior and erosion‐driven degradability. In vitro, no cytotoxic effect is detected on C2C12 murine myoblasts, while myogenic differentiation is successfully accomplished on hydrogel‐seeded cells in then low serum culture medium. In vivo, 7 d subcutaneous implantation of selected thiol‐ene hydrogel in rats reveal higher cell infiltration, blood vessel formation, and denser tissue interface compared to a clinical gold standard collagen matrix (Mucograft, a trademark of Geistlich Biomaterials). These results, therefore, support the applicability and further development of this hydrogel platform for skeletal muscle regeneration.