Volumetric muscle loss (VML) leads to severe skeletal muscle dysfunction. While muscle tissue engineering offers a promising strategy, challenges persist due to insufficient neuromuscular innervation and poor reconstruction of neuromuscular junctions (NMJs). Conductive hydrogels can mimic the electrophysiological microenvironment and thus promote structural and functional regeneration, yet commonly used conductive materials still suffer from poor hydrophilicity, non-degradability, and potential cytotoxicity, while their underlying mechanisms remain unclear. Ti3C2Tx MXene, a class of two-dimensional nanomaterials with high conductivity and biocompatibility, shows potential for repairing electroactive tissues. In this study, we developed a novel biomimetic electroactive hydrogel by incorporating Ti3C2Tx MXene nanosheets into adipose-derived decellularized extracellular matrix (adECM). This study aimed to investigate the effects and mechanisms of MXene/adECM hydrogel on muscle regeneration and innervation. MXene/adECM hydrogel demonstrated excellent biocompatibility, biodegradability, and conductivity. Compared to the adECM hydrogel, the incorporation of MXene promoted myogenesis, along with increased expression of Desmin, MyoD1, and Myf5. Furthermore, the MXene/adECM hydrogel at the optimal concentration increased the average neurite length by 47.29μm (p < 0.05) relative to the adECM group. Transcriptomic analysis combined with a neuromuscular co-culture system indicated that the MXene/adECM hydrogel promoted the formation of neuromuscular junctions (NMJs). The incorporation of MXene upregulated the expression of specific voltage-gated calcium channels at the motor endplate, with transcript levels of Cacna1a and Cacna1s increased to 2.1-fold and 3.1-fold, respectively. It was further observed that calcium signaling was enhanced in the MXene/adECM group, with the peak calcium signal intensity being 2.40 times that of the adECM group. In vivo rat VML model confirmed that, compared to the adECM hydrogel, the MXene/adECM hydrogel promoted an increase in regenerated muscle fiber area, reduced collagen deposition, and elevated the fluorescence intensity of CD31 and Tuj. The co-localization percentage of presynaptic and postsynaptic NMJ markers increased from 27.85 ± 8.69% to 42.21 ± 15.52%. Gait analysis showed significant improvements in print area, swing/stance ratio, and movement velocity. In the MXene/adECM group, the isometric tetanic force (ITF) upon sciatic nerve stimulation was significantly higher than that of the adECM group (0.082 ± 0.012N vs. 0.057 ± 0.014N, p < 0.05), approaching the level of the uninjured group. Together, these findings demonstrate that the incorporation of MXenes into adECM provides a promising strategy that integrates microenvironmental support with endogenous electrical cues to modulate calcium influx and promote NMJ formation, offering a new paradigm for the treatment of VML.

Stage-specific transcriptional atlas of goose satellite cells uncovers molecular dynamics driving embryonic skeletal muscle development.

Genetic and epigenetic determinants of injury risk and recovery in elite athletes: toward precision sports medicine.

Versatile role of fibro-adipogenic progenitors in neuromuscular junction homeostasis and pathology.

Dual role of macrophages in skeletal muscle atrophy: Mechanisms and therapeutic strategies.

A newly identified specific category of non-coding RNA (ncRNA), circRNAs, is drawing interest for their role in controlling several biological processes including muscle regeneration, aging, and adaptation to physical activity. Unlike linear RNAs, circRNAs are very stable and can have long-lasting regulatory impact since they create a covalently closed loop structure. Emerging evidence indicates that circRNAs play a pivotal role in skeletal muscle biology by regulating myogenesis, satellite cell activation, protein synthesis, and cellular senescence-processes significantly influenced by aging. These molecules are crucial for muscle function and regeneration, acting as microRNA sponges, interacting with RNA-binding proteins, and modulating gene expression and translation. Exercise-especially resistance and endurance training-has been shown to change circRNA expression in skeletal muscle, therefore possibly reducing age-related muscle loss and improving regenerative capacity. Though encouraging, much of the circRNA in muscle biology research is still in its early stages, with few functional studies and varying outcomes across various species and exercise models. Moreover, the exact ways circRNAs affect muscular adaptation to exercise and stop aging-related degeneration are still not completely known. This review addresses the existing knowledge gaps regarding the potential therapeutic applications of circRNAs in combating muscle degeneration and sarcopenia, as well as their role in muscle health and aging.

Aging is characterized by a decline in the ability of tissue repair and regeneration after injury. In skeletal muscle, this decline is largely driven by impaired function of muscle stem cells (MuSCs) to efficiently contribute to muscle regeneration. We uncovered a cause of this aging-associated dysfunction: a cellular survivorship bias that prioritizes stem cell persistence at the expense of functionality. With age, MuSCs increased expression of a tumor suppressor, N-myc down-regulated gene 1 (NDRG1), which, by suppressing the mammalian target of rapamycin (mTOR) pathway, increased their long-term survival potential but at the cost of their ability to promptly activate and contribute to muscle regeneration. This delayed muscle regeneration with age may result from a trade-off that favors long-term stem cell survival over immediate regenerative capacity.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. Fusion of satellite cell-derived myoblasts (SCMs) is indispensable for generating the multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain incompletely understood. Here, we reveal a critical role for branched actin polymerization in SCM fusion during mouse skeletal muscle regeneration. Using conditional knockouts of the Arp2/3 complex and its actin nucleation-promoting factors N-WASP and WAVE, we demonstrate that branched actin polymerization is specifically required for SCM fusion but dispensable for satellite cell proliferation, differentiation, and migration. We show that the N-WASP and WAVE complexes have partially redundant functions in regulating SCM fusion and that branched actin polymerization is essential for generating invasive protrusions at fusogenic synapses in SCMs. Together, our study identifies branched-actin regulators as key components of the myoblast fusion machinery and establishes invasive protrusion formation as a critical mechanism enabling myoblast fusion during skeletal muscle regeneration.

Zinc chelation therapy mitigates muscle fibrosis and improves functional outcomes in a murine model of crush syndrome.

Muscle RING finger-1 facilitates skeletal muscle regeneration via regulating myoblast proliferation and differentiation.

The extracellular matrix (ECM) protease Adamts5 and its ECM substrates are critical regulators of inflammation and fibrosis; whether Adamts5 also regulates muscle regeneration is not known. Right tibialis anterior (TA) muscles from adult Adamts5--/- mice or wild type mice were injected with glycerol to induce injury. In uninjured muscles and at 7- and 14-days post injury, TA contractile function was determined in situ, followed by an assessment of pathology using histology and immunohistochemistry. Immunoblotting was performed for the versikine fragment which is generated when Adamts5 cleaves its substrate versican. Versikine protein, which correlates with Adamts5 proteolytic activity, was lower in uninjured and injured TA muscles from Adamts5-/- mice versus wild type mice. In uninjured TA muscles, Adamts5 deletion of the catalytic and ancillary domains decreased the absolute (Po) and normalized to muscle size (sPo) force output, with no significant effect on muscle mass and myofiber size. Adamts5 deletion compromised regeneration with greater impairment evident at the later timepoint. Force output (Po and sPo) was lower in Adamts5-/- mice at 7- and 14-days post injury. TA mass and myofiber size were only decreased at 14-days post injury, while embryonic myosin heavy chain expression did not significantly differ between genotypes. Degeneration, mononuclear infiltrates, and ECM deposition including fibronectin protein were greater in injured TA muscles from Adamts5-/- mice. Resolution of inflammation was also delayed in Adamts5-/- mice, with more infiltrating macrophages observed at 14-days post injury. In conclusion, Adamts5 regulates the balance between muscle regeneration, fibrosis, and inflammation following glycerol injury.

Dysregulation of stem cell properties is a hallmark of many pathologies, but the dynamic behaviour of stem cells in their microenvironment during disease progression remains poorly understood. Using the mdx mouse model of Duchenne Muscular Dystrophy, we developed innovative live imaging of muscle stem cells (MuSCs) in vivo, and ex vivo on isolated myofibres. We show that mdx MuSCs have impaired migration and precocious differentiation through unbalanced symmetric divisions, driven by p38 and PI3K signalling pathways, in contrast to the p38-only dependence of healthy MuSCs. Cross-grafting shows that MuSC fate decisions are governed by fibre-independent cues, whereas their migration behaviour is determined by the myofibre niche. This study provides the first dynamic analysis of dystrophic MuSC properties in vivo, reconciling conflicting reports on their function. Our findings establish DMD as a MuSC disease with niche dysfunctions, offering strategies to restore stem cell functions for improved muscle regeneration.

Mitocytosis, mitophagy, and apoptosis coordinately drive mitochondrial clearance to regulate myogenic differentiation.

MRG15 decline in aged/injured MuSCs hinders regeneration via differentiation defects

To observe the effect of electroacupuncture (EA) on the expressions of paired box transcription factor 7 (Pax7), myogenic differentiation antigen (MyoD), myogenin (MyoG) and myosin heavy chain (MyHC) in the multifidus muscle, and CD63, programmed cell death protein 6 interacting protein (Alix) and tumor susceptibility gene 101 (TSG101) proteins in the serum exosomes in rats with lumbar multifidus muscle injury (MFMI), so as to explore the effect of exosomes in acupoint areas on EA improvement of muscular regeneration and repair. Forty male SD rats were randomly divided into normal control, model, EA and EA+exosome inhibitor (EA+inhibitor) groups, with 10 rats in each group. The MFMI model was established by injection of 0.5% bupivacaine (150 μL × 4) into the 4 points of the multifidus muscle along the bilateral lumbar (L)4-L5 spinous processes. EA (2 Hz/10 Hz, 1 mA) was applied to bilateral "Weizhong" (BL40) and "Shenshu" (BL23) for 20 min, once a day for 7 d. For rats of the EA+inhibitor group, exosome inhibitor GW4869 (3 mg/mL, 50 μL/acupoint) was injected into bilateral BL40 and BL23 1 h before each EA intervention. The morphological changes of the multifidus muscle were observed after H.E. staining and Masson staining. The immunoactivity of Pax7 and MyoD was observed by immunohistochemistry. The serum exosomes were extracted and identified by transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). The expression levels of MyoG and MyHC in the multifidus muscle tissue and CD63, Alix and TSG101 proteins in the serum exosomes were detected by Western blot. Morphological results showed that in the model group, most of the muscle fibers were degenerated and necrotic, a large number of inflammatory cells infiltrated around the muscle fibers and more blue-stained collagen fibers were observed. In the EA group, the morphology of muscle fibers was relatively complete, with more new muscle fibers and reduced inflammatory cells in the injured area, and the collagen fibers were significantly reduced. In the EA+inhibitor group, there were still more muscle fiber destruction and inflammatory cell infiltration, new muscle fibers with uneven diameter and more collagen fibers. Compared with the normal control group, the immunoactivity of Pax7 in the multifidus muscle, the expression of Alix and CD63 proteins in the serum exosomes were significantly increased in the model group (P<0.01, P<0.05, P<0.001). In comparison with the model group, the immunoactivity of Pax7 and MyoD, the expression levels of Alix and TSG101 in the serum exosomes and MyHC and MyoG proteins in the multifidus muscle were considerably up-regulated in the EA group (P<0.01, P<0.05). After local injection of GW4869 at BL40 and BL23, the immunoactivity of Pax7 and MyoD, the protein expression levels of TSG101, CD63, MyHC and MyoG were significantly lower in the EA+inhibitor group than those of the EA group (P<0.01, P<0.05, P<0.001). The results of TEM and NTA showed that the exosomes were successfully extracted. The morphology of the exosomes was typical saucer-like under electron microscope, and the particle size range was concentrated in 70-200 nm. EA of BL40 and BL23 can significantly up-regulate the expressions of Pax7, MyoD, MyoG and MyHC in the injured multifidus muscle, and promote the regeneration and repair of lumbar multifidus muscle, which may be related to its functions in promoting the release of exosomes in the acupoint area.

Currently, effective treatments for skeletal muscle injury remain limited. The self-repair of skeletal muscle relies on the activation and differentiation of satellite cells (SCs), which fuse with damaged myofibers to form new fibers and thereby support muscle regeneration. However, in cases of severe injury, it is difficult for muscle tissue to fully restore its original structure and function, and its regenerative capacity is often markedly reduced. Thus, there is an urgent need to develop therapies that enhance muscle repair and restore physiological function. In this study, we investigated extracellular vesicles derived from neonatal mouse skeletal muscle (NMM-EVs), which are enriched in cargo from Pax7⁺ myogenic progenitor cells. We hypothesized that NMM-EVs could enhance SC activation and improve muscle regeneration following injury. Using glycerol-induced tibialis anterior (TA) muscle injury model, we evaluated the effects of intramuscular NMM-EV administration on skeletal muscle regeneration by histological, immunofluorescence, and functional analyses. In vivo, NMM-EVs significantly promoted skeletal muscle regeneration and functional recovery, upregulated Pax7 expression, increased the cross-sectional area and muscle mass of regenerated TA, and reduced fibrosis and fat infiltration. In vitro, NMM-EVs enhanced the proliferation and myogenic differentiation of mouse SCs and increased the expression of myogenic regulatory factors at both the mRNA and protein levels. In conclusion, this study demonstrates that NMM-EVs activate SCs within injured muscle, promote their proliferation and differentiation, and thereby accelerate injury repair and myofiber regeneration while attenuating fibrotic and adipogenic remodeling. These findings provide a scientific basis for the development of neonatal muscle–derived extracellular vesicle–based, cell-free therapeutic strategies for skeletal muscle injury.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00274-6.

Immunometabolic reprogramming of macrophages: Emerging roles in skeletal muscle regeneration and therapeutic perspectives.

Skeletal muscle plays a crucial role in human life, contributing to posture, movement, nutrient storage, and body temperature regulation. Development and regeneration of skeletal muscles rely on embryonic myogenic progenitors and postnatal satellite cells (MuSCs), respectively. Identification of new molecular markers and elucidating their functions in MuSCs will provide better understanding of muscle development and regeneration. We surveyed single cell RNA-seq (scRNA-seq) data (Tabula Muris and GSE150366) to identify ASB5 (Ankyrin repeat and Suppressor of cytokine signaling Box containing 5) as a marker of MuSCs. We also used CRISPR-CAS9 genome editing and oviduct electroporation to generate a germline knockout (KO) mouse line of Asb5. We then analyzed the muscle growth and regeneration of the KO mice. We further analyzed proliferation and differentiation of MuSCs attached on myofibers. We finally performed Realtime PCR (qPCR) to examine how Asb5 KO affects gene expression in the skeletal muscle. Analysis of data publicly available at Tabula Muris identified Asb5 as a specific marker of MuSCs. Further analysis of scRNA-seq data on FACS-purified MuSCs at various regeneration time points revealed that Asb5 is highly expressed in MuSCs and their progenies across various stages of muscle regeneration. We then generated a novel Asb5 KO mouse line through CRISPR-Cas9 deletion of Exon 4. The Asb5-KO mice were born normally and exhibited normal postnatal growth. In addition, Asb5-KO MuSCs proliferated, differentiated and self-renewed normally on myofiber explants. Furthermore, the skeletal muscles of Asb5-KO mice regenerated normally after acute injury. qPCR analysis showed that Asb5 KO reduces the expression levels of Tnfa (Tumor Necrosis Factor Alpha) in the skeletal muscles. These data together identify ASB5 as an abundantly expressed and specific marker of MuSCs and myogenic progenitors. However, Asb5 loss-of-function has no effects on embryonic development and postnatal growth of skeletal muscles, or behavior and regenerative functions of MuSCs under normal physiological conditions.

Aged skeletal muscle satellite cells (MuSCs) exhibit impaired autophagy-related activity, reduced proliferative capacity, and compromised myogenic differentiation, which collectively contribute to defective muscle regeneration during aging. However, whether hypoxia-driven modulation of autophagy-related activity can improve aged MuSC function and the underlying molecular mechanisms remain incompletely understood. In this study, aged MuSCs were divided into three groups: normoxia, hypoxia, and hypoxia combined with an autophagy inhibitor. Aged MuSCs exhibited a decreased LC3B-II/LC3B-I ratio and Beclin-1 expression, together with elevated p62 levels, indicating altered autophagy-related activity. Hypoxic culture was associated with enhanced autophagy-related activity in aged MuSCs, accompanied by HIF-1α stabilization, BNIP3 upregulation, and reduced p62 accumulation. Functionally, hypoxia significantly promoted the proliferation and myogenic differentiation of aged MuSCs. Pharmacological inhibition of autophagy using 3-methyladenine, as well as BNIP3 suppression, markedly attenuated these hypoxia-induced functional improvements. Collectively, these findings suggest that hypoxia is associated with improved proliferative and myogenic capacities of aged MuSCs, potentially involving autophagy-related activity regulated by the HIF-1α/BNIP3 pathway. This study provides insight into the relationship between hypoxic signaling and autophagy in aged MuSCs and may inform future strategies aimed at improving muscle regeneration during aging.

MUSTN1 prevents muscle atrophy through ferroptosis suppression: A ACO1-dependent and exosome-mediated mechanism.