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.

Thyroid hormone (TH) secreted by the thyroid gland plays essential roles in regulating metabolism, development, and nervous system function. Thyroid hormone receptor-associated protein 3 (THRAP3) is a nuclear coactivator that interacts with the thyroid hormone receptor (TR) and facilitates target gene regulation through the mediator complex. Although this mechanism has been well studied in other tissues, the specific role of THRAP3 in skeletal muscle remains unclear. Here we investigated the function of THRAP3 in skeletal muscle using Thrap3 knockout (KO) C2C12 cells. Loss of THRAP3 significantly suppressed the expression of key myogenic regulatory factors, including Myod1, Mef2c, and myosin heavy chain genes, resulting in impaired myogenic differentiation and muscle diameter. Furthermore, we found that THRAP3 influences triiodothyronine (T3)-induced gene expression, suggesting that it cooperatively modulates thyroid hormone signaling in muscle cells. Taken together, our findings identify THRAP3 as a novel regulator of myogenesis and indicate that it supports T3 activity by coordinating thyroid hormone–responsive gene expression in skeletal muscle.

Genome editing of late myogenic regulators provides a way to dissect the mechanisms through which transcriptional programs and growth-related signaling pathways shape muscle gene expression programs in farmed fish. This study disrupted myogenic regulatory factor 4 (MRF4) in Nile tilapia using CRISPR/Cas9 to examine downstream transcriptional changes in fast skeletal muscle across the trunk, belly, and head regions. Adult F0 crispants carried a frameshift mutation that truncated the basic helix–loop–helix domain and showed an approximate 80–85% reduction in MRF4 mRNA across the trunk, belly, and head muscles. The expression of 23 genes representing myogenic regulatory factors, MEF2 paralogs, structural and contractile components, non-myotomal regulators, cell adhesion and fusion-related transcripts, and growth-related genes within the GH–IGF–MSTN axis was quantified and compared between wild-type and MRF4-crispants. Expressions of major structural genes remained unchanged despite MRF4 depletion, whereas MyoG and MyoD were upregulated together with MEF2B and MEF2D, indicating strong transcriptional compensation. Twist1, ID1, PLAU, CDH15, CHRNG, NCAM1, MYMK, GHR, and FGF6 were also significantly elevated, while IGF1 was reduced, and MSTN remained stable. Together, these results show that MRF4 loss is associated with coordinated transcriptional changes in regulatory and growth-related pathways, while major fast-muscle structural and contractile transcript levels remain stable, thereby highlighting candidate transcriptional targets for future studies that will evaluate links to muscle phenotype and growth performance in Nile tilapia.

Bioactive fractions of Swertia species protect myotubes and improve insulin sensitivity by regulating myogenic factors in differentiated skeletal muscle cells.

Effect of the sdc4 gene knockdown on muscle development in zebrafish.

Musculoskeletal injuries involving volumetric muscle loss remain difficult to treat due to limited regenerative capacity and the lack of physiologically relevant experimental models. This study introduces a computer-controlled ex vivo mouse hindlimb culturing platform that applies dynamic mechanical loading to evaluate muscle regeneration in a critical-size tibialis anterior (TA) defect. The defect was treated with an injectable myoblast-laden nanofibrous scaffold composed of polycaprolactone nanofibers and collagen (PNCOL). The ex vivo mouse hindlimb culturing platform maintained tissue viability and transmitted physiological strain across bone and muscle without disrupting the unity of the bone–muscle structure. PNCOL treatment under mechanical loading enhanced muscle fiber organization, extracellular matrix regeneration, and anti-inflammatory responses (CD206) while upregulating paired box 7 (PAX7), myogenic factor 5 (MYF5), myogenic regulatory factor 4 (MRF4), and transforming growth factor beta1 (TGFβ1) expression. Cytokine profiling revealed an anabolic shift involving wingless/integrated (WNT) and insulin-like growth factor-1 (IGF-1) signaling, indicating a pro-regenerative microenvironment. Overall, the combination of mechanical stimulation and biomaterial-based therapy significantly improved muscle regeneration within a controlled ex vivo model. This multidimensional approach provides a reproducible and ethical platform that advances musculoskeletal regenerative research while reducing animal use.

Evidence from genome-wide association studies (GWAS) links genetic variants near the LYPLA1 locus to variations in sheep muscle development. To further investigate whether LYPLA1 expression plays a role in fetal sheep myoblast development, we performed gain-of-function/loss-of-function analyses targeting LYPLA1 using siRNA and recombinant vectors. CCK-8 assays, EdU staining, and flow cytometry were used to investigate the effects of both LYPLA1 knockdown and overexpression on the proliferation of fetal sheep myoblasts. Furthermore, this study investigated the effect of LYPLA1 on myogenic differentiation by means of indirect immunofluorescence staining with MyHC and quantitative analysis of myogenic regulatory factor (MRF) expression. The results showed that downregulation of LYPLA1 significantly reduced cell viability and proliferation rate, arrested myoblasts in the G2 phase, and inhibited proliferation. These effects were reversed when LYPLA1 was overexpressed. Furthermore, LYPLA1 knockdown significantly impaired myotube formation during terminal differentiation (days 5-7). This defect was correlated with a marked reduction in the expression of myogenic regulatory factors (MRFs). This effect was reversed when LYPLA1 was highly expressed. In summary, this study demonstrates that LYPLA1 promotes the proliferation and differentiation of fetal sheep myoblasts, establishing its potential as a target for the molecular breeding of sheep.

Gene expression levels of myogenic regulatory factors (MRFs) (myf5, myod2, and myog), myostatin (mstn1a and mstn1b), and the muscle protein myosin (myhc) in muscles were studied in the rainbow trout Oncorhynchus mykiss Walb. Study fish of three size groups (SGs) were fed with two commercial feeds differing in compositions. Expression levels of myhc, myf5, myog, and mstn1a in muscles were found to differ depending on the feed, especially in smaller fish (<1000 g). The finding suggests that the feed composition, including the quantitative ratio of ingredients and the sources of proteins, fats, and carbohydrates in the feed, affects the muscle growth regulation in early trout growth and development. The results complement the literature data on features of the regulation of myogenesis in response to external factors.

Thyroid hormone levels decrease with aging, and low thyroxine levels are correlated with sarcopenia development. While thyroid hormone stimulates myogenesis in young subjects, its effect on aged muscle regeneration is unclear. We aimed to investigate the impact of a low dose of thyroxine (T4) replacement therapy (7.5 ng/g body weight) on tibial anterior regeneration 7 days after injury by 1.2% BaCl2 injection in 24-27-month-old male mice. Our primary data suggest that regenerating aged skeletal muscle exhibits local resistance to thyroid hormone action without altering myogenic regulatory factors expression. However, T4 treatment decreases the number of central nuclei, indicative of newly formed fibers. In addition, we observed a decrease in cross-sectional area and an increase in myonuclei domain, cell death, and laminin expression in T4-treatment injured muscles. Rather than improving regeneration, T4 replacement therapy appears to induce atrophy and tissue remodeling. Our data highlight the need to understand aging physiology since thyroid hormones are crucial for muscle regeneration in young animals, although T4 replacement therapy does not improve muscle regeneration post-injury in elderly mice. This research may support clinical recommendations against treating sarcopenic patients with subclinical hypothyroidism, especially following fall-related injuries.

Obesity resulting from chronic overnutrition and physical inactivity promotes the development of metabolic disorders by disrupting physiological processes in metabolically active organs, including skeletal muscles. To investigate whether skeletal muscle stem cells (satellite cells, SCs) are affected by systemic metabolic stress, we established primary SC cultures from male mice fed a high-fat diet (HFD) for 8wk, and from control mice fed a standard chow (CTL). This model allowed us to assess diet-induced obesity (DIO)-related changes in SC-specific molecular and cellular signatures. Although body weight, body fat composition, and adipose tissue-associated macrophages differed significantly between DIO and CTL ex vivo, we observed no differences in the in vitro behaviour of primary SC-derived myoblasts from either group. Parameters such as proliferation and differentiation following serum deprivation were comparable. Expression levels and distribution patterns of myogenic regulatory factors (MRF), SC-specific markers (Pax7, CD56, Itga7), and hallmarks for senescence (GLB1), autophagy (p62, LC3B), and oxidative stress (ALDH1A1, ALDH1A3) remained unchanged. Thus, potential differences in the signatures of SC-derived myoblasts after 8wk of a high-fat diet cannot be depicted in vitro. However, future experiments should address whether prolonged and metabolically more susceptible diets will exert long-term effects on myogenesis in vitro or not. Overall, we propose that primary SC cultures are better suited for acute in vitro testing regarding the molecular and cellular plasticity in metabolic shifts as induced by pharmacological treatments or genetical modifications, rather than for modeling long-term dietary effects.

Low-serum culture systems offer enhanced controllability, improved safety, and increased cost-effectiveness for applications in tissue engineering, regenerative medicine, drug screening, and cultured meat production. In this study, we developed a novel proliferation synergy factor cocktail (PSFC) consisting of IGF-1, bFGF, TGF-β, IL-6, and G-CSF under low-serum (5% FBS) conditions. This system not only sustained robust proliferation of porcine muscle satellite cells (PSCs) and porcine kidney fibroblasts (PKFs), but also exhibited broad applicability in C2C12 myoblasts and mouse skeletal muscle satellite cells (SSCs). RT-qPCR and Western blot showed that there were no significant differences in the expression levels of the proliferation marker Ki67, as well as the myogenic regulatory factors MyoG and MyHC, between the 5% FBS-PSFC culture system and the conventional serum culture system. Notably, PSFC supplementation enhanced the average transfection efficiency by 16.9% across all tested cell types. Furthermore, the 5% FBS-PSFC platform facilitated three-dimensional (3D) culture within gelatin methacryloyl (GelMA) hydrogels, enabling scalable cultured meat production while reducing serum costs by 75%. Further RNA-seq analysis revealed that the there was no significant changes in the expression of cell proliferation-related genes which may be crucial for maintaining cell proliferation of this system, while the upregulation of genes associated with membrane fluidity and endocytosis, such as ITGA3, SEMA7A, ADAM8 and AREG, may lead to the enhancement of transfection efficiency. Collectively, these findings establish a cost-effective and versatile culture platform that addresses critical challenges in cell expansion for cellular agriculture, while providing a scalable approach to enhance transfection efficiency for gene editing applications.

Skeletal muscle tissue consists of not only myofibers, i.e., muscle cells, but also intramuscular adipocytes. Our previous study demonstrated that adipocytes produce secretory factors during differentiation, leading us to hypothesize that soluble factors derived from adipocytes regulate gene expression and cellular function in muscle cells. Yet the mechanism by which coexisting adipocytes influence muscle cells remains unclear. Here, microarray analysis was used to examine transcriptional changes in muscle cells under two co-culture conditions: myoblasts co-cultured with differentiated adipocytes and myotubes co-cultured with preadipocytes. Gene Ontology terms related to cell adhesion, extracellular matrix (ECM) organization, and metabolic processes were significantly enriched in both conditions. We also assessed the influence of adipocyte co-culture on myogenic differentiation and fiber type-specific gene expression. In myoblasts, co-culture with differentiated adipocytes had no significant effect on the expression of myogenic regulatory factors, whereas Myh2 and Myh4 expression was markedly increased in myotubes co-cultured with preadipocytes. These results indicate that adipocyte-derived soluble factors alter the transcriptional landscape of muscle cells, especially genes involved in ECM remodeling and metabolic regulation. This intercellular communication likely contributes to structural and metabolic adaptations in skeletal muscle tissue in vivo.

Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma with a skeletal muscle phenotype, accounting for approximately 50% of all pediatric soft tissue sarcomas and 8% of all childhood cancers. Although RMS cells express myogenic regulatory factors, they fail to undergo terminal differentiation into mature muscle cells. Transforming growth factor β-activated kinase 1 (TAK1) is a major signaling protein that activates multiple intracellular pathways in response to growth factors, cytokines, and microbial products. Emerging evidence suggests that TAK1 is also an important regulator of self-renewal, proliferation, and differentiation of muscle progenitor cells. However, the role and mechanisms of action of TAK1 in RMS remain completely unknown. In this study, we demonstrate that TAK1 expression and activity are markedly elevated in a panel of RMS cell lines and in patient tumor specimens. Reverse phase protein array (RPPA) analyses revealed that TAK1 regulates the expression and activity of many molecules involved in cell cycle control, cell proliferation, and oncogenic signaling. Genetic knockdown or pharmacological inhibition of TAK1 suppresses RMS cell proliferation, migration, and invasiveness, while also promoting terminal myogenic differentiation. TAK1 inhibits differentiation in RMS, at least in part, through up-regulating YAP1 signaling. Our results also demonstrate that inducible knockdown of TAK1 in human RMS xenografts retards tumor growth and enhances myogenic differentiation in vivo. Collectively, these findings uncover a previously unrecognized role for TAK1 in RMS growth and differentiation, and suggest that TAK1 can be a potential therapeutic target for the treatment of RMS.

Development and characterization of a new muscle cell line developed from pearl spot, Etroplus suratensis (Bloch 1790).

This study aimed to evaluate the regenerative effects of various microcurrent waveforms in cast-induced gastrocnemius muscle atrophy in rabbits, integrating both in vitro and in vivo analyses. After two weeks of enforced hindlimb immobilization via casting, twenty-four rabbits were divided into four groups and treated for two weeks: Group-1 (control) received sham microcurrent, Group-2 was treated with a square waveform microcurrent, Group-3 with a sine waveform, and Group-4 with a triangular waveform. Treatments were administered daily for one hour. Calf circumference, muscle thickness (via ultrasound), tibial nerve CMAP, muscle fiber CSA, and protein expression (via Western blot analysis) were assessed. Among the groups, the sine waveform microcurrent resulted in significantly enhanced recovery across all measured parameters (p < 0.05), showing superior improvements in muscle thickness, CMAP amplitude, and fiber CSA. Immunohistochemical analysis revealed increased expression of proliferation and angiogenesis markers, including BrdU, PCNA, VEGF, and PECAM-1, while Western blotting demonstrated robust upregulation of myogenic regulatory factors such as MyoD and myogenin. Furthermore, levels of inflammatory and apoptotic markers, including TNF-α, NF-κB, and cleaved caspase-3, and stress response proteins, including p-CHK1 and p-CHK2, were markedly reduced. Collectively, these findings indicate that sine waveform microcurrent stimulation most effectively promotes muscle regeneration in both dexamethasone-induced C2C12 myoblasts and cast-induced muscle atrophy, underscoring its therapeutic potential and warranting further studies to optimize clinical application parameters.

The effects of high plant-based proteins (PP) used as alternative protein sources in aquafeeds on muscle cellularity and myogenic factors of rainbow trout, Oncorhynchus mykiss, remain unclear. This study explored muscle fibre growth phases and the impact of two additive mixtures (A) in high-PP diets on muscle physiology. Over a seven-month trial, 2000 fish (2·22 g) were divided into four groups (five replicates each) and fed isonitrogenous (fry, 46 %; fingerling, 44 %; and grow-out, 42 % crude protein) and isolipidic (20 % lipid) diets: control (30 % fishmeal), PP, PP + A1 (krill meal, taurine, selenium) and PP + A2 (proline, hydroxyproline, vitamin C). Sampling for muscle histology and myogenic gene expression was conducted at ten sampling points from Day 0 to Day 214. Muscle histology (fibre distribution: small, 0–20 μm; small-medium, 20–60 μm; large-medium, 60–100 μm and large, ≥ 100 μm diameter) revealed four growth phases: hyperplasia (2·2–15 g), hypertrophy (15–50 g), hyperplasia (50–150 g) and hypertrophy (150–350 g). MyoD2 and myogenic regulatory factor 4 (MRF4) were upregulated during hyperplasia, while myostatin 1 (MSTN1)/myostatin 2 (2) and reduced Paired box 7 indicating growth inhibition and fewer satellite cells. The PP diet without additives altered fibre recruitment, while PP + A2 enhanced hypertrophy, increasing large (> 100 μm) fibres. Additive mixtures modulated myogenic gene expression, with PP + A2 promoting MyoD2, myogenin and MRF4 and reducing MEF2A/C, contrary to known hypertrophy markers. PP + A1 and PP + A2 diets reduced MSTN1 expression, potentially mitigating growth inhibition. Additive supplementation in PP diets alleviates negative impacts on muscle cellularity and myogenic regulation. The identified growth phases provide insights for precision nutrition, supporting improved feeding strategies for sustainable aquaculture.

Spotted seabass (Lateolabrax maculatus) is an economically important fish species in China. In this study, the muscle of L. maculatus was used as a material, and the muscle cell line was successfully established using the tissue block method. The established muscle cell line exhibited vigorous growth and had been successfully passaged for more than 100 generations, maintaining stable polygonal cell morphology. The cell viability of L. maculatus muscle cell line exhibited a notable increase following a 24-h treatment with 30 M taurine, and the relative expression of myogenic regulatory factors myf5 and myf6 was significantly increased. Therefore, the muscle cell line of L. maculatus was successfully established and served as a valuable resource for conducting fundamental research in L. maculatus, providing essential materials for gene function analysis, cytogenetics, and nutritional metabolism mechanism in L. maculatus.

Balanced nutrient composition of larval fish diet significantly affects growth and survival. Stunted growth and high mortality are common issues in conventional carp hatcheries. To explore the causes, we analyzed the proximate and biochemical composition of live feed (LF) in nursery ponds and compared it to a formulated nano-diet (FND). Rohu (Labeo rohita (Hamilton,1822)) larvae 3 DAH (days after hatching) were divided into ten tanks, with five receiving LF and five FND until 35 DAH. Larvae were periodically analyzed for growth and gene expression at specific intervals (3, 10, 15, 20, 25, 30, and 35 DAH). Significant differences in body composition were observed between the two groups. Certain essential fatty acids (arachidonic acid, EPA, DHA) and amino acids (methionine, lysine, phenylalanine) were lower in both LF and LF-fed larvae compared to FND and FND-fed larvae. The FND group showed higher survival, specific growth rate, and net weight gain. Additionally, higher expression of GH, IGF-1, and myogenic regulatory factors in the FND group suggest that nutrient composition influences molecular growth regulation. In contrast, elevated Myostatin expression in LF-fed larvae suggests a potential inhibitory effect on early myogenesis, which might indicate the limited nutrient availability in LF.

Objective: Severe skeletal muscle contusions result in significant inflammation, fibrotic scarring, and incomplete functional recovery. Betulin, a natural triterpenoid, has demonstrated anti-inflammatory and regenerative properties. This study aimed to investigate the efficacy of betulin in promoting molecular and functional recovery in a murine model of muscle contusion. Methods: A standardized contusion injury was induced in the tibialis anterior muscle of C57BL/6 mice. Mice were treated daily with either betulin (50 mg/kg, IP) or a vehicle control. Functional recovery was assessed at days 3, 7, 14, and 28 post-injury using in vivo grip strength tests. Muscle tissues were harvested at the same time points for analysis. Histological evaluation was performed using Hematoxylin &amp; Eosin (H&amp;E) for general morphology and inflammation, and Masson’s Trichrome for fibrosis. Myogenic regeneration and key molecular markers of inflammation and fibrosis were quantified using immunohistochemistry and quantitative real-time PCR (qRT-PCR). Results: Betulin treatment was associated with a significant acceleration in the recovery of muscle strength compared to vehicle-treated controls. Histological analysis revealed that betulin markedly reduced inflammatory cell infiltration at early time points and decreased the total area of fibrotic tissue by day 28. This was correlated with a significant downregulation of pro-inflammatory (Tnf-α, Il-6) and pro-fibrotic (Tgf-β1, Col1a1) gene expression. Furthermore, betulin-treated muscles exhibited an increased number of newly formed, centrally nucleated myofibers. This enhanced myogenesis was supported by the upregulation of myogenic regulatory factors, including Myod1 and Myog, indicating an augmented satellite cell-mediated repair process. Conclusion: Betulin appears to promote comprehensive recovery following muscle contusion by targeting multiple pathological processes. It is associated with mitigated acute inflammation and downstream fibrosis while simultaneously enhancing the intrinsic myogenic regenerative capacity of the tissue. These findings establish betulin as a potent therapeutic candidate for improving outcomes after severe muscle injuries.

Objective: Severe skeletal muscle contusions result in significant inflammation, fibrotic scarring, and incomplete functional recovery. Betulin, a natural triterpenoid, has demonstrated anti-inflammatory and regenerative properties. This study aimed to investigate the efficacy of betulin in promoting molecular and functional recovery in a murine model of muscle contusion. Methods: A standardized contusion injury was induced in the tibialis anterior muscle of C57BL/6 mice. Mice were treated daily with either betulin (50 mg/kg, IP) or a vehicle control. Functional recovery was assessed at days 3, 7, 14, and 28 post-injury using in vivo grip strength tests. Muscle tissues were harvested at the same time points for analysis. Histological evaluation was performed using Hematoxylin &amp; Eosin (H&amp;E) for general morphology and inflammation, and Masson’s Trichrome for fibrosis. Myogenic regeneration and key molecular markers of inflammation and fibrosis were quantified using immunohistochemistry and quantitative real-time PCR (qRT-PCR). Results: Betulin treatment was associated with a significant acceleration in the recovery of muscle strength compared to vehicle-treated controls. Histological analysis revealed that betulin markedly reduced inflammatory cell infiltration at early time points and decreased the total area of fibrotic tissue by day 28. This was correlated with a significant downregulation of pro-inflammatory (Tnf-α, Il-6) and pro-fibrotic (Tgf-β1, Col1a1) gene expression. Furthermore, betulin-treated muscles exhibited an increased number of newly formed, centrally nucleated myofibers. This enhanced myogenesis was supported by the upregulation of myogenic regulatory factors, including Myod1 and Myog, indicating an augmented satellite cell-mediated repair process. Conclusion: Betulin appears to promote comprehensive recovery following muscle contusion by targeting multiple pathological processes. It is associated with mitigated acute inflammation and downstream fibrosis while simultaneously enhancing the intrinsic myogenic regenerative capacity of the tissue. These findings establish betulin as a potent therapeutic candidate for improving outcomes after severe muscle injuries.