Inhibition of Sesn2 has negative regulatory effects on the myogenic differentiation of C2C12 myoblasts.

Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.

Establishment and Characterization of a Chicken Myoblast Cell Line.

Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.

Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Expression mechanisms of mir-486-5p and its host gene sANK1 in porcine muscle.

BACKGROUND: MiR-486-5p has been identified as a crucial regulator of the PI3K/AKT signalling pathway, which plays a significant role in skeletal muscle development. Its host gene, sANK1, is also essential for skeletal muscle development. However, the understanding of porcine miR-486-5p and sANK1 has been limited. METHODS AND RESULTS: In this study, PCR analyses revealed a positive correlation between the expression of miR-486-5p and sANK1 in the longissimus dorsi muscle of the Bama mini-pig and Landrace-pig, as well as during myoblast differentiation. Furthermore, the expression of miR-486-5p/sANK1 was higher in the Bama mini-pig compared to the Landrace-pig. There was a total of 18 single nucleotide polymorphisms (SNP) present in the sANK1 promoter region. Among these SNPs, 14 of them resulted in alterations in transcription factor binding sites (TFBs). Additionally, the promoter fluorescence assay demonstrated that the activity of the sANK1 promoter derived from the Bama mini-pig was significantly higher compared to Landrace-pig. It is worth noting that ten regulatory SNPs have the potential to influence the activity of the sANK1 promoter. A nuclear mutation A-G located at position - 401 (relative to the transcription start site) in the Bama mini-pig was identified, which creates a putative TFB motif for MyoD. CONCLUSIONS: The findings presented in this study offer fundamental molecular knowledge and expression patterns of miR-486-5p/sANK1, which can be valuable for gaining a deeper understanding of the gene's involvement in porcine skeletal muscle development, and meat quality.

Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands.

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

Development of a local controlled release system for therapeutic proteins in the treatment of skeletal muscle injuries and diseases.

The present study aims to develop and characterize a controlled-release delivery system for protein therapeutics in skeletal muscle regeneration following an acute injury. The therapeutic protein, a membrane-GPI anchored protein called Cripto, was immobilized in an injectable hydrogel delivery vehicle for local administration and sustained release. The hydrogel was made of poly(ethylene glycol)-fibrinogen (PEG-Fibrinogen, PF), in the form of injectable microspheres. The PF microspheres exhibited a spherical morphology with an average diameter of approximately 100 micrometers, and the Cripto protein was uniformly entrapped within them. The release rate of Cripto from the PF microspheres was controlled by tuning the crosslinking density of the hydrogel, which was varied by changing the concentration of poly(ethylene glycol) diacrylate (PEG-DA) crosslinker. In vitro experiments confirmed a sustained-release profile of Cripto from the PF microspheres for up to 27 days. The released Cripto was biologically active and promoted the in vitro proliferation of mouse myoblasts. The therapeutic effect of PF-mediated delivery of Cripto in vivo was tested in a cardiotoxin (CTX)-induced muscle injury model in mice. The Cripto caused an increase in the in vivo expression of the myogenic markers Pax7, the differentiation makers eMHC and Desmin, higher numbers of centro-nucleated myofibers and greater areas of regenerated muscle tissue. Collectively, these results establish the PF microspheres as a potential delivery system for the localized, sustained release of therapeutic proteins toward the accelerated repair of damaged muscle tissue following acute injuries.

Tumor Necrosis Factor-Alpha Modulates Expression of Genes Involved in Cytokines and Chemokine Pathways in Proliferative Myoblast Cells.

Skeletal muscle regeneration after injury is a complex process involving inflammatory signaling and myoblast activation. Pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) are key mediators, but their effects on gene expression in proliferating myoblasts are unclear. We performed the RNA sequencing of TNF-α treated C2C12 myoblasts to elucidate the signaling pathways and gene networks regulated by TNF-α during myoblast proliferation. The TNF-α (10 ng/mL) treatment of C2C12 cells led to 958 differentially expressed genes compared to the controls. Pathway analysis revealed significant regulation of TNF-α signaling, along with the chemokine and IL-17 pathways. Key upregulated genes included cytokines (e.g., IL-6), chemokines (e.g., CCL7), and matrix metalloproteinases (MMPs). TNF-α increased myogenic factor 5 (Myf5) but decreased MyoD protein levels and stimulated the release of MMP-9, MMP-10, and MMP-13. TNF-α also upregulates versican and myostatin mRNA. Overall, our study demonstrates the TNF-α modulation of distinct gene expression patterns and signaling pathways that likely contribute to enhanced myoblast proliferation while suppressing premature differentiation after muscle injury. Elucidating the mechanisms involved in skeletal muscle regeneration can aid in the development of regeneration-enhancing therapeutics.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

OTUB1 regulates ferroptosis to inhibit myoblast differentiation into myotubes by deubiquitinating P62.

As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.

Taurine Alleviates Ferroptosis-Induced Metabolic Impairments in C2C12 Myoblasts by Stabilizing the Labile Iron Pool and Improving Redox Homeostasis.

Ferroptosis adversely affects the viability, differentiation, and metabolic integrity of C2C12 myoblasts, contributing to the decline in skeletal muscle health. The intricate mechanisms behind this process are not fully understood. In this study, we induced ferroptosis in myoblasts using targeted inducers and found a marked decrease in specific redox metabolites, particularly taurine. Taurine supplementation effectively reversed the deleterious effects of ferroptosis, significantly increased cellular glutathione levels, reduced MDA and ROS levels, and rejuvenated impaired myogenic differentiation. Furthermore, taurine downregulated HO-1 expression and decreased intracellular Fe2+ levels, thereby stabilizing the labile iron pool. Using NMR metabolomic analysis, we observed that taurine profoundly promoted glycerophospholipid metabolism, which is critical for cell membrane repair, and enhanced mitochondrial bioenergetics, thereby increasing the energy reserves essential for muscle satellite cell regeneration. These results suggest that taurine is a potent ferroptosis inhibitor that attenuates key drivers of this process, strengthens oxidative defenses, and improves redox homeostasis. This combined effect protects cells from ferroptosis-induced damage. This study highlights the potential of taurine as a valuable ferroptosis inhibitor that protects skeletal muscle from ferroptosis-induced damage and provides a basis for therapeutic strategies to rejuvenate and facilitate the regeneration of aging skeletal muscle.

Novel Circular RNA CircSLC2A13 Regulates Chicken Muscle Development by Sponging MiR-34a-3p.

The skeletal muscle is the major muscle tissue in animals, and its production is subject to a complex and strict regulation. The proliferation and differentiation of myoblasts are important factors determining chicken muscle development. Circular RNAs (circRNAs) are endogenous RNAs that are widely present in various tissues of organisms. Recent studies have shown that circRNA plays key roles in the development of skeletal muscles. The solute carrier (SLC) family functions in the transport of metabolites such as amino acids, glucose, nucleotides, and essential nutrients and is widely involved in various basic physiological metabolic processes within the body. In this study, we have cloned a novel chicken circular RNA circSLC2A13 generated from the solute carrier family 2 member 13 gene (SLC2A13). Also, circSLC2A1 was confirmed by sequencing verification, RNase R treatment, and reverse transcription analysis. Currently, our results show that circSLC2A13 promoted the proliferation and differentiation of chicken myoblasts. The double luciferase reporter system revealed that circSLC2A13 regulated the proliferation and differentiation of myoblasts by competitive binding with miR-34a-3p. In addition, results indicated that circSLC2A13 acts as a miR-34a-3p sponge to relieve its inhibitory effect on the target SMAD3 gene. In summary, this study found that chicken circSLC2A13 can bind to miR-34a-3p and weaken its inhibitory effect on the SMAD family member 3 gene (SMAD3), thereby promoting the proliferation and differentiation of myoblasts. This study laid foundations for broiler industry and muscle development research.

Tumor necrosis factor α deficiency promotes myogenesis and muscle regeneration.

Tumor necrosis factor α (TNFα) exhibits diverse biological functions; however, its regulatory roles in myogenesis are not fully understood. In the present study, we explored the function of TNFα in myoblast proliferation, differentiation, migration, and myotube fusion in primary myoblasts and C2C12 cells. To this end, we constructed TNFα muscle-conditional knockout ( TNFα-CKO) mice and compared them with flox mice to assess the effects of TNFα knockout on skeletal muscles. Results indicated that TNFα-CKO mice displayed phenotypes such as accelerated muscle development, enhanced regenerative capacity, and improved exercise endurance compared to flox mice, with no significant differences observed in major visceral organs or skeletal structure. Using label-free proteomic analysis, we found that TNFα-CKO altered the distribution of several muscle development-related proteins, such as Hira, Casz1, Casp7, Arhgap10, Gas1, Diaph1, Map3k20, Cfl2, and Igf2, in the nucleus and cytoplasm. Gene set enrichment analysis (GSEA) further revealed that TNFα deficiency resulted in positive enrichment in oxidative phosphorylation and MyoD targets and negative enrichment in JAK-STAT signaling. These findings suggest that TNFα-CKO positively regulates muscle growth and development, possibly via these newly identified targets and pathways.

The long noncoding RNA lncMPD2 inhibits myogenesis by targeting the miR-34a-5p/THBS1 axis.

Long noncoding RNAs (lncRNAs) participate in regulating skeletal muscle development. However, little is known about their role in regulating chicken myogenesis. In this study, we identified a novel lncRNA, lncMPD2, through transcriptome sequencing of chicken myoblasts at different developmental stages. Functionally, gain- and loss-of-function experiments showed that lncMPD2 inhibited myoblast proliferation and differentiation. Mechanistically, lncMPD2 directly bound to miR-34a-5p, and miR-34a-5p promoted myoblasts proliferation and differentiation and inhibited the mRNA and protein expression of its target gene THBS1. THBS1 inhibited myoblast proliferation and differentiation in vitro and delayed muscle regeneration in vivo. Furthermore, rescue experiments showed that lncMPD2 counteracted the inhibitory effects of miR-34a-5p on THBS1 and myogenesis-related gene mRNA and protein expression. In conclusion, lncMPD2 regulates the miR-34a-5p/THBS1 axis to inhibit the proliferation and differentiation of myoblasts and skeletal muscle regeneration. This study provides more insight into the molecular regulatory network of skeletal muscle development, identifying novel potential biomarkers for improving chicken quality and increasing chicken yield. In addition, this study provides a potential goal for breeding strategies that minimize muscle damage in chickens.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells.

We have recently identified the uncharacterized ZNF555 protein as a component of a productive complex involved in the morbid function of the 4qA locus in facioscapulohumeral dystrophy. Subsequently named DiPRO1 (Death, Differentiation, and PROliferation related PROtein 1), our study provides substantial evidence of its role in the differentiation and proliferation of human myoblasts. DiPRO1 operates through the regulatory binding regions of SIX1, a master regulator of myogenesis. Its relevance extends to mesenchymal tumors, such as rhabdomyosarcoma (RMS) and Ewing sarcoma, where DiPRO1 acts as a repressor via the epigenetic regulators TIF1B and UHRF1, maintaining methylation of cis-regulatory elements and gene promoters. Loss of DiPRO1 mimics the host defense response to virus, awakening retrotransposable repeats and the ZNF/KZFP gene family. This enables the eradication of cancer cells, reprogramming the cellular decision balance towards inflammation and/or apoptosis by controlling TNF-α via NF-kappaB signaling. Finally, our results highlight the vulnerability of mesenchymal cancer tumors to si/shDiPRO1-based nanomedicines, positioning DiPRO1 as a potential therapeutic target.

Inhibition of skeletal muscle differentiation by calciprotein particles in human primary myoblasts.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

Unveiling the role of circRBBP7 in myoblast proliferation and differentiation: A novel regulator of muscle development.

Muscle development is a multistep process regulated by diverse gene networks, and circRNAs are considered novel regulators mediating myogenesis. Here, we systematically analyzed the role and underlying regulatory mechanisms of circRBBP7 in myoblast proliferation and differentiation. Results showed that circRBBP7 has a typical circular structure and encodes a 13 -kDa protein. By performing circRBBP7 overexpression and RNA interference, we found that the function of circRBBP7 was positively correlated with the proliferation and differentiation of myoblasts. Using RNA sequencing, we identified 1633 and 532 differentially expressed genes (DEGs) during myoblast proliferation or differentiation, respectively. The DEGs were found mainly enriched in cell cycle- and skeletal muscle development-related pathways, such as the MDM2/p53 and PI3K-Akt signaling pathways. Further co-IP and IF co-localization analysis revealed that VEGFR-1 is a target of circRBBP7 in myoblasts. qRT-PCR and WB analysis further confirmed the positive correlation between VEGFR-1 and circRBBP7. Moreover, we found that in vivo transfection of circRBBP7 into injured muscle tissues significantly promoted the regeneration and repair of myofibers in mice. Therefore, we speculate that circRBBP7 may affect the activity of MDM2 by targeting VEGFR-1, altering the expression of muscle development-related genes by mediating p53 degradation, and ultimately promoting myoblast development and muscle regeneration. This study provides essential evidence that circRBBP7 can serve as a potential target for myogenesis regulation and a reference for the application of circRBBP7 in cattle genetic breeding and muscle injury treatment.

Skeletal Muscle UCHL1 Negatively Regulates Muscle Development and Recovery after Muscle Injury.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme originally found in the brain. Our previous work revealed that UCHL1 was also expressed in skeletal muscle and affected myoblast differentiation and metabolism. In this study, we further tested the role of UCHL1 in myogenesis and muscle regeneration following muscle ischemia-reperfusion (IR) injury. In the C2C12 myoblast, UCHL1 knockdown upregulated MyoD and myogenin and promoted myotube formation. The skeletal muscle-specific knockout (smKO) of UCHL1 increased muscle fiber sizes in young mice (1 to 2 months old) but not in adult mice (3 months old). In IR-injured hindlimb muscle, UCHL1 was upregulated. UCHL1 smKO ameliorated tissue damage and injury-induced inflammation. UCHL1 smKO also upregulated myogenic factors and promoted functional recovery in IR injury muscle. Moreover, UCHL1 smKO increased Akt and Pink1/Parkin activities. The overall results suggest that skeletal muscle UCHL1 is a negative factor in skeletal muscle development and recovery following IR injury and therefore is a potential therapeutic target to improve muscle regeneration and functional recovery following injuries.

Muscle cell-derived Ccl8 is a negative regulator of skeletal muscle regeneration.

Skeletal muscles undergo robust regeneration upon injury, and infiltrating immune cells play a major role in not only clearing damaged tissues but also regulating the myogenic process through secreted cytokines. Chemokine C-C motif ligand 8 (Ccl8), along with Ccl2 and Ccl7, has been reported to mediate inflammatory responses to suppress muscle regeneration. Ccl8 is also expressed by muscle cells, but a role of the muscle cell-derived Ccl8 in myogenesis has not been reported. In this study, we found that knockdown of Ccl8, but not Ccl2 or Ccl7, led to increased differentiation of C2C12 myoblasts. Analysis of existing single-cell transcriptomic datasets revealed that both immune cells and muscle stem cells (MuSCs) in regenerating muscles express Ccl8, with the expression by MuSCs at a much lower level, and that the temporal patterns of Ccl8 expression were different in MuSCs and macrophages. To probe a function of muscle cell-derived Ccl8 in vivo, we utilized a mouse system in which Cas9 was expressed in Pax7+ myogenic progenitor cells (MPCs) and Ccl8 gene editing was induced by AAV9-delivered sgRNA. Depletion of Ccl8 in Pax7+ MPCs resulted in accelerated muscle regeneration after barium chloride-induced injury in both young and middle-aged mice, and intramuscular administration of a recombinant Ccl8 reversed the phenotype. Accelerated regeneration was also observed when Ccl8 was depleted in Myf5+ or MyoD+ MPCs by similar approaches. Our results suggest that muscle cell-derived Ccl8 plays a unique role in regulating the initiation of myogenic differentiation during injury-induced muscle regeneration.

Dasatinib promotes muscle differentiation and disrupts normal muscle regeneration.

Dasatinib is one of the second-generation tyrosine kinase inhibitors used to treat chronic myeloid leukemia and has a broad target spectrum, including KIT, PDGFR, and SRC family kinases. Due to its broad drug spectrum, dasatinib has been reported at the basic research level to improve athletic performance by eliminating senescent cell removal and to have an effect on muscle diseases such as Duchenne muscular dystrophy, but its effect on myoblasts has not been investigated. In this study, we evaluated the effects of dasatinib on skeletal muscle both under normal conditions and in the regenerating state. Dasatinib suppressed the proliferation and promoted the fusion of C2C12 myoblasts. During muscle regeneration, dasatinib increased the gene expressions of myogenic-related genes (Myod, Myog, and Mymx), and caused abnormally thin muscle fibers on the CTX-induced muscle injury mouse model. From these results, dasatinib changes the closely regulated gene expression pattern of myogenic regulatory factors during muscle differentiation and disrupts normal muscle regeneration. Our data suggest that when using dasatinib, its effects on skeletal muscle should be considered, particularly at regenerating stages.