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Skeletal muscle is crucial for animal movement and posture maintenance, and it serves as a significant source of meat in the livestock and poultry industry. The number of muscle fibers differentiated from myoblast in the embryonic stage is one of the factors determining the content of skeletal muscle. Insulin‐like growth factor 2 (IGF2), a well‐known growth‐promoting hormone, is crucial for embryonic and skeletal muscle growth and development. However, the specific molecular mechanism underlying its impact on chicken embryonic myoblast differentiation remains unclear. To elucidate the molecular mechanism by which IGF2 regulates chicken myoblast differentiation, we manipulated IGF2 expression in chicken embryonic myoblast. The results demonstrated that IGF2 was upregulated during chicken skeletal muscle development and myoblast differentiation. On the one hand, we found that IGF2 promotes mitochondrial biogenesis through the PGC1/NRF1/TFAM pathway, thereby enhancing mitochondrial membrane potential, oxidative phosphorylation, and ATP synthesis during myoblast differentiation. This process is mediated by the PI3K/AKT pathway. On the other hand, IGF2 regulates BNIP3‐mediated mitophagy, clearing dysfunctional mitochondria. Collectively, our findings confirmed that IGF2 cooperatively regulates mitochondrial biogenesis and mitophagy to remodel the mitochondrial network and enhance mitochondrial function, ultimately promoting myoblast differentiation. [ABSTRACT FROM AUTHOR]

Abstract Background Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. Methods Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10 weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20 μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80 mg/L) and fed to the mice (50 mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT‐qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. Results During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P

Abstract Background The clinical efficacy of Jinchuang Ointment, a traditional Chinese medicine (TCM), in treating chronic non-healing diabetic wounds has been demonstrated over the past decades. Both in vitro and in vivo angiogenic activities have been reported for its herbal ingredients, including dragon blood from the palm tree Daemonorops draco and catechu from Uncaria gambir Roxb. Additionally, crude extracts of dragon blood have exhibited hypoglycemic effects not only in animal studies but also in cell-based in vitro assays. Results Our findings indicate that crude dragon blood extract promotes the differentiation of myoblasts into myotubes. Partially purified fractions of dragon blood crude extract significantly enhance the expression of muscle cell differentiation-related genes such as myoG, myoD, and myoHC. Our results also demonstrate that crude extracts of dragon blood can inhibit platelet-derived growth factor-induced PAI-1 expression in primary rat vascular smooth muscle cells, thereby favoring changes in hemostasis towards fibrinolysis. Consistent with previous reports, reduced expression of plasminogen activator inhibitor 1 (PAI-1) accelerates wound healing. However, further separation resulted in a significant loss of both activities, indicating the involvement of more than one compound in these processes. Stem cells play a crucial role in muscle injury repair. Neither dragon blood nor catechu alone stimulated the proliferation of human telomerase reverse transcriptase (hTERT)-immortalized and umbilical cord mesenchymal stem cells. Interestingly, the proliferation of both types of stem cells was observed when crude extracts of dragon blood and catechu were present together in the stem cell growth medium. Conclusions Dragon blood from D. draco offers multifaceted therapeutic benefits for treating chronic nonhealing diabetic wounds from various perspectives. Most drugs in Western medicine consist of small molecules with defined ingredients. However, this is not the case in TCM, as the activities of dragon blood reported in this study. Surprisingly, the activities documented here align with descriptions in ancient Chinese medical texts dating back to A.D. 1625.

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. [ABSTRACT FROM AUTHOR]

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle‐ and liver/erythrocyte‐specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle‐specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA‐seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub‐family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation. [ABSTRACT FROM AUTHOR]

Background: The clinical efficacy of Jinchuang Ointment, a traditional Chinese medicine (TCM), in treating chronic non-healing diabetic wounds has been demonstrated over the past decades. Both in vitro and in vivo angiogenic activities have been reported for its herbal ingredients, including dragon blood from the palm tree Daemonorops draco and catechu from Uncaria gambir Roxb. Additionally, crude extracts of dragon blood have exhibited hypoglycemic effects not only in animal studies but also in cell-based in vitro assays. Results: Our findings indicate that crude dragon blood extract promotes the differentiation of myoblasts into myotubes. Partially purified fractions of dragon blood crude extract significantly enhance the expression of muscle cell differentiation-related genes such as myoG, myoD, and myoHC. Our results also demonstrate that crude extracts of dragon blood can inhibit platelet-derived growth factor-induced PAI-1 expression in primary rat vascular smooth muscle cells, thereby favoring changes in hemostasis towards fibrinolysis. Consistent with previous reports, reduced expression of plasminogen activator inhibitor 1 (PAI-1) accelerates wound healing. However, further separation resulted in a significant loss of both activities, indicating the involvement of more than one compound in these processes. Stem cells play a crucial role in muscle injury repair. Neither dragon blood nor catechu alone stimulated the proliferation of human telomerase reverse transcriptase (hTERT)-immortalized and umbilical cord mesenchymal stem cells. Interestingly, the proliferation of both types of stem cells was observed when crude extracts of dragon blood and catechu were present together in the stem cell growth medium. Conclusions: Dragon blood from D. draco offers multifaceted therapeutic benefits for treating chronic nonhealing diabetic wounds from various perspectives. Most drugs in Western medicine consist of small molecules with defined ingredients. However, this is not the case in TCM, as the activities of dragon blood reported in this study. Surprisingly, the activities documented here align with descriptions in ancient Chinese medical texts dating back to A.D. 1625. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy was one of main complications of type 2 diabetes mellitus. Hydrogen sulfide (H2S) is involved in various physiological functions, such as anti-hypertension and anti-oxidant. Skeletal muscle atrophy caused by type 2 diabetes could lead to the regeneration of muscle fibers. Wnt signaling pathway plays a crucial important role in this process. H2S maybe regulate the Wnt signaling pathway to alleviate skeletal muscle atrophy, however, this role has not been clarified. The aim of this study is to investigate the potential regulatory role of H2S in the Wnt signaling pathway. C2C12 myoblasts treated with 500 μmol palmitate as an in vitro model. Western blot was used to detect the levels of CSE, PKM1, β-catenin, MuRF1, MYOG, MYF6 and MYOD1. In addition, MuRF1 was mutated at Cys44 and MuRF1 S-sulfhydration was detected by biotin switch assay. The interaction between PKM1 and MuRF1 was assessed via Co-immunoprecipitation. Differentiation of C2C12 myoblasts was evaluated using LAMININ staining. These data showed the levels of CSE, β-catenin, PKM1, MYOG, MYF6 and MYOD1 were decreased in pal group, compared with control and pal + NaHS groups. MuRF1 Cys44 mutants increased the protein levels of β-catenin, MYOG, MYF6 and MYOD1 in pal group. Our results suggest that H2S regulates the S-sulfhydration levels of MuRF1 at Cys44, influencing the ubiquitination levels of PKM1 and ultimately promoting myoblast differentiation.

Abstract Background We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. Results We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. Conclusions For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.

Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C, specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo. Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.

Background: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. Results: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. Conclusions: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype. [ABSTRACT FROM AUTHOR]

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein–protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development. [ABSTRACT FROM AUTHOR]

High doses or prolonged use of the exogenous synthetic glucocorticoid dexamethasone (Dex) can lead to muscle atrophy. In this study, the anti-atrophic effects of ginsenosides Rh1, Rg2, and Rg3 on Dex-induced C2C12 myotube atrophy were assessed by XTT, myotube diameter, fusion index, and western blot analysis. The XTT assay results showed that treatment with Rh1, Rg2, and Rg3 enhanced cell viability in Dex-injured C2C12 myotubes. Compared with the control group, the myotube diameter and fusion index were both reduced in Dex-treated cells, but treatment with Rh1, Rg2, and Rg3 increased these parameters. Furthermore, Rh1, Rg2, and Rg3 significantly downregulated the protein expression of FoxO3a, MuRF1, and Fbx32, while also upregulating mitochondrial biogenesis through the SIRT1/PGC-1α pathway. It also prevents myotube atrophy by regulating the IGF-1/Akt/ mTOR signaling pathway. These findings indicate that Rh1, Rg2, and Rg3 have great potential as useful agents for the prevention and treatment of muscle atrophy. [ABSTRACT FROM AUTHOR]

Abstract Background Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive. Recently, lactate‐mediated histone lactylation is identified as a novel epigenetic modification that promotes gene transcription. Methods We used mouse C2C12 cell line and 2‐month‐old male mice as in vitro and in vivo models, respectively. These models were treated with lactate to explore the biological function and latent mechanism of lactate‐derived histone lactylation on myogenic differentiation by quantitative real‐time PCR, western blotting, immunofluorescence staining, chromatin immunoprecipitation, cleavage under targets and tagmentation assay and RNA sequencing. Results Using immunofluorescence staining and western blotting, we proposed that lactylation might occur in the histones. Inhibition of lactate production or intake both impaired myoblast differentiation, accompanied by diminished lactylation in the histones. Using lactylation site‐specific antibodies, we demonstrated that lactate preferentially increased H3K9 lactylation (H3K9la) during myoblast differentiation (CT VS 5, 10, 15, 20, 25 mM lactate treatment, P = 0.0012, P = 0.0007, and the rest of all P

Abstract Background The circadian clock is an evolutionarily conserved mechanism that exerts pervasive temporal control in stem cell behavior. This time-keeping machinery is required for orchestrating myogenic progenitor properties in regenerative myogenesis that ameliorates muscular dystrophy. Here we report a screening platform to discover circadian clock modulators that promote myogenesis and identify chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic activities. Methods A high-throughput molecular docking pipeline was applied to identify compounds with a structural fit for a hydrophobic pocket within the key circadian transcription factor protein, Circadian Locomotor Output Cycles Kaput (CLOCK). These identified molecules were further screened for clock-modulatory activities and functional validations for pro-myogenic properties. Results CHX was identified as a clock activator that promotes distinct aspects of myogenesis. CHX activated circadian clock that reduced cycling period length and augmented amplitude. This action was mediated by the targeted CLOCK structure via augmented interaction with heterodimer partner Bmal1, leading to enhanced CLOCK/Bmal1-controlled transcription with upregulation of core clock genes. Consistent with its clock-activating function, CHX displayed robust effects on stimulating myogenic differentiation in a clock-dependent manner. In addition, CHX augmented the proliferative and migratory activities of myoblasts. Conclusion Our findings demonstrate the feasibility of a screening platform to discover clock modulators with myogenic regulatory activities. Discovery of CHX as a pro-myogenic molecule could be applicable to promote regenerative capacities in ameliorating dystrophic or degenerative muscle diseases.

Abstract Background Myoblast differentiation requires metabolic reprogramming driven by increased mitochondrial biogenesis and oxidative phosphorylation. The canonical GH-GHR-IGFs axis in liver exhibits a great complexity in response to somatic growth. However, the underlying mechanism of whether local GHR acts as a control valve to regulate mitochondrial function through mitochondrial biogenesis during myoblast differentiation remains unknown. Methods We manipulated the GHR expression in chicken primary myoblast to investigate its roles in mitochondrial biogenesis and function during myoblast differentiation. Results We reported that GHR is induced during myoblast differentiation. Local GHR promoted mitochondrial biogenesis during myoblast differentiation, as determined by the fluorescence intensity of Mito-Tracker Green staining and MitoTimer reporter system, the expression of mitochondrial biogenesis markers (PGC1α, NRF1, TFAM) and mtDNA encoded gene (ND1, CYTB, COX1, ATP6), as well as mtDNA content. Consistently, local GHR enhanced mitochondrial function during myoblast differentiation, as determined by the oxygen consumption rate, mitochondrial membrane potential, ATP level and ROS production. We next revealed that the regulation of mitochondrial biogenesis and function by GHR depends on IGF1. In terms of the underlying mechanism, we demonstrated that IGF1 regulates mitochondrial biogenesis via PI3K/AKT/CREB pathway. Additionally, GHR knockdown repressed myoblast differentiation. Conclusions In conclusion, our data corroborate that local GHR acts as a control valve to enhance mitochondrial function by promoting mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway during myoblast differentiation. Video Abstract

Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense‐mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide‐3‐kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up‐frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle‐related injuries and diseases. [ABSTRACT FROM AUTHOR]

BACKGROUND: The dangerous inducers of muscle atrophy are inflammatory reaction, oxidative stress, and cachexia, etc. β‐Glucan, an important food derived active ingredient, has been reported to exert anti‐inflammatory effects, however, its effects on regulating myoblast differentiation and protein degradation are unclear. This study is aimed to investigate the mechanism of oat β‐glucan on alleviating muscle atrophy. RESULTS: The results showed that oat β‐glucan treatment reversed tumor necrosis factor‐α (TNF‐α) induced abnormal myoblast differentiation and reduced muscle atrophy related MuRF‐1 and Atrogin‐1 protein expression. The similar phenomenon was observed after using MCC950 (NLRP3 specific inhibitor) or AS1842856 (FoxO1 specific inhibitor) to suppress NLRP3 and FoxO1 expression, respectively. Exposure to β‐glucan or AS1842856 also inhibited TNF‐α induced the activation of TLR4/NF‐κB pathway by inactivating FoxO1, and subsequently suppressed the expression of NLRP3. CONCLUSION: Our results indicate that oat β‐glucan exerts essential roles in promoting myoblast differentiation and alleviating muscle atrophy via inactivating FoxO1 and NLRP3 inflammasome signal pathway. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Objective It was found that gypenoside not only have anti-inflammatory and anti-aging effects, but also can promote the proliferation and differentiation of osteoblasts and protect their oxidative stress damage. This study aims to investigate the effect of gypenoside on myoblast differentiation of C2C12 cells. Methods C2C12 cells were cultured in complete medium. After cell proliferation and adherent to the wall, differentiation medium containing different concentrations of gypenoside was used to induce cell differentiation, and three repores were set for each concentration. On the fifth day, observed the morphology of cell differentiation and calculated the activity of cell differentiation. Western Blot was used to detect the expression of myoblast markers MYF5 and MyoD1, immunofluorescence staining was used to detect the expression of MyoD1, and Image J software was used to calculate the fusion index of myotube. Results On the fifth day of induction of C2C12 cell differentiation, the cells could differentiate into typical myotube morphology. MYF5 and MyoD1 were significantly expressed under the intervention of 10 mg/L and 5 mg/L gypenoside concentrations, and the fusion index of myotube was also higher with statistically significant differences compared with the control group(P＜0.05). Conclusion Gypenoside can promote myoblast differentiation of C2C12 cells, and the concentration of 10 mg/L and 5 mg/L is better. [ABSTRACT FROM AUTHOR]

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia. [ABSTRACT FROM AUTHOR]

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1–4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior–posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases. [ABSTRACT FROM AUTHOR]

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein–protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.

Background: The circadian clock is an evolutionarily conserved mechanism that exerts pervasive temporal control in stem cell behavior. This time-keeping machinery is required for orchestrating myogenic progenitor properties in regenerative myogenesis that ameliorates muscular dystrophy. Here we report a screening platform to discover circadian clock modulators that promote myogenesis and identify chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic activities. Methods: A high-throughput molecular docking pipeline was applied to identify compounds with a structural fit for a hydrophobic pocket within the key circadian transcription factor protein, Circadian Locomotor Output Cycles Kaput (CLOCK). These identified molecules were further screened for clock-modulatory activities and functional validations for pro-myogenic properties. Results: CHX was identified as a clock activator that promotes distinct aspects of myogenesis. CHX activated circadian clock that reduced cycling period length and augmented amplitude. This action was mediated by the targeted CLOCK structure via augmented interaction with heterodimer partner Bmal1, leading to enhanced CLOCK/Bmal1-controlled transcription with upregulation of core clock genes. Consistent with its clock-activating function, CHX displayed robust effects on stimulating myogenic differentiation in a clock-dependent manner. In addition, CHX augmented the proliferative and migratory activities of myoblasts. Conclusion: Our findings demonstrate the feasibility of a screening platform to discover clock modulators with myogenic regulatory activities. Discovery of CHX as a pro-myogenic molecule could be applicable to promote regenerative capacities in ameliorating dystrophic or degenerative muscle diseases. [ABSTRACT FROM AUTHOR]

Background: Myoblast differentiation requires metabolic reprogramming driven by increased mitochondrial biogenesis and oxidative phosphorylation. The canonical GH-GHR-IGFs axis in liver exhibits a great complexity in response to somatic growth. However, the underlying mechanism of whether local GHR acts as a control valve to regulate mitochondrial function through mitochondrial biogenesis during myoblast differentiation remains unknown. Methods: We manipulated the GHR expression in chicken primary myoblast to investigate its roles in mitochondrial biogenesis and function during myoblast differentiation. Results: We reported that GHR is induced during myoblast differentiation. Local GHR promoted mitochondrial biogenesis during myoblast differentiation, as determined by the fluorescence intensity of Mito-Tracker Green staining and MitoTimer reporter system, the expression of mitochondrial biogenesis markers (PGC1α, NRF1, TFAM) and mtDNA encoded gene (ND1, CYTB, COX1, ATP6), as well as mtDNA content. Consistently, local GHR enhanced mitochondrial function during myoblast differentiation, as determined by the oxygen consumption rate, mitochondrial membrane potential, ATP level and ROS production. We next revealed that the regulation of mitochondrial biogenesis and function by GHR depends on IGF1. In terms of the underlying mechanism, we demonstrated that IGF1 regulates mitochondrial biogenesis via PI3K/AKT/CREB pathway. Additionally, GHR knockdown repressed myoblast differentiation. Conclusions: In conclusion, our data corroborate that local GHR acts as a control valve to enhance mitochondrial function by promoting mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway during myoblast differentiation. 2uqMpL47bHNL2ReoTRE-H3 Video Abstract [ABSTRACT FROM AUTHOR]

Jie Ren,1,* Jingzhou Wang,2,* Xingchen Yao,1 Yue Wu,1 Ming Shi,1 Xiangjun Shi,3 Xinru Du1 1Department of Orthopaedics, Beijing Chao-yang Hospital, Beijing, 100020, People’s Republic of China; 2Department of Orthopaedics, Beijing Daxing District People’s Hospital, Beijing, 102600, People’s Republic of China; 3Department of Hematology, Beijing Chao-yang Hospital, Beijing, 100020, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xinru Du, Department of orthopaedics, Beijing Chao-yang Hospital, No. 8 of Gongti South Road, Chaoyang District, Beijing, 100020, People’s Republic of China, Tel +86 13683156652, Email duxinru@163.comObjective: To explore the role of sclerosteosis (SOST) gene expression in the occurrence and development of multiple myeloma (MM) complicated with sarcopenia.Methods: Analysis of the SOST expression in skeletal muscle tissue of patients with MM using high-throughput sequencing combined with transcriptomics; observation of morphological changes of the mouse C2C12 myoblasts co-cultured with SP2/0 myeloma cells in Transwell; observation of the SOST expression in the C2C12 myoblasts using the immunofluorescence labeling method; and assessment of the changes in exercise capacity of mice with MM using ethology; and the measurement of the SOST expression in muscles of mice using immunohistochemistry.Results: The transcription level of the SOST gene in the muscle tissue was significantly higher in patients with MM and sarcopenia than in patients with MM without sarcopenia and elderly patients with sarcopenia; the area of C2C12 mouse myoblasts co-cultured with SP2/0 myeloma cells was 167,904 ± 8653.7 pix; this was significantly lower than the area of 402,994 ± 13,575.0 pix in the control group (CG); the fluorescence intensity of SOST in the cells of the experimental group (EG) was 159,389 ± 10,534 AU; this was significantly higher than the intensity of 26,338 ± 6059 AU in the CG; the differences in results of the coat-hanger test, the tail suspension test, the weight-bearing forced swimming test, and the grip strength test between the tumor-bearing mice in the EG and the CG were statistically significant; and the quantitative result of SOST expression in the muscle tissue of the EG mice was 11,515 ± 1573 pix; this was significantly higher than the result of 3399 ± 798.8 pix in the CG.Conclusion: The SOST gene expression was significantly higher in muscle of mice in EG than in CG; and increased SOST gene expression might be a pathogenesis of MM complicated with sarcopenia.Keywords: multiple myeloma, SOST, multiple myeloma mouse model, ethology, sarcopenia, myoblast differentiation

Abstract Background Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and ‘proteostasis collapse’. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age‐related declines in muscle homeostasis. Methods The Absolute Dynamic Profiling Technique for Proteomics (Proteo‐ADPT) was used to investigate proteostasis in young (passage 6–10) and replicatively aged (passage 48–50) C2C12 myoblast cultures supplemented with deuterium oxide (D2O) during early (0–24 h) or late (72–96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Results Young cells exhibited a consistent ~25% rise in protein accretion over the 96‐h experimental period. In aged cells, protein accretion increased by 32% (P

DNA methylation and demethylation are crucial epigenetic modification and regulation for animal development, and their dynamic changes may affect skeletal muscle development. The ten-eleven translocation (Tet) family proteins are demethylases which are involved in the dynamic changes of DNA methylation. However, the expression pattern of Tet family genes and their role in myogenesis in fish remains unclear. In this study, the temporal and spatial expression profiles of Tet1, Tet2 and Tet3 were assayed with RT-qPCR techniques in Chinese perch, Siniperca chuatsi. The obtained data showed that three Tet family genes were differentially expressed at different development stages. Tet1 was expressed low at blastula stage, but highly expressed at gastrula stage, then remained low until hatching. The expressions of Tet2 and Tet3 were significantly increased at late gastrula and kept high expression before hatching stage. At the spatial level, the Tet1 expression was highest in gill tissue, moderate level in brain and slow muscle. Tet2 was similar to that of Tet1 except that it was expressed at a lower level in slow muscle, and Tet3 exhibited a higher expression level in gill and brain, a moderate level in fast muscle. Cosinor analysis turned out that the expression of Tet1 and Tet2 displayed a significant daily rhythm in fast muscle, but Tet3 did not show daily rhythmicity. Inhibiting the activity of Tet1/2 proteins by injecting Bobcat339 significantly reduced the expression of MyoD and MRF4, but not MyoG and Myf5, by which leads to the increase of the number of satellite cells and proliferating myoblasts. Together, the results suggest that Tet1/2 may target to MyoD and MRF4 resulted in DNA demethylation and promoting their expression, thus stimulating myoblast differentiation.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation. [ABSTRACT FROM AUTHOR]

Aging, and other disease-related muscle disorders are serious health problems. Dexamethasone (DEX), a synthetic glucocorticoid, can trigger skeletal muscle atrophy. This study examined the effects of mealworm (Tenebrio molitor larva) ethanol extract (TME) on C2C12 myoblast differentiation and DEX-induced myotube atrophy. TME induced myotube formation compared to the differentiation medium (DM) group. TME also significantly increased the mRNA expression of muscle creatine kinase (CKm) and myogenic regulatory factors (MRFs), such as myogenin (MyoG), myogenic factor (Myf)5, and MRF4 (Myf6). TME dramatically increased the muscle-specific protein, MyoG, compared to the control, whereas the expression of myogenic differentiation 1 (MyoD) remained unchanged. It also activated the mammalian target of rapamycin (mTOR) signaling pathway. In the DEX-induced muscle atrophy C2C12 model, TME reduced the gene expression of atrogin-1, muscle RING finger protein-1 (MuRF-1), and myostatin, which are involved in protein degradation in skeletal muscles. Furthermore, TME elevated the phosphorylation of forkhead box O3 (FoxO3α) and protein kinase B (Akt). These findings suggest that TME can enhance myotube hypertrophy by regulating the mTOR signaling pathway, and can rescue DEX-induced muscle atrophy by alleviating atrophic muscle markers mediated by Akt activation. Thus, TME can be a potential therapeutic agent for treating muscle weakness and atrophy. [ABSTRACT FROM AUTHOR]

DNA methylation (5mC) and mRNA N 6 -methyladenosine (m6A) play an essential role in gene transcriptional regulation. DNA methylation has been well established to be involved in skeletal muscle development. Interacting regulatory mechanisms between DNA methylation and mRNA m6A modification have been identified in a variety of biological processes. However, the effect of m6A on skeletal muscle differentiation and the underlying mechanisms are still unclear. It is also unknown whether there is an interaction between DNA methylation and mRNA m6A modification in skeletal myogenesis. In the present study, we used m6A-IP-qPCR, LC–MS/MS and dot blot assays to determine that the DNA demethylase gene, TET1 , exhibited increased m6A levels and decreased mRNA expression during bovine skeletal myoblast differentiation. Dual-luciferase reporter assays and RIP experiments demonstrated that METTL3 suppressed TET1 expression by regulating TET1 mRNA stability in a m6A-YTHDF2-dependent manner. Furthermore, TET1 mediated DNA demethylation of itself, MYOD1 and MYOG , thereby stimulating their expression to promote myogenic differentiation. Ectopic expression of TET1 rescued the effect of METTL3 knockdown on reduced myotubes. In contrast, TET1 knockdown impaired the myogenic differentiation promoted by METTL3 overexpression. Moreover, ChIP experiments found that TET1 could bind and demethylate METTL3 DNA, which enhanced METTL3 expression. In addition, TET1 knockdown decreased m6A levels. ChIP assays also showed that TET1 knockdown contributed to the binding of H3K4me3 and H3K27me3 to METTL3 DNA. Our results revealed a negative feedback regulatory loop between TET1 and METTL3 in myoblast differentiation, which unveiled the interplay among DNA methylation, RNA methylation and histone methylation in skeletal myogenesis. • The mRNA m6A level of TET1 is increased and its expression is decreased during myoblast differentiation. • METTL3 inhibits TET1 expression in an m6A-YTHDF2-dependent manner. • TET1 mediates DNA demethylation of METTL3 to regulate m6A levels. • TET1 binds to the METTL3 promoter and inhibits the binding of H3K4me3 and H3K4me3 to METTL3. [ABSTRACT FROM AUTHOR]

High glucose levels are a primary cause of diabetes-associated cellular dysfunction and tissue damage. Muscles are the key insulin target organ and therefore, have a high level of sensitivity to hyperglycemia. Our previous study revealed that 20(S)-ginsenoside Rg3 (S-Rg3) is a monomer with a good myogenic differentiation effect in ginsenoside. Furthermore, it can alleviate dexamethasone-induced muscle atrophy by protecting mitochondrial function. However, whether S-Rg3 is effective for diabetic-induced muscle atrophy has not been reported. This study aimed to investigate the protective effect of S-Rg3 on diabetic-induced muscle atrophy. C2C12 myoblasts, Drosophila , and mice were used as model systems, and the protective effect of S-Rg3 on diabetes was evaluated by assessing the levels of glucose and lipids. Furthermore, H&E, toluidine blue, Giemsa, and immunofluorescence staining were performed to detect the effects of S-Rg3 on muscle atrophy and myogenic differentiation. Moreover, the effects of S-Rg3 on mitochondrial morphology and function were also evaluated by electron microscopy, flow cytometry, and Seahorse. In addition, the underlying pathways of S-Rg3 effects were detected by Western blot. The related inhibitors and gene mutations in Drosophila were used for validation. The analysis of diabetic mice model fed with a high-fat diet (HFD) and high glucose (HG) revealed that in the injured C2C12 myoblasts, S-Rg3 treatment significantly reduced the levels of triglycerides and glucose. Furthermore, it promoted the differentiation of myoblasts and inhibited mitochondrial dysfunction. In the Drosophila HG and HFD diabetic model, S-Rg3 reduced triglyceride and trehalose levels, increased climbing distance values, promoted myoblasts differentiation, preserved mitochondrial function, and inhibited muscle atrophy. Mechanistically, the beneficial effects of S-Rg3 were at least partially associated with the phosphorylation of AMPK and FoxO3 together with the inhibition of Smad3 phosphorylation, this pathway was validated by the UAS-AMPKα-RNAi Drosophila model. In summary, this study revealed mechanistic insights into how S-Rg3 protects against diabetes-associated muscle atrophy in cells, Drosophila, and mice. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA) has been shown to promote muscle fiber development in fish, but its specific involvement in the fusion of myoblasts into myotubes remains uncertain. In this study, we first isolated grass carp (Ctenopharyngodon idellus) myoblasts and established the differentiation system using 2% horse serum taking advantage of cell morphological observation, transcriptomic sequencing, and expression analysis of myogenic differentiation marker genes. Mitochondrial mass and the expression of genes related to mitochondrial division, fusion, mitophagy, and mitochondrial biogenesis also increased during myoblast differentiation, indicating that mitochondrial quality control (MQC) is involved in the myoblast-to-myotube formation in grass carp (P < 0.05). Since we previously demonstrated that 50 μM DHA promotes myoblast proliferation and that dietary 1% DHA significantly promoted the growth performance in grass carp, we subsequently explored the effect of 50 μM DHA on myoblast-to-myotube formation. DHA promoted the mRNA and protein expression of the key genes of differentiation, especially after four days of treatment (P < 0.05). Moreover, DHA also increased most marker genes of mitochondrial fission, fusion, autophagy, and biogenesis during myoblast-to-myotube formation (P < 0.05). Importantly, trametinib (Tra), an MEK inhibitor, significantly disrupted the advantages of DHA in improving MQC and energy metabolism in vivo (P < 0.05). In vitro, the Tra also significantly reduced the effect of DHA on both the promotion of differentiation and the improvement of MQC, indicating that the MEK/ERK-mediated MQC pathway plays a vital role in the effect of DHA during myogenesis (P < 0.05). The establishment of the grass carp myoblast culture system provides a valuable platform for further research on the development of fish muscle fibers. And this study may contribute to the understanding of the molecular mechanism underlying DHA's function in promoting vertebrate myogenesis. • Obtained and differentiated grass carp myoblasts by tissue block culture firstly. • Transcriptome sequencing validated the establishment of differentiation lines. • DHA improved mitochondrial quality control to promote myoblast differentiation. • DHA activated MEK/ERK to regulate mitochondrial quality control in grass carp. [ABSTRACT FROM AUTHOR]

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia.

Skeletal muscle development is closely related with the amount of meat production and its quality in chickens. Natriuretic peptides (NPs) play an important role in myotube formation and fat oxidation of skeletal muscle in animals. The effect of C-type natriuretic peptide (CNP), an important member of the NPs, and its underlying molecular mechanisms in skeletal muscle are incompletely understood. Treatment of myoblasts with CNP led to enhanced proliferation/differentiation and significantly upregulated (P

In the myoblasts differentiation process of C2C12 cells, it is imperative to research the expression and synthesis of the myogenic regulatory factors in extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) exposure. In the present study, using a cell cultivation method of C2C12 cells, the subcultured cells were divided into control and ELF-PEMF treated: A (0 mT), B (0.5 mT), C (1.0 mT) and D (1.5 mT) groups for five consecutive days training. Furthermore, cells were analyzed using immunochemical staining to detect the cell phenotypic changes, and RT-PCR and Western blot were used to test the expression levels of the myogenic regulatory factor D (MyoD) and the myogenic regulatory factor 5 (Myf5) genes and proteins synthesis in the process of myblasts differentiation. The results indicated that compared with the control group, different intensities of extremely low-frequency pulse electromagnetic fields could induce myoblast nuclear fusion, amplify MyoD and Mfy5 expression, and particularly the 1.5 mT group at the 5th day exhibited the highest expression of these genes. Our study can pave a way for the development of a non-invasive and effective clinical prevention and treatment strategy in serious disorders like skeletal muscle atrophy. [ABSTRACT FROM AUTHOR]

Agricultural meat composition and quality are not independent of the effects of skeletal muscle growth and development in animals. Cofilin is distributed extensively in muscle and non-muscle cells, and its function is tightly regulated in the cell. Cofilin has two variants in mammals, cofilin-1 (CFL1, non-muscle type) and cofilin-2 (CFL2, muscle type), and has a dual function on skeletal muscle fibers. Our study examined the expression pattern of CFL1 and CFL2 in different fetal bovine, calf, and adult cattle tissues. The content of the CFL2 gene increased significantly with the increase in cattle age in muscle tissues; CFL1 showed the opposite trend. In muscle tissues, DNA methylation levels of CFL1 and CFL2 were high in fetal bovine, and the mRNA level of CFL2 was significantly lower compared to CFL1. However, DNA methylation levels of CFL2 were lower than CFL1, and the mRNA level of CFL2 was remarkably higher compared to CFL1 in adult cattle. Overexpression of CFL1 or knockdown CFL2 reduced the expression levels of muscle differentiation markers, i.e., MYOD, MYOG, and MYH3. Overexpression of CFL2 or knockdown CFL1 stimulated the expression of these marker genes. Therefore, CFL2 may be superior to CFL1 as a candidate gene for subsequent research on cattle genetics and breeding. [ABSTRACT FROM AUTHOR]

Our previous studies have demonstrated the important roles of androgen receptor (AR) in myoblast proliferation regulated by 15 % (mimic appropriate exercise) and 20 % (mimic excessive exercise) mechanical stretches. Except for myoblast proliferation, differentiation is also an important factor affecting muscle mass and strength. But the role of AR in stretch-regulated myoblast differentiation and AR's upstream and downstream signals remain unknown. In the present study, firstly the differences of myogenic differentiation between C2C12 (with AR expression) and L6 (without AR expression) myoblasts induced by 15 % and 20 % mechanical stretches were compared; secondly, AR antagonist flutamide and AR agonist GTx-007 were used in 15 % and 20 % stretched myoblasts respectively to confirm AR's roles in stretch-regulated myoblast differentiation; thirdly, RNA-seq, molecular dynamic simulation (MD) and co-immunoprecipitation were performed to screen the downstream and upstream molecules of AR during stretches. We found that (1) 15 % stretch increased while 20 % stretch decreased myotube number in differentiating C2C12 and L6 myoblasts, with more significant changes in C2C12 cells than L6 cells; (2) in stretched C2C12 myoblasts, AR antagonist flutamide inhibited 15 % stretch-promoted differentiation while AR agonist GTx-007 reversed 20 % stretch-inhibited differentiation (reflected by changes in myotube number, MHC contents of fast-twitch and slow-twitch fiber, and the levels of myogenic regulatory factors (MRFs) such as MyoD and myogenin); (3) KEGG analysis of RNA-seq showed that the differently expressed genes (DEGs) in C2C12 cells induced by 15 % stretch were enriched in FoxO and JAK-STAT signaling pathways, while DEGs by 20 % stretch were enriched in FoxO and MAPK signaling pathways; (4) MD and co-immunoprecipitation showed that β1 integrin could interact with AR and influence AR's activity in C2C12 cells. In conclusion, AR plays important roles in myoblast differentiation promoted by 15 % stretch while inhibited by 20 % stretch, which was fulfilled through FoxO-MRFs. In addition, α7β1 integrin may be a bridge linking mechanical stretch and AR. This study is beneficial to deeply understand the roles and mechanisms of AR in stretch-regulated muscle mass and strength; and reports firstly that myoblasts sense mechanical stimulus and transmit into intracellular AR via α7β1 integrin., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca 2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca 2+ channel., Methods: Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10 weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20 μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80 mg/L) and fed to the mice (50 mg/kg). Fluorescence microscopy was used to determine the concentration of Ca 2+ . Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining., Results: During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P < 0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.05), and conversely, knockdown of Wnt5a inhibited their expression (P < 0.001). The Wnt5a pathway mediated the opening of Ca 2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P < 0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P < 0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated., Conclusions: Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca 2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle., (© 2024 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Skeletal muscle atrophy was one of main complications of type 2 diabetes mellitus. Hydrogen sulfide (H 2 S) is involved in various physiological functions, such as anti-hypertension and anti-oxidant. Skeletal muscle atrophy caused by type 2 diabetes could lead to the regeneration of muscle fibers. Wnt signaling pathway plays a crucial important role in this process. H 2 S maybe regulate the Wnt signaling pathway to alleviate skeletal muscle atrophy, however, this role has not been clarified. The aim of this study is to investigate the potential regulatory role of H 2 S in the Wnt signaling pathway. C2C12 myoblasts treated with 500 μmol palmitate as an in vitro model. Western blot was used to detect the levels of CSE, PKM1, β-catenin, MuRF1, MYOG, MYF6 and MYOD1. In addition, MuRF1 was mutated at Cys44 and MuRF1 S-sulfhydration was detected by biotin switch assay. The interaction between PKM1 and MuRF1 was assessed via Co-immunoprecipitation. Differentiation of C2C12 myoblasts was evaluated using LAMININ staining. These data showed the levels of CSE, β-catenin, PKM1, MYOG, MYF6 and MYOD1 were decreased in pal group, compared with control and pal + NaHS groups. MuRF1 Cys44 mutants increased the protein levels of β-catenin, MYOG, MYF6 and MYOD1 in pal group. Our results suggest that H 2 S regulates the S-sulfhydration levels of MuRF1 at Cys44, influencing the ubiquitination levels of PKM1 and ultimately promoting myoblast differentiation., Competing Interests: The authors declare no conflict of interest., (© 2024 Published by Elsevier Ltd.)

During skeletal myogenesis, the zinc‐finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro‐differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA‐mediated gene silencing in C2C12 myoblasts and whole‐transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts. [ABSTRACT FROM AUTHOR]

Abstract Background Sarcopenia is a common skeletal disease related to myogenic disorders and muscle atrophy. Current clinical management has limited effectiveness. We sought to investigate the role of miR-1290 in myoblast differentiation and muscle atrophy. Methods By transfecting miR-1290 into C2C12 cells, we investigated whether miR-1290 regulates myogenesis and myotube atrophy via AKT/P70 signaling pathway. MHC staining was performed to assess myoblast differentiation. Differentiation-related MHC, Myod, and Myog protein levels, and atrophy-related MuRF1 and atrogin-1 were explored by western blot. An LPS-induced muscle atrophy rat model was developed. RT-PCR was conducted to analyze miR-1290 serum levels in muscle atrophy patients and normal controls (NCs). Results The miR-1290 transfection increased MHC-positive cells and MHC, Myod, and Myog protein levels in the miR-1290 transfection group, demonstrating that miR-1290 promoted C2C12 myoblast differentiation. Myotube diameter in the miR-1290 transfection group was higher than in the TNF-α-induced model group. Western blot analysis showed decreased MuRF1 and atrogin-1 levels in the miR-1290 transfection group compared with the model group, demonstrating that miR-1290 protected against myoblast cellular atrophy. Luciferase assay and western blot analysis showed that miR-1290 regulation was likely caused by AKT/p70/FOXO3 phosphorylation activation. In the LPS-induced muscle atrophy rat model, miR-1290 mimics ameliorated gastrocnemius muscle loss and increased muscle fiber cross-sectional area. Clinically, miR-1290 serum level was significantly decreased in muscle atrophy patients. Conclusions We found that miR-1290 enhances myoblast differentiation and inhibits myotube atrophy through Akt/p70/FoxO3 signaling in vitro and in vivo. In addition, miR-1290 may be a potential therapeutic target for sarcopenia treatment.

Myoblast differentiation depends on fatty acid oxidation (FAO)，and its rate-limiting enzyme acetyl-CoA carboxylase 2 (ACC2) participate in the regulation skeletal muscle development. However, the precise regulatory mechanism is still unknown. Using previous RNA-sequencing data from our laboratory, we explored the effect of ACC2 on myoblast differentiation, as a candidate gene, since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). Our findings show that siACC2 inhibited myoblast proliferation, promoted differentiation, and boosted mitochondrial and fatty acid oxidation activities. The effect of ACC2 on goat muscle cell differentiation was modulated by Etomoxir, a CPT1A inhibitor. Notably, the AMPK/ACC2 pathway was found to regulate fatty acid oxidation and goat muscle cell differentiation. Inhibiting the AMPK/ACC2 pathway significantly reduced CPT1A expression. These findings indicate that AMPK/ACC2 regulate goat myoblast differentiation via fatty acid oxidation, contributing to understanding the mechanism of goat skeletal muscle development. [ABSTRACT FROM AUTHOR]

Whey protein has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that whey protein would regulate muscle cell differentiation. Adding various concentrations of whey protein to C2C12 myoblasts induced cell differentiation and MyoD (myogenic differentiation protein) expression as well as the phosphorylation of AKT (protein kinase B). Whey protein-induced differentiation-specific markers increased the enzymatic activities of creatine kinase and citrate synthase and the expression of muscle-specific microRNA. Whey protein elevated AKT phosphorylation on Thr308 and Ser473, which was inhibited by LY294002 (a nonselective phosphoinositide 3-kinases inhibitor), suggesting that whey protein acts via PI3K (phosphatidylinositol 3-kinase). Blocking of the PI3K/AKT pathway with specific inhibitors revealed its requirement in mediating the promotive effects of whey protein on C2C12 cell differentiation. These effects of whey protein on myoblast differentiation suggest its positive influence in preventing muscle atrophy. [ABSTRACT FROM AUTHOR]

Exercise, especially anaerobic one, can gradually increase muscle mass over time as a result of adaptive responses of muscle cells to ensure metabolic homeostasis in the tissue. Low‐level light therapy (LLLT) or photobiomodulation exhibits beneficial effects on promoting muscular functions, regeneration, and recovery from exhausting exercise, although the underlying cellular mechanisms remain poorly understood. We found that hypoxia, a condition following anaerobic exercise, significantly impeded myotube differentiation from myoblasts. However, this adverse effect was blunted greatly by pre‐exposure of myoblast cells to a 980 nm laser at 0.1 J/cm2, resulting in almost nearly normal myotube differentiation. LLL pre‐treatment enhanced myotube formation by 80%, with a tubular diameter of 4.28 ± 0.11 μm on average, representative of a 53.4% increase over sham light treatment. The normalized myoblast differentiation concurred with 68% more mitochondrial mass and myogenin expression over controls. Moreover, LLL pre‐treatment appeared to enhance glucose uptake, prevent energy metabolic switch from oxidative phosphorylation to glycolysis, and diminish lactate production under hypoxic conditions. The observation provides valuable guidance with respect to the timing of LLLT and its potential effects on muscle strengths in concert with anaerobic exercise. [ABSTRACT FROM AUTHOR]

Objective: Paravertebral muscle asymmetry may be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS), and the Tent5a protein was recently identified as a novel active noncanonical poly(A) polymerase. We, therefore, explored the function of the AIS susceptibility gene Tent5a in myoblasts. Materials and methods: RNA‐seq of AIS paravertebral muscle was performed, and the molecular differences in paravertebral muscle were investigated. Twenty‐four AIS susceptibility genes were screened, and differential expression of Tent5a in paravertebral muscles was confirmed with qPCR and Western blot. After the knockdown of Tent5a, the functional effects of Tent5a on C2C12 cell proliferation, migration, and apoptosis were detected by Cell Counting Kit‐8 assay, wound‐healing assay, and TUNEL assay, respectively. Myogenic differentiation markers were tested with immunofluorescence and qPCR in vitro, and muscle fiber formation was compared in vivo. Results: The AIS susceptibility gene Tent5a was differentially expressed in AIS paravertebral muscles. Tent5a knockdown inhibited the proliferation and migration of C2C12 cells and inhibited the maturation of type I muscle fibers in vitro and in vivo. Mechanistically, the expression of myogenin was decreased along with the suppression of Tent5a. Conclusions: Tent5a plays an important role in the proliferation and migration of myoblasts, and it regulates muscle fiber maturation by maintaining the stability of myogenin. Tent5a may be involved in the pathogenesis of AIS by regulating the formation of muscle fiber type I. [ABSTRACT FROM AUTHOR]

In the modern poultry industry, with increasing product demand, muscle growth rate and meat yield in chickens have tremendously changed. Understanding the regulation of muscle development is important to maintain efficient growth and development in meat-type chickens. 20(S)-hydroxycholesterol (20S) is known as one of the naturally occurring osteogenic cholesterol derivatives due to its ability to induce osteogenic differentiation; however, no studies have evaluated myogenic response to 20S in chicken muscle cells. To determine the use of 20S in vitro for the proliferation and differentiation of chicken satellite cells, satellite cells were isolated from pectoralis major muscle of 4-week-old Ross 708 male chickens and subjected to 0.25, 0.5, and 1.0 μmol of 20S during their proliferation and differentiation stages. Cell proliferation and differentiation were measured every 24 h for 72 h by determining DNA concentration, the activity of creatine kinase, and the expressions of myogenic regulatory transcription factors. Together these results suggested that a lower concentration of 20S did not affect myogenesis but a high concentration of 1.0 μmol 20S can negatively affect proliferation and differentiation in chicken satellite cells.

N6-methyladenosine (m6A) plays an essential role in regulating gene expression. However, the effect of m6A on skeletal myoblast differentiation and the underlying mechanisms are still unclear. Here, we ascertained mRNA m6A methylation exhibited declined changes during bovine skeletal myoblast differentiation, and both MEF2C mRNA expression and m6A levels were significantly increased during myoblast differentiation. We found that MEF2C with mutated m6A sites significantly inhibited myoblast differentiation compared with wild-type MEF2C. METTL3 promoted MEF2C protein expression through posttranscriptional modification in an m6A-YTHDF1-dependent manner. Moreover, MEF2C promoted the expression of METTL3 by binding to its promoter. These results revealed that there is a positive feedback loop between these molecules in myoblast differentiation. Our study provided new insights into skeletal muscle differentiation and fusion, which may provide an RNA methylation-based approach for molecular genetics and breeding in livestock as well as for the treatment of muscle-related diseases.

Metabolic dysfunction, dysregulated differentiation, and atrophy of skeletal muscle occur as part of a cluster of abnormalities associated with the development of Type 2 diabetes mellitus (T2DM). Recent interest has turned to the attention of the role of 1-deoxysphingolipids (1-DSL), atypical class of sphingolipids which are found significantly elevated in patients diagnosed with T2DM but also in the asymptomatic population who later develop T2DM. In vitro studies demonstrated that 1-DSL have cytotoxic properties and compromise the secretion of insulin from pancreatic beta cells. However, the role of 1-DSL on the functionality of skeletal muscle cells in the pathophysiology of T2DM still remains unclear. This study aimed to investigate whether 1-DSL are cytotoxic and disrupt the cellular processes of skeletal muscle precursors (myoblasts) and differentiated cells (myotubes) by performing a battery of in vitro assays including cell viability adenosine triphosphate assay, migration assay, myoblast fusion assay, glucose uptake assay, and immunocytochemistry. Our results demonstrated that 1-DSL significantly reduced the viability of myoblasts in a concentration and time-dependent manner, and induced apoptosis as well as cellular necrosis. Importantly, myoblasts were more sensitive to the cytotoxic effects induced by 1-DSL rather than by saturated fatty acids, such as palmitate, which are critical mediators of skeletal muscle dysfunction in T2DM. Additionally, 1-DSL significantly reduced the migration ability of myoblasts and the differentiation process of myoblasts into myotubes. 1-DSL also triggered autophagy in myoblasts and significantly reduced insulin-stimulated glucose uptake in myotubes. These findings demonstrate that 1-DSL directly compromise the functionality of skeletal muscle cells and suggest that increased levels of 1-DSL observed during the development of T2DM are likely to contribute to the pathophysiology of muscle dysfunction detected in this disease. [ABSTRACT FROM AUTHOR]

Complete skeletal muscle repair and regeneration due to severe large injury or disease is still a challenge. Biochemical cues are critical to control myoblast cell function and can be utilized to develop smart biomaterials for skeletal muscle engineering. Citric acid-based biodegradable polymers have received much attention on tissue engineering, however, their regulation on myoblast cell differentiation and mechanism was few investigated. Here, we find that citrate-based polycitrate-polyethylene glycol-polyethylenimine (POCG-PEI600) nanoclusters can significantly enhance the in vitro myoblast proliferation by probably reinforcing the mitochondrial number, promote the myotube formation and full-thickness skeletal muscle regeneration in vivo by activating the myogenic biomarker genes expression of Myod and Mhc. POCG-PEI600 nanoclusters could also promote the phosphorylation of p38 in MAP kinases (MAPK) signaling pathway, which led to the promotion of the myoblast differentiation. The in vivo skeletal muscle loss rat model also confirmed that POCG-PEI600 nanoclusters could significantly improve the angiogenesis, myofibers formation and complete skeletal muscle regeneration. POCG-PEI600 nanocluster could be also biodegraded into small molecules and eliminated in vivo, suggesting their high biocompatibility and biosafety. This study could provide a bioactive biomaterial-based strategy to repair and regenerate skeletal muscle tissue.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.