Myoblast Differentiation Research Articles

The influence of Type I and III collagen on the proliferation, migration and differentiation of myoblasts

Myoblast is a kind of activated muscle stem cell. Its biological activities, such as proliferation, migration, differentiation, and fusion, play a crucial role in maintaining the integrity of the skeletal muscle system. These activities of myoblasts can be significantly influenced by the extracellular matrix. Collagen, being a principal constituent of the extracellular matrix, substantially influences these biological activities. In skeletal muscle, collagen I and III are two kinds of primary collagen types. Their influence on myoblasts and the difference between them remain ambiguous. The purpose of this study is to discover the influence of collagen I and III on biological function of myoblasts and compare their differences. We used C2C12 cell line and primary myoblasts to discover the effect of collagen I and III on proliferation, migration and differentiation of myoblasts and then performed the transcriptome sequencing and analysis. The results showed that both collagen I and III enhanced the proliferation of myoblasts, with no statistical difference between them. Similarly, collagen I and III enhanced the migration of myoblasts, with collagen I was more pronounced in Transwell assay. On the contrary, collagen I and III inhibited myoblasts differentiation, with collagen III was more pronounced at gene expression level. The transcriptome sequencing identified DEGs and enrichment analysis elucidated different terms between Type I and III collagen. Collectively, our research preliminarily elucidated the influence of collagen I and III on myoblasts and their difference and provided the preliminary experimental foundation for subsequent research.

Effect of castration method on porcine skeletal muscle fiber traits and transcriptome profiles

This study examined the effects of immunocastration and surgical castration on the histomorphometric and transcriptome traits of the porcine skeletal muscle. We hypothesized that the differences in duration of androgen deprivation resulting from different castration methods influence skeletal muscle biology in a muscle-specific manner. This was tested by analyzing samples of m. longissimus dorsi (LD) and m. semispinalis capitis (SSC) from immunocastrated (IC; n = 12), entire male (EM; n = 12), and surgically castrated (SC; n = 12) pigs using enzyme/immunohistochemical classification and histomorphometric analysis of myofibers, quantitative PCR, and RNA sequencing. The results confirmed the distinctive histomorphometric profiles of LD and SSC and the castration method related muscle-specific effects at the histomorphometric and transcriptome levels. Long-term androgen deficiency (surgical castration) significantly reduced the proportion of fast-twitch type IIa myofibers in LD (P<0.05), whereas short-term androgen deprivation (immunocastration) reduced the cross-sectional area of oxidative type I myofibers in SSC (P<0.05). At the transcriptional level, glycolytic LD adapted to long- and short-term androgen deprivation by upregulating genes controlling myoblast proliferation and differentiation to maintain fiber size. In contrast, increased protein degradation through the ubiquitin ligase-mediated atrophy pathway (significantly increased TRIM63 and FBXO32 expression; P<0.05) could underly reduced cross-sectional area of type I myofibers in the oxidative SSC in IC. Potential candidate genes (HK2, ARID5B, SERPINE1, and SCD) linked to specific metabolic profiles and meat quality traits were also identified in IC, providing a foundation for studying the effects of immunocastration on skeletal muscle fiber and carcass/meat quality traits.

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 invivo, 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.

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

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. 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. 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.

Delta-like noncanonical notch ligand 2 regulates the proliferation and differentiation of sheep myoblasts through the Wnt/β-catenin signaling pathway.

This study delved into the role ofdelta-like noncanonical notch ligand 2(DLK2) in the cell cycle, proliferation, apoptosis, and differentiation of myoblasts, as well as its interaction with the classical Wnt/β-catenin signaling pathway in regulating myoblast function. The research revealed that upregulation of DLK2 in myoblasts during the proliferation phase enhanced myoblast proliferation, facilitated cell cycle progression, and reduced apoptosis. Conversely, downregulation of DLK2 expression using siRNA during the differentiation phase promoted myoblast hypertrophy and fusion, suppressed the expression of muscle fiber degradation factors, and expedited the differentiation process. DLK2 regulates myoblasts function by influencing the expression of various factors associated with the Wnt/β-catenin signaling pathway, including CTNNB1, FZD1, FZD6, RSPO1, RSPO4, WNT4, WNT5A, andadenomatous polyposis coli. In essence, DLK2, with the involvement of the Wnt/β-catenin signaling pathway, plays a crucial regulatory role in the cell cycle, proliferation, apoptosis, and differentiation of myoblasts.

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.

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.

Platelet-Rich Plasma Promotes the Expansion of Human Myoblasts and Favors the In Vitro Generation of Human Muscle Reserve Cells in a Deeper State of Quiescence

Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.Graphical

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 invivo 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.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

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. Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20μL). 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 (80mg/L) and fed to the mice (50mg/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. 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 Ca2+ 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. Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

Ankrd1 inhibits the FAK/Rho-GTPase/F-actin pathway by downregulating ITGA6 transcriptional to regulate myoblast functions.

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA-seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F-actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual-luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho-GTPase/F-actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle-related diseases.

High Muscle Expression of IGF2BP1 Gene Promotes Proliferation and Differentiation of Chicken Primary Myoblasts: Results of Transcriptome Analysis.

Muscle development is a multifaceted process influenced by numerous genes and regulatory networks. Currently, the regulatory network of chicken muscle development remains incompletely elucidated, and its molecular genetic mechanisms require further investigation. The Longsheng-Feng chicken, one of the elite local breeds in Guangxi, serves as an excellent resource for the selection and breeding of high-quality broiler chickens. In this study, we conducted transcriptome sequencing of the pectoral muscles of Longsheng-Feng chickens and AA broiler chickens with different growth rates. Through comprehensive bioinformatics analysis, we identified differentially expressed genes that affect muscle growth and showed that IGF2BP1 is a key participant in chicken muscle development. Subsequently, we employed QRT-PCR, EdU staining, and flow cytometry to further investigate the role of IGF2BP1 in the proliferation and differentiation of chicken myogenic cells. We identified 1143 differentially expressed genes, among which IGF2BP1 is intimately related to the muscle development process and is highly expressed in muscle tissues. Overexpression of IGF2BP1 significantly promotes the proliferation and differentiation of chicken primary myoblasts, while knockdown of IGF2BP1 significantly inhibits these processes. In summary, these results provide valuable preliminary insights into the regulatory roles of IGF2BP1 in chicken growth and development.

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.

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

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.

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.

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.

Effect of Glycyrrhiza uralensis crude water extract on the expression of Nitric Oxide Synthase 2 gene during myogenesis

Glycyrrhiza uralensis is a traditional herbal medicine with significant bioactivity. This study investigated the effect of G. uralensis crude water extract (GU-CWE) on nitric oxide synthase 2 (NOS2) expression during myogenesis. GU-CWE treatment increased myoblast differentiation by downregulating NOS2 and upregulating myogenic regulatory factors (MYOD, MYOG, and MYH). Notably, this effect was supported by an observed decrease in NOS2 expression in the gastrocnemius tissues of mice treated with GU-CWE. In addition, GU-CWE treatment and NOS2 knockdown were associated with reductions in reactive oxygen species levels. We further elucidate the role of the NOS2 gene in myoblast differentiation, demonstrating that its role was expression dependent, being beneficial at low expression but detrimental at high expression. High NOS2 gene expression induced oxidative stress, whereas its low expression impaired myotube formation. These findings highlight that the modulation of NOS2 expression by G. uralensis can potentially be use for managing muscle wasting disorders.

Effects of Melilotus Albus Extract on Myoblast Differentiation and Muscle Atrophy in a Type 2 Diabetes Model

Effects of Melilotus Albus Extract on Myoblast Differentiation and Muscle Atrophy in a Type 2 Diabetes Model