Filbertone Reduces Senescence in C2C12 Myotubes Treated with Doxorubicin or H2O2 through MuRF1 and Myogenin.

It has been demonstrated that filbertone, the principal flavor compound of hazelnuts, exhibits preventive effects against hypothalamic inflammation, obesity, neurodegenerative diseases, and muscle lipid accumulation. However, its influence on muscle aging has yet to be elucidated. The objective of this study was to investigate the effects of filbertone on muscle aging in C2C12 myotubes subjected to senescence induction by either doxorubicin or hydrogen peroxide. To ascertain the mechanisms by which filbertone exerts its effects, we conducted a series of experiments, including Western blot analysis, reverse transcription quantitative polymerase chain reaction (qRT-PCR), and senescence-associated ß-galactosidase (SA-ß-gal) staining. Filbertone was markedly observed to decrease not only the protein levels of p53 (p < 0.01) in senescence-induced skeletal muscle cells, but also the gene expression levels of p21 (p < 0.05), a direct target of p53. The expression of muscle-related genes, including myogenin and muscle RING-finger protein-1 (MuRF1), was found to be significantly enhanced in senescent muscle cells following treatment with filbertone (p < 0.05). In addition, the number of senescent skeletal muscle cells exhibiting ß-galactosidase activity was found to be markedly reduced in the presence of filbertone (p < 0.01). Collectively, these findings suggest that filbertone plays a pivotal role in the regulation of muscle aging.

Evaluation of muscular apoptotic changes and myogenin gene expression in experimental trichinosis after stem cells and atorvastatin added to ivermectin treatment.

Trichinosis is a common parasitic disease that affects the striated skeletal muscles, causing apoptotic and degenerative changes associated with myogenin expression in the affected myocytes. Hence, this study aimed to assess the ameliorative effects of stem cells and atorvastatin added to ivermectin on the infected myocytes during the muscular phase of murine trichinosis. 120 laboratory Swiss albino male mice were divided into 10 groups, and each group was subdivided into intestinal and muscular phases (each n = 6); uninfected control; untreated infected control; infected received ivermectin monotherapy; infected received atorvastatin monotherapy; infected received stem cells monotherapy; infected received ivermectin and atorvastatin dual therapy; infected received ivermectin and stem cells dual therapy; infected received atorvastatin and stem cells dual therapy; infected received ivermectin 0.2, atorvastatin 40, and stem cells triple therapy; and infected received ivermectin 0.1, atorvastatin 20, and stem cells triple therapy. Intestinal phase mice were sacrificed on the 5th day post-infection, while those of the muscular phase were sacrificed on the 35th day post-infection. Parasitological, histopathological, ultrastructural, histochemical, biochemical, and myogenin gene expression assessments were performed. The results revealed that mice that received ivermectin, atorvastatin, and stem cell triple therapies showed the maximum reduction in the adult worm and larvae burden, marked improvement in the underlying muscular degenerative changes (as was noticed by histopathological, ultrastructural, and histochemical Feulgen stain assessment), lower biochemical levels of serum NK-κB and tissue NO, and lower myogenin expression. Accordingly, the combination of stem cells, atorvastatin, and ivermectin affords a potential synergistic activity against trichinosis with considerable healing of the underlying degenerative sequel.

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

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

A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.

Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.

Muscle regeneration therapy using dedifferentiated fat cell (DFAT) for anal sphincter dysfunction.

PURPOSE: We investigated the effects of mouse-derived DFAT on the myogenic differentiation of a mouse-derived myoblast cell line (C2C12) and examined the therapeutic effects of rat-derived DFAT on anal sphincter injury using a rat model. METHODS: C2C12 cells were cultured using DMEM and DFAT-conditioned medium (DFAT-CM), evaluating MyoD and Myogenin gene expression via RT-PCR. DFAT was locally administered to model rats with anorectal sphincter dysfunction 3 days post-CTX injection. Therapeutic effects were assessed through functional assessment, including anal pressure measurement using solid-state manometry pre/post-CTX, and on days 1, 3, 7, 10, 14, 17, and 21 post-DFAT administration. Histological evaluation involved anal canal excision on days 1, 3, 7, 14, and 21 after CTX administration, followed by hematoxylin-eosin staining. RESULTS: C2C12 cells cultured with DFAT-CM exhibited increased MyoD and Myogenin gene expression compared to control. Anal pressure measurements revealed early recovery of resting pressure in the DFAT-treated group. Histologically, DFAT-treated rats demonstrated an increase in mature muscle cells within newly formed muscle fibers on days 14 and 21 after CTX administration, indicating enhanced muscle tissue repair. CONCLUSION: DFAT demonstrated the potential to enhance histological and functional muscle tissue repair. These findings propose DFAT as a novel therapeutic approach for anorectal sphincter dysfunction treatment.

The role of ZEB1 in mediating the protective effects of metformin on skeletal muscle atrophy.

Metformin is an important antidiabetic drug that has the potential to reduce skeletal muscle atrophy and promote the differentiation of muscle cells. However, the exact molecular mechanism underlying these functions remains unclear. Previous studies revealed that the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), which participates in tumor progression, inhibits muscle atrophy. Therefore, we hypothesized that the protective effect of metformin might be related to ZEB1. We investigated the positive effect of metformin on IL-1ß-induced skeletal muscle atrophy by regulating ZEB1 in vitro and in vivo. Compared with the normal cell differentiation group, the metformin-treated group presented increased myotube diameters and reduced expression levels of atrophy-marker proteins. Moreover, muscle cell differentiation was hindered, when we artificially interfered with ZEB1 expression in mouse skeletal myoblast (C2C12) cells via ZEB1-specific small interfering RNA (si-ZEB1). In response to inflammatory stimulation, metformin treatment increased the expression levels of ZEB1 and three differentiation proteins, MHC, MyoD, and myogenin, whereas si-ZEB1 partially counteracted these effects. Moreover, marked atrophy was induced in a mouse model via the administration of lipopolysaccharide (LPS) to the skeletal muscles of the lower limbs. Over a 4-week period of intragastric administration, metformin treatment ameliorated muscle atrophy and increased the expression levels of ZEB1. Metformin treatment partially alleviated muscle atrophy and stimulated differentiation. Overall, our findings may provide a better understanding of the mechanism underlying the effects of metformin treatment on skeletal muscle atrophy and suggest the potential of metformin as a therapeutic drug.

Photobiomodulation mitigates Bothrops jararacussu venom-induced damage in myoblast cells by enhancing myogenic factors and reducing cytokine production.

BACKGROUND: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: C2C12 myoblast cells were exposed to BjsuV (12.5 µg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кß, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-ß, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-ß, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. CONCLUSION/SIGNIFICANCE: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

Comparing the efficacy of concomitant treatment of resistance exercise and creatine monohydrate versus multiple individual therapies in age related sarcopenia.

Aging-related sarcopenia is a degenerative loss of strength and skeletal muscle mass that impairs quality of life. Evaluating NUDT3 gene and myogenin expression as new diagnostic tools in sarcopenia. Also, comparing the concomitant treatment of resistance exercise (EX) and creatine monohydrate (CrM) versus single therapy by EX, coenzyme Q10 (CoQ10), and CrM using aged rats. Sixty male rats were equally divided into groups. The control group, aging group, EX-treated group, the CoQ10 group were administered (500 mg/kg) of CoQ10, the CrM group supplied (0.3 mg/kg of CrM), and a group of CrM concomitant with resistance exercise. Serum lipid profiles, certain antioxidant markers, electromyography (EMG), nudix hydrolase 3 (NUDT3) expression, creatine kinase (CK), and sarcopenic index markers were measured after 12 weeks. The gastrocnemius muscle was stained with hematoxylin-eosin (H&E) and myogenin. The EX-CrM combination showed significant improvement in serum lipid profile, antioxidant markers, EMG, NUDT3 gene, myogenin expression, CK, and sarcopenic index markers from other groups. The NUDT3 gene and myogenin expression have proven efficient as diagnostic tools for sarcopenia. Concomitant treatment of CrM and EX is preferable to individual therapy because it reduces inflammation, improves the lipid serum profile, promotes muscle regeneration, and thus has the potential to improve sarcopenia.

Leu promotes C2C12 cell differentiation by regulating the GSK3ß/ß-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

BACKGROUND: Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. RESULTS: Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3ß and increased nuclear ß-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3ß and decreased nuclear ß-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear ß-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. CONCLUSION: Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3ß/ß-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

Myogenin Regulates DUSP13 to Inhibit Apoptosis Induced by Reactive Oxygen Species.

BACKGROUND: Myogenin is well known as a crucial transcription factor in skeletal muscle development, yet its other biological functions remain unexplored. Previous research showed that myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes, and offered a new perspective on myogenin's role in cardioprotection. However, the detailed mechanism of this cardioprotection, especially under oxidative stress, is still unclear. METHODS: In this study, hydrogen peroxide (H2O2) was used to generate reactive oxygen species in myogenin-overexpressing cardiomyocytes. The apoptosis was examined by flow cytometry. Transcriptome sequencing (RNA-seq) was performed to identify genes regulated by myogenin. Western blotting was used to detect the protein level of DUSP13 and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). The dual-luciferase reporter assay and ChIP assay were used to confirm the binding of myogenin to the promoter region of DUSP13. DUSP13 overexpression and knockdown assays were performed to study its anti-apoptotic role. RESULTS: Flow cytometry analysis of apoptosis showed that overexpressing myogenin for 24 and 48 hours decreased the apoptotic ratio by 47.9% and 63.5%, respectively, compared with untreated controls. Transcriptome sequencing performed on cardiomyocytes that expressed myogenin for different amounts of time (6, 12, 24, and 48 hours) identified DUSP13 as being up-regulated by myogenin. Western blotting showed that overexpression of myogenin increased the expression of DUSP13 and decreased the phosphorylation level of p38 MAPK. A dual-luciferase reporter assay proved that myogenin bound directly to the promoter region of DUSP13 and led to strong relative luciferase activity. Direct expression of DUSP13A and DUSP13B significantly reduced the rates of apoptosis and necrosis in cells treated with H2O2. Knockdown of DUSP13B significantly increased the rate of apoptosis in cells treated with H2O2. CONCLUSIONS: The present findings suggest that myogenin might attenuate apoptosis induced by reactive oxygen species by up-regulating DUSP13 and inactivating the p38 MAPK pathway.

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation.

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

Myosin heavy chain-perinatal regulates skeletal muscle differentiation, oxidative phenotype and regeneration.

Myosin heavy chain-perinatal (MyHC-perinatal) is one of two development-specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC-perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus-pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC-perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC-perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro. Proteomic analysis revealed that loss of MyHC-perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC-perinatal function on myogenic differentiation, possibly mediated by non-apoptotic caspase-3 signaling along with enhanced levels of the pro-survival apoptosis regulator Bcl2 and nuclear factor kappa-B (NF-κB). Knockdown of MyHC-perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC-perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.

Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.

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.

DNA-PKcs regulates myogenesis in an Akt-dependent manner independent of induced DNA damage.

Skeletal muscle regeneration relies on muscle stem (satellite) cells. We previously demonstrated that satellite cells efficiently and accurately repair radiation-induced DNA double-strand breaks (DSBs) via the DNA-dependent kinase DNA-PKcs. We show here that DNA-PKcs affects myogenesis independently of its role in DSB repair. Consequently, this process does not require the accumulation of DSBs and it is also independent of caspase-induced DNA damage. We report that in myogenic cells DNA-PKcs is essential for the expression of the differentiation factor Myogenin in an Akt2-dependent manner. DNA-PKcs interacts with the p300-containing complex that activates Myogenin transcription. We show also that SCID mice that are deficient in DNA-PKcs, and are used for transplantation and muscle regeneration studies, display altered myofiber composition and delayed myogenesis upon injury. These defects are exacerbated after repeated injury/regeneration events resulting in reduced muscle size. We thus identify a novel, caspase-independent, regulation of myogenic differentiation, and define a differentiation phase that does not involve the DNA damage/repair process.

A dysfunctional miR-1-TRPS1-MYOG axis drives ERMS by suppressing terminal myogenic differentiation.

Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma in vitro as well as in vivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.

SPSB1-mediated inhibition of TGF-ß receptor-II impairs myogenesis in inflammation.

BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/ß-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

LncRNA TCONS_00323213 Promotes Myogenic Differentiation by Interacting with PKNOX2 to Upregulate MyoG in Porcine Satellite Cells.

Myogenic differentiation is a complex biological process that is regulated by multiple factors, among which long noncoding RNAs (lncRNAs) play an essential role. However, in-depth studies on the regulatory mechanisms of long noncoding RNAs (lncRNAs) in myogenic differentiation are limited. In this study, we characterized the role of the novel lncRNA TCONS_00323213, which is upregulated during porcine skeletal muscle satellite cell (PSC) differentiation in myogenesis. We found that TCONS_00323213 affected the proliferation and differentiation of PSC in vitro. We performed quantitative polymerase chain reaction (qPCR), 5-ethynyl-20-deoxyuridine (EdU), western blotting, immunofluorescence staining, pull-down assays, and cleavage under targets and tagmentation (CUT and Tag) assays to clarify the effects and action mechanisms of TCONS_00323213. LncRNA TCONS_00323213 inhibited myoblast proliferation based on analyses of cell survival rates during PSC proliferation. Functional analyses revealed that TCONS_00323213 promotes cell differentiation and enhances myogenin (MyoG), myosin heavy chain (MyHC), and myocyte enhancer factor 2 (MEF2C) during myoblast differentiation. As determined by pull-down and RNA immunoprecipitation (RIP) assays, the lncRNA TCONS_00323213 interacted with PBX/Knotted Homeobox 2 (PKNOX2). CUT and Tag assays showed that PKNOX2 was significantly enriched on the MyoG promoter after lncRNA TCONS_00323213 knockdown. Our findings demonstrate that the interaction between lncRNA TCONS_00323213 and PKNOX2 relieves the inhibitory effect of PKNOX2 on the MyoG promoter, increases its expression, and promotes PSC differentiation. This novel role of lncRNA TCONS_00323213 sheds light on the molecular mechanisms by which lncRNAs regulate porcine myogenesis.