Skeletal Muscle Myoblasts Research Articles

Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism.

The gut microbiome supplies essential metabolites such as short-chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota-derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN-M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose- and insulin-treated myotubes. One of the most effective was isovanillic acid 3 -O-sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3 -O-glucoside, a major compound in berry fruits. IVAS stimulated a dose-dependent increase in glucose transport through glucose transporter GLUT4- and PI3K-dependent mechanisms. IVAS also up-regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.-Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism.

Overexpression of the Rybp Gene Inhibits Differentiation of Bovine Myoblasts into Myotubes.

RING1 and YY1 binding protein (Rybp) genes inhibit myogenesis in mice, but there are no reports on the effects of these genes in cattle. The aim of this study is to investigate the roles of the Rybp gene on bovine skeletal muscle development and myoblast differentiation. In the present study, the Rybp gene was overexpressed in bovine myoblasts via adenovirus. RNA-seq was performed to screen differentially expressed genes (DEGs). The results showed that overexpressing the Rybp gene inhibits the formation of myotubes. The morphological differences in myoblasts began on the second day and were very significant 6 days after adenovirus induction. A total of 1311 (707 upregulated and 604 downregulated) DEGs were screened using RNA-seq between myoblasts with added negative control adenoviruses (AD-NC) and Rybp adenoviruses (AD-Rybp) after 6 days of induction. Gene ontology (GO) and KEGG analysis revealed that the downregulated DEGs were mainly involved in biological functions related to muscle, and, of the 32 pathways, those associated with muscle development were significantly enriched for the identified DEGs. This study can not only provide a theoretical basis for the regulation of skeletal muscle development in cattle by exploring the roles of the Rybp gene in myoblast differentiation, but it can also lay a theoretical foundation for molecular breeding of beef cattle.

Novel ultrashort self-assembling peptide bioinks for 3D culture of muscle myoblast cells.

The ability of skeletal muscle to self-repair after a traumatic injury, tumor ablation, or muscular disease is slow and limited, and the capacity of skeletal muscle to self-regenerate declines steeply with age. Tissue engineering of functional skeletal muscle using 3D bioprinting technology is promising for creating tissue constructs that repair and promote regeneration of damaged tissue. Hydrogel scaffolds used as biomaterials for skeletal muscle tissue engineering can provide chemical, physical and mechanical cues to the cells in three dimensions thus promoting regeneration. Herein, we have developed two synthetically designed novel tetramer peptide biomaterials. These peptides are self-assembling into a nanofibrous 3D network, entrapping 99.9% water and mimicking the native collagen of an extracellular matrix. Different biocompatibility assays including MTT, 3D cell viability assay, cytotoxicity assay and live-dead assay confirm the biocompatibility of these peptide hydrogels for mouse myoblast cells (C2C12). Immunofluorescence analysis of cell-laden hydrogels revealed that the proliferation of C2C12 cells was well-aligned in the peptide hydrogels compared to the alginategelatin control. These results indicate that these peptide hydrogels are suitable for skeletal muscle tissue engineering. Finally, we tested the printability of the peptide bioinks using a commercially available 3D bioprinter. The ability to print these hydrogels will enable future development of 3D bioprinted scaffolds containing skeletal muscle myoblasts for tissue engineering applications.

Novel ultrashort self-assembling peptide bioinks for 3D culture of muscle myoblast cells

The ability of skeletal muscle to self-repair after a traumatic injury, tumor ablation, or muscular disease is slow and limited, and the capacity of skeletal muscle to self-regenerate declines steeply with age. Tissue engineering of functional skeletal muscle using 3D bioprinting technology is promising for creating tissue constructs that repair and promote regeneration of damaged tissue. Hydrogel scaffolds used as biomaterials for skeletal muscle tissue engineering can provide chemical, physical and mechanical cues to the cells in three dimensions thus promoting regeneration. Herein, we have developed two synthetically designed novel tetramer peptide biomaterials. These peptides are self-assembling into a nanofibrous 3D network, entrapping 99.9% water and mimicking the native collagen of an extracellular matrix. Different biocompatibility assays including MTT, 3D cell viability assay, cytotoxicity assay and live-dead assay confirm the biocompatibility of these peptide hydrogels for mouse myoblast cells (C2C12). Immunofluorescence analysis of cell-laden hydrogels revealed that the proliferation of C2C12 cells was well-aligned in the peptide hydrogels compared to the alginategelatin control. These results indicate that these peptide hydrogels are suitable for skeletal muscle tissue engineering. Finally, we tested the printability of the peptide bioinks using a commercially available 3D bioprinter. The ability to print these hydrogels will enable future development of 3D bioprinted scaffolds containing skeletal muscle myoblasts for tissue engineering applications.

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to β-adrenergic stimulation.

Nanodrug delivery platform for glucocorticoid use in skeletal muscle injury.

Glucocorticoids are utilized for their anti-inflammatory properties in the skeletal muscle and arthritis. However, the major drawback of use of glucocorticoids is that it leads to senescence and toxicity. Therefore, based on the idea that decreasing particle size allows for increased surface area and bioavailability of the drug, in the present study, we hypothesized that nanodelivery of dexamethasone will offer increased efficacy and decreased toxicity. The dexamethasone-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared using nanoprecipitation method. The morphological characteristics of the nanoparticles were studied under scanning electron microscope. The particle size of nanoparticles was 217.5 ± 19.99 nm with polydispersity index of 0.14 ± 0.07. The nanoparticles encapsulation efficiency was 34.57% ± 1.99% with in vitro drug release profile exhibiting a sustained release pattern over 10 days. We identified improved skeletal muscle myoblast performance with improved closure of the wound along with increased cell viability at 10 nmol/L nano-dexamethasone-PLGA. However, dexamethasone solution (1 μmol/L) was injurious to cells because the migration efficiency was decreased. In addition, the use of dexamethasone nanoparticles decreased lipopolysaccharide-induced lactate dehydrogenase release compared with dexamethasone solution. Taken together, the present study clearly demonstrates that delivery of PLGA-dexamethasone nanoparticles to the skeletal muscle cells is beneficial for treating inflammation and skeletal muscle function.

The Effect of Creatine and Creatinine on Myocellular Injury in Doxorubicin-Treated Skeletal Muscle Myoblasts

Doxorubicin (DOX) is a powerful chemotherapy agent that is associated with a number of deleterious side effects including skeletal muscle dysfunction and atrophy. Although the exact mechanisms behind the observed myotoxicity are not fully understood, DOX treatment has been shown to result in the generation of reactive oxygen species and changes in short-term energy metabolism. Conversely, creatine (Cr) supplementation has been shown to have a therapeutic role in several disease states characterized by muscle atrophy, which is a hallmark of DOX treatment. PURPOSE: To examine the ability of Cr and CrN to attenuate the decline in Cr metabolism and minimize DOX-induced apoptosis and necrosis in skeletal muscle myoblasts. METHODS: Rat skeletal muscle myoblasts were cultured until they reached 85-90% confluency using rat skeletal muscle growth media (GM). Cells were subcultured and treated with one of the following for 12 hours: normal GM (control); Cr (10mM Cr+GM); CrN (10mM CrN+GM); DOX (25μM DOX+GM); DOX+Cr+GM; and DOX+CrN+GM. After incubation, protein analysis was performed using western blotting and rates of apoptosis and necrosis were assessed using an Annexin V apoptosis detection kit and high contrast staining. A one-way ANOVA with Tukey’s post-hoc testing was used to detect significance. RESULTS: There was a significant change relative to GAPDH in creatine kinase (CK) expression between the control and DOX-treated cells (15±18.2% vs. 90±8.7%, p=0.03). In addition,13.2±7.5% of DOX-treated cells were undergoing apoptosis, which was significantly higher than the 3.26±5.5% in the control cells (p=0.04). No significant differences in rates of apoptosis were found between control samples and cells treated with DOX+Cr or DOX+CrN. CONCLUSION: These findings suggest that CK expression is significantly altered in skeletal muscle myoblasts treated with DOX relative to control cells and that DOX treatment results in higher rates of necrosis and apoptosis. Treatment with Cr or CrN minimized the DOX-induced change in CK expression, as well as the rate of apoptosis and necrosis. These findings suggest that Cr and CrN may attenuate the degree of skeletal muscle dysfunction and atrophy during chemotherapy with DOX.

250 - Effect of fibroblast population on vegf secretion in human skeletal muscle myoblast sheet

Human skeletal muscle myoblast (HSMM) sheet transplantation is a novel and promising approach to restores the function of a damaged tissue for myocardial infarction treatment. Since the HSMM sheet has ability to secrete multiple cytokines that improve heart function through the facilitation of angiogenesis, here we are focusing on understanding of cytokines regulation in the HSMM sheet, which is necessary for development of more functional and complex engineered tissue.

Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast.

Myocyte enhancer factor 2A (MEF2A) is widely distributed in various tissues or organs and plays crucial roles in multiple biological processes. To examine the potential effects of MEF2A on skeletal muscle myoblast, the functional role of MFE2A in myoblast proliferation and differentiation was investigated. In this study, we found that the mRNA expression level of Mef2a was dramatically increased during the myogenesis of bovine skeletal muscle primary myoblast. Overexpression of MEF2A significantly promoted myoblast proliferation, while knockdown of MEF2A inhibited the proliferation and differentiation of myoblast. RT-PCR and western blot analysis revealed that this positive effect of MEF2A on the proliferation of myoblast was carried out by triggering cell cycle progression by activating CDK2 protein expression. Besides, MEF2A was found to be an important transcription factor that bound to the myozenin 2 (MyoZ2) proximal promoter and performed upstream of MyoZ2 during myoblast differentiation. This study provides the first experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that MEF2A regulates myoblast differentiation via regulating MyoZ2.

The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myotubes.

Skeletal muscle is an insulin sensitive tissue and accounts for approximately 80% of post-prandial glucose disposal. This study describes the effects of insulin, delivered for 72 h, to skeletal muscle myoblasts during differentiation or to skeletal muscle myotubes. After chronic treatment, cultures were acutely stimulated with insulin and analyzed for total and phosphorylated Akt (Ser473 ), mRNA expression of metabolic and myogenic markers and insulin-stimulated glucose uptake. Skeletal muscle cells differentiated in the presence of insulin chronically, reduced acute insulin stimulated phosphorylation of Akt Ser473 . In addition, there was a reduction in mRNA expression of Hexokinase II (HKII), GLUT4 and PGC-1α. Insulin-stimulated glucose uptake was attenuated when cells were differentiated in the presence of insulin. In contrast, myotubes exposed to chronic insulin showed no alterations in phosphorylation of Akt Ser473 . Both HKII and GLUT4 mRNA expression were reduced by chronic exposure to insulin; while PGC-1α was not different between culture conditions and was increased by acute insulin stimulation. These data suggest that there are differential responses in insulin signalling, transcription, and glucose uptake of skeletal muscle cells when cultured in either the presence of insulin during differentiation or in myotube cultures.

Hsp90\u03b2 knockdown in DIO mice reverses insulin resistance and improves glucose tolerance

BackgroundInhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism.MethodsIn a diet-induced obese (DIO) mouse model of diabetes, expression of various Hsp90 isoforms in skeletal tissue was examined. Subsequent experiments characterized the role of Hsp90ab1 isoform in glucose metabolism and insulin signaling in primary human skeletal muscle myoblasts (HSMM) and a DIO mouse model.ResultsIn DIO mice Hsp90ab1 mRNA was upregulated in skeletal muscle compared to lean mice and knockdown using anti-sense oligonucleotide (ASO) resulted in reduced expression in skeletal muscle that was associated with improved glucose tolerance, reduced fed glucose and fed insulin levels compared to DIO mice that were treated with a negative control oligonucleotide. In addition, knockdown of HSP90ab1 in DIO mice was associated with reduced pyruvate dehydrogenase kinase-4 mRNA and phosphorylation of the muscle pyruvate dehydrogenase complex (at serine 232, 293 and 300), but increased phosphofructokinase 1, glycogen synthase 1 and long-chain specific acyl-CoA dehydrogenase mRNA. In HSMM, siRNA knockdown of Hsp90ab1 induced an increase in substrate metabolism, mitochondrial respiration capacity, and insulin sensitivity, providing further evidence for the role of Hsp90ab1 in metabolism.ConclusionsThe data support a novel role for Hsp90ab1 in arbitrating skeletal muscle plasticity via modulation of substrate utilization including glucose and fatty acids in normal and disease conditions. Hsp90ab1 represents a novel target for potential treatment of metabolic disease including diabetes.

1α,25-dihydroxyvitamin D3 mitigates cancer cell mediated mitochondrial dysfunction in human skeletal muscle cells

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which has previously been shown to increase skeletal myoblast oxygen consumption rate, could reverse the deleterious effects of tumor cell conditioned medium on myoblast function. Conditioned medium from Lewis lung carcinoma (LLC1) cells inhibits oxygen consumption, increases mitochondrial fragmentation, inhibits pyruvate dehydrogenase activity, and enhances proteasomal activity in human skeletal muscle myoblasts. 1α,25(OH)2D3 reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)2D3 might be useful in treatment of weakness seen in association with CC.

GH directly stimulates UCP3 expression

ObjectiveWe evaluated the direct action of GH signaling in energy homeostasis in myocytes. DesignWe investigated the GH-induced expression of UCP3 in human embryonic kidney 293 cells, human H-EMC-SS chondrosarcoma cells, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal muscle cells, as well as its direct effect on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. ResultsWe demonstrated that the regulation of energy metabolism by GH involves UCP3 via activated STAT5, a signal transducer downstream of GH. UCP3 expression increased with STAT5 in a dose-dependent manner and was higher than that of UCP2. We confirmed the functional STAT5 binding site consensus sequences at −861 and −507 bp in the UCP3 promoter region. ConclusionThe results suggest that GH stimulates UCP3 directly and that UCP2 and that UCP3 participate in the signal transduction pathway that functions downstream of the GHR/JAK/STAT.

Coffee consumption promotes skeletal muscle hypertrophy and myoblast differentiation.

Coffee is a widely consumed beverage worldwide and is believed to help prevent the occurrence of various chronic diseases. However, the effect of coffee on skeletal muscle hypertrophy, differentiation and the mechanisms of action responsible have remained unclear. To investigate the effect of coffee on skeletal muscle hypertrophy, mice were fed a normal diet or a normal diet supplemented with 0.3% coffee or 1% coffee. Coffee supplementation was observed to increase skeletal muscle hypertrophy, while simultaneously upregulating protein expression of total MHC, MHC2A, and MHC2B in quadricep muscle. Myostatin expression was also attenuated, and IGF1 was upregulated with subsequent phosphorylation of Akt and mTOR, while AMPK phosphorylation was attenuated. Coffee also increased the grip strength and PGC-1α protein expression, and decreased the expressions of TGF-β and myostatin in tricep muscle. Coffee activated the MKK3/6-p38 pathway and upregulated PGC-1α, which may play a role in promoting myogenic differentiation and myogenin expression in C2C12 cells. These results suggest that coffee increases skeletal muscle function and hypertrophy by regulating the TGF-β/myostatin - Akt - mTORC1.

Silencing of gelatinase expression delays myoblast differentiation in vitro.

Skeletal muscle growth and regeneration relies on the activation of muscle specific stem cells, that is, satellite cells. The activation and differentiation of satellite cells into myoblasts, as well as their migration, proliferation, and fusion of mononuclear myoblasts into a functional multi-nucleated muscle fiber, are associated with extracellular matrix (ECM) protein synthesis and degradation. The extracellular environment is dynamically adapting to the changes accompanying skeletal muscle growth or repair. Enzymes engaged in many biological processes that involve ECM remodeling are matrix metalloproteinases (MMPs). Among metalloproteinases crucial for skeletal muscles are two gelatinases-MMP-9 and MMP-2. In the current study we test the effect of silencing the MMP-9 and MMP-2 expression on the proliferation and differentiation of in vitro cultured skeletal muscle myoblasts. We show that downregulating gelatinase MMP-9 expression results in a delayed myoblast differentiation.

53 - Lipid peroxidation impairs proliferation and differentiation of skeletal muscle myoblasts in vitro

Background Oxidative stress can lead to loss of muscle mass and function, which contributes to age-mediated skeletal muscle complications such as sarcopenia, impaired satellite cell regeneration, and reduced energy production. Here, we hypothesized that lipid peroxidation, a key determinant of oxidative stress, has distinct roles on survival, differentiation and regeneration of myoblasts. Methods C2C12 Myoblasts were cultured in normal growth medium and differentiation was induced by exposing the cells to low nutrient media supplemented with 2% horse serum for 5 days. Cells were treated with varying doses of malondialdehyde (MDA) (0.25 to 100µM) and cell survival was analyzed using an MTT assay. We selected 1 and 10µM of MDA to examine myoblast proliferation and differentiation using bright field microscopy and immunoblotting. Lastly, a scratch assay was performed to visualize MDA mediated effects on regeneration. All experiments were performed in replicates (n=3-4/group). Results 12.5µM of MDA significantly (p 50%) at 24 and 48hr indicating a pathological relevance of oxidative stress in myoblasts. Although proliferation was unchanged in MDA treated cells at 24hr, both 1 and 10µM MDA were sufficient to impair proliferation at 48hr. Despite similar effects on proliferation, 1 and 10µM MDA induced distinct changes in myotube differentiation. While similar myotube structure was seen at day 1, 10µM MDA delayed differentiation progress at day 2 and appeared to produce a dedifferentiated phenotype at day 3. Cell morphology in the 10µM condition was unique from 1µM as ~50% differentiation occurred in these cells. These results indicate that a pro-oxidative (1µM of MDA) environment might be beneficial to the differentiating cells, but the severe oxidative condition (10µM of MDA) is detrimental and compromises the differentiation process. Indeed, immunoblot analysis for Myogenin, a differentiation marker, was undetectable in 10µM MDA treated cells on day 3, confirming that severe lipid peroxidation inhibits the myotube formation. Moreover, the scratch assay indicated that wound closure in MDA treated cells was significantly (p Conclusion Our results reveal the unique effects of lipid peroxidation on myoblast proliferation and differentiation. In particular, we postulate that a pro-oxidative environment is crucial for the myoblast differentiation process.

Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population.

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

Injectable Anisotropic Nanocomposite Hydrogels Direct in Situ Growth and Alignment of Myotubes.

While injectable in situ cross-linking hydrogels have attracted increasing attention as minimally invasive tissue scaffolds and controlled delivery systems, their inherently disorganized and isotropic network structure limits their utility in engineering oriented biological tissues. Traditional methods to prepare anisotropic hydrogels are not easily translatable to injectable systems given the need for external equipment to direct anisotropic gel fabrication and/or the required use of temperatures or solvents incompatible with biological systems. Herein, we report a new class of injectable nanocomposite hydrogels based on hydrazone cross-linked poly(oligoethylene glycol methacrylate) and magnetically aligned cellulose nanocrystals (CNCs) capable of encapsulating skeletal muscle myoblasts and promoting their differentiation into highly oriented myotubes in situ. CNC alignment occurs on the same time scale as network gelation and remains fixed after the removal of the magnetic field, enabling concurrent CNC orientation and hydrogel injection. The aligned hydrogels show mechanical and swelling profiles that can be rationally modulated by the degree of CNC alignment and can direct myotube alignment both in two- and three-dimensions following coinjection of the myoblasts with the gel precursor components. As such, these hydrogels represent a critical advancement in anisotropic biomimetic scaffolds that can be generated noninvasively in vivo following simple injection.

Apigenin enhances skeletal muscle hypertrophy and myoblast differentiation by regulating Prmt7.

Apigenin, a natural flavone abundant in various plant-derived foods including parsley and celery, has been shown to prevent inflammation and inflammatory diseases. However, the effect of apigenin on skeletal muscle hypertrophy and myogenic differentiation has not previously been elucidated. Here, we investigated the effects of apigenin on quadricep muscle weight and running distance using C57BL/6 mice on an accelerating treadmill. Apigenin stimulated mRNA expression of MHC (myosin heavy chain) 1, MHC2A, and MHC2B in the quadricep muscles of these animals. GPR56 (G protein-coupled receptor 56) and its ligand collagen III were upregulated by apigenin supplementation, together with enhanced PGC-1α, PGC-1α1, PGC-1α4, IGF1, and IGF2 expression. Prmt7 protein expression increased in conjunction with Akt and mTORC1 activation. Apigenin treatment also upregulated FNDC5 (fibronectin type III domain containing 5) mRNA expression and serum irisin levels. Furthermore, apigenin stimulated C2C12 myogenic differentiation and upregulated total MHC, MHC2A, and MHC2B expression. These events were attributable to an increase in Prmt7-p38-myoD expression and Akt and S6K1 phosphorylation. We also observed that Prmt7 regulates both PGC-1α1 and PGC-1α4 expression, resulting in a subsequent increase in GPR56 expression and mTORC1 activation. Taken together, these findings suggest that apigenin supplementation can promote skeletal muscle hypertrophy and myogenic differentiation by regulating the Prmt7-PGC-1α-GPR56 pathway, as well as the Prmt7-p38-myoD pathway, which may contribute toward the prevention of skeletal muscle weakness.

Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts.

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP+ /DsRed+ hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products.