Proliferation Of C2C12 Myoblast Cells Research Articles

A standardized herbal combination of Astragalus membranaceus and Paeonia japonica promotes skeletal muscle hypertrophy in a treadmill exercise mouse model.

Maintaining a normal range of muscle mass and function is crucial not only for sustaining a healthy life but also for preventing various disorders. Numerous nutritional or natural resources are being explored for their potential muscle hypertrophic properties. We aimed to evaluate the muscle hypertrophic effects of APX, a 1:1 mixture of Astragalus membranaceus and Paeonia japonica. In addition to the myotube differentiation cell assay, we utilized a weighted exercise-based animal model and evaluated changes in muscle hypertrophy using dual-energy X-ray absorptiometry (DXA) and histological analysis. The 8-week treadmill exercise led to notable decreases in body weight and fat mass but an increase in muscle mass compared to the control group. Administration of APX significantly accelerated muscle mass gain (p < 0.05) without altering body weight or fat mass compared to the exercise-only group. This muscle hypertrophic effect of APX was consistent with the histologic size of muscle fibers in the gastrocnemius (p > 0.05) and rectus femoris (p < 0.05), as well as the regulation of myogenic transcription factors (MyoD and myogenin), respectively. Furthermore, APX demonstrated a similar action to insulin-like growth factor 1, influencing the proliferation of C2C12 myoblast cells (p < 0.01) and their differentiation into myotubes (p < 0.05) compared to the control group. The present study provides experimental evidence that APX has muscle hypertrophic effects, and its underlying mechanisms would involve the modulation of MyoD and myogenin.

Design of Hybrid Polymer Nanofiber/Collagen Patches Releasing IGF and HGF to Promote Cardiac Regeneration.

Cardiovascular diseases are the leading cause of death globally. Myocardial infarction in particular leads to a high rate of mortality, and in the case of survival, to a loss of myocardial functionality due to post-infarction necrosis. This functionality can be restored by cell therapy or biomaterial implantation, and the need for a rapid regeneration has led to the development of bioactive patches, in particular through the incorporation of growth factors (GF). In this work, we designed hybrid patches composed of polymer nanofibers loaded with HGF and IGF and associated with a collagen membrane. Among the different copolymers studied, the polymers and their porogens PLA-Pluronic-PLA + PEG and PCL + Pluronic were selected to encapsulate HGF and IGF. While 89 and 92% of IGF were released in 2 days, HGF was released up to 58% and 50% in 35 days from PLA-Pluronic-PLA + PEG and PCL + Pluronic nanofibers, respectively. We also compared two ways of association for the loaded nanofibers and the collagen membrane, namely a direct deposition of the nanofibers on a moisturized collagen membrane (wet association), or entrapment between collagen layers (sandwich association). The interfacial cohesion and the degradation properties of the patches were evaluated. We also show that the sandwich association decreases the burst release of HGF while increasing the release efficiency. Finally, we show that the patches are cytocompatible and that the presence of collagen and IGF promotes the proliferation of C2C12 myoblast cells for 11 days. Taken together, these results show that these hybrid patches are of interest for cardiac muscle regeneration.

Taurine stimulates protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway.

Taurine (Tau) has many profound physiological functions, but its role and molecular mechanism in muscle cells are still not fully understood. In this study, we investigated the role and underlying molecular mechanism of Tau on protein synthesis and proliferation of C2C12 myoblast cells. Cells were treated with Tau (0, 60, 120, 180 and 240 μM) for 24 h. Tau dose-dependently promoted protein synthesis, cell proliferation, mechanistic target of rapamycin protein (mTOR) phosphorylation and also AT-rich interaction domain 4B (ARID4B) expression, with the best stimulatory effects at 120 μM. LY 294002 treatment showed that Tau promoted ARID4B expression in a phosphoinositide 3-kinase (PI3K)-dependent manner. ARID4B knockdown (by small interfering RNA transfection for 24 h) prevented Tau from stimulating protein synthesis and cell proliferation, whereas ARID4B gene activation (using the CRISPR/dCas9 technology) had stimulatory effects. ARID4B knockdown abolished Tau signalling to mRNA expression and protein phosphorylation of mTOR, whereas ARID4B gene activation had stimulatory effects. Chromatin immunoprecipitation (ChIP)-PCR identified that all of ARID4B, H3K27ac and H3K27me3 bound to the -4368 to -4591 bp site in the mTOR promoter, and ChIP-quantitative PCR (qPCR) further detected that Tau stimulated ARID4B binding to this site. ARID4B knockdown or gene activation did not affect H3K27me3 binding to the mTOR promoter but decreased or increased H3K27ac binding, respectively. Furthermore, ARID4B knockdown abolished the stimulation of Tau on H3K27ac binding to the mTOR promoter. In summary, these data uncover that Tau promotes protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway, providing a deep understanding of how Tau regulates anabolism in muscle cells.

The structure and properties of MFG-E8 and the In vitro assessment of its toxic effects on myoblast cells

Four high-molecular-weight protein fractions of milk fat globule membrane (MFGM) were isolated from bovine milk. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF/TOF™ and Liquid chromatography electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) were used to measure the molecular sizes of the MFGM. Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) were performed to determine the conformations of the MFGM. The results showed that the main protein (98.33%) in MFGM protein fraction 2 was Milk fat globule epidermal growth factor-VIII (MFG-E8), with a molecular weight of 47.82 kDa. The secondary structural component measurements showed that the MFG-E8 consisted of 5% helix, 70% sheet and 25% random coil, and the results matched well with the prediction by SSPro 5.1 bioinformatic analysis. The thermograms analysis revealed that Td and△H of MFG-E8 were 60.50°Cand 132.29 kJ/mol. The in vitro digestibility of MFG-E8 showed that it can be enzymatically hydrolyzed in the stomach and relatively stable in the intestinal fluid. The in vitro C2C12 and Caco2 cell activity tests indicated that MFG-E8 promoted the proliferation of C2C12 myoblast cells without cytotoxicity. The biological functional properties of MFG-E8 may be related to the fact that MFG-E8 possesses a high level of β-sheet structure. Our results suggested that MFG-E8 possesses broad prospects not only for use in functional food products but also as a source of natural anti-sarcopenia drugs.

Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development

N6-methyladenosine (m6A) represents the most abundantly occurring mRNA modification and is involved in the regulation of skeletal muscle development. However, the status and function of m6A methylation in prenatal myogenesis remains unclear. In this study, we first demonstrated that knockdown of METTL14, an m6A methyltransferase, inhibited the differentiation and promoted the proliferation of C2C12 myoblast cells. Then, using a refined m6A-specific methylated RNA immunoprecipitation (RIP) with next generation sequencing (MeRIP-seq) method that is optimal for use with samples containing small amounts of RNA, we performed transcriptome-wide m6A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis. The results revealed that, along with a continuous decrease in the mRNA expression of the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), the m6A methylome underwent highly dynamic changes across different development stages, with most of the affected genes being enriched in pathways related to skeletal muscle development. RNA immunoprecipitation confirmed that IGF2BP1 targets 76 genes involved in pathways associated with muscle development, including the key marker genes MYH2 and MyoG. Moreover, small interfering RNA (siRNA)-mediated knockdown of IGF2BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14. In conclusion, we clarified the dynamics of m6A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.

Low Levels of Serum Tryptophan Underlie Skeletal Muscle Atrophy.

Sarcopenia is a poor prognosis factor in some cancer patients, but little is known about the mechanisms by which malignant tumors cause skeletal muscle atrophy. Tryptophan metabolism mediated by indoleamine 2,3-dioxygenase is one of the most important amino acid changes associated with cancer progression. Herein, we demonstrate the relationship between skeletal muscles and low levels of tryptophan. A positive correlation was observed between the volume of skeletal muscles and serum tryptophan levels in patients with diffuse large B-cell lymphoma. Low levels of tryptophan reduced C2C12 myoblast cell proliferation and differentiation. Fiber diameters in the tibialis anterior of C57BL/6 mice fed a tryptophan-deficient diet were smaller than those in mice fed a standard diet. Metabolomics analysis revealed that tryptophan-deficient diet downregulated glycolysis in the gastrocnemius and upregulated the concentrations of amino acids associated with the tricarboxylic acid cycle. The weights and muscle fiber diameters of mice fed the tryptophan-deficient diet recovered after switching to the standard diet. Our data showed a critical role for tryptophan in regulating skeletal muscle mass. Thus, the tryptophan metabolism pathway may be a promising target for preventing or treating skeletal muscle atrophies.

Effect of aqueous extract of ajwa dates on C2C12 myoblast cell proliferation, migration and differentiation

Chemotherapeutic agents generate side effects or cytotoxicity to other cells that include muscle cells. Doxorubicin, one of the mostly used chemotherapeutic agents, has been known to cause a loss of skeletal muscle mass which known as cachexia. On the other hand, chemopreventive agents, which mostly derived from phytochemicals, have been known to show less cytotoxity to cancer cells than chemotherapeutic agents. However, their effects on the muscle cells remain largely unknown. In this study, we investigated the effect of ajwa dates extract in C2C12 myoblast cells. C2C12 cells are satellite cells or adult muscle stem cells which can differentiate to from multinucleated myotube. MTT assays had been performed to determine the effect of ajwa dates extract on cell viability. In addition, effects on cell migration were examined by wound healing method. Proliferation assay was done to investigate the effect of ajwa dates extract to cell growth. Furthermore, C2C12 differentiation into myotubes was also investigated. The results of the cell viability assay against C2C12 cells showed that low concentration of ajwa dates was not toxic for C2C12 cells, whereas higher concentration of ajwa dates (10 µg/µl) decreased cell viability. In addition, the effect of ajwa dates extract (1 µg/µl) on cell migration depended on cell seeding density. At a lower cell density, the extract might promote myoblasts migration, whereas at a higher cell density it inhibited the cell migration that may be related to the induction of myocytes differentiation. Overall, we found that ajwa dates extract exhibited low or no cytotoxicity on C2C12 myoblast cells.

Binary polyhydroxyalkanoate systems for soft tissue engineering

Progress in tissue engineering is dependent on the availability of suitable biomaterials. In an effort to overcome the brittleness of poly(3-hydroxybutyrate), P(3HB), a natural biodegradable polyester, and widen its biomedical applications, plasticising of P(3HB) with oligomeric substances of related structure has been studied. A biosynthesised medium-chain-length polyhydroxyalkanoate (mcl-PHA) copolymer, the plasticiser precursor, was obtained using vegetable waste frying oil as a sole carbon source. The mcl-PHA was transformed into an oligomeric derivative by acid hydrolysis. The plasticising effect of the oligomeric mcl-PHA on P(3HB) was studied via characterisation of thermal and mechanical properties of the blends in the course of ageing at ambient conditions. Addition of oligomeric mcl-PHA to P(3HB) resulted in softer and more flexible materials based entirely on PHAs. It was shown that the oligomeric mcl-PHA transformed highly crystalline P(3HB) into materials with a dominant amorphous phase when the content of oligomeric mcl-PHA exceeded 10 wt%. In vitro biocompatibility studies of the new binary PHA materials showed high viability and proliferation of C2C12 myoblast cells. Thus, the proposed approach for P(3HB) plasticisation has the potential for the generation of more pliable biomaterials based on P(3HB) which can find application in unique soft tissue engineering applications where a balance between stiffness, tensile strength and ductility is required. Statement of SignificancePolyhydroxyalkanoates, a broad family of natural biodegradable and biocompatible polymers, have emerged as highly promising biomaterials both for bulk and biomedical applications. Here we describe an approach to tune the mechanical properties of stiff and brittle poly(3-hydroxybutyrate) and thereby to expand its potential biomedical applications. Plasticisation, a common practice in the plastic industry to modify polymer mechanical properties, has been used very cautiously for biomedical applications due to plasticiser toxicity and migration. We have developed a plasticiser for poly(3-hydroxybutyrate) based on a structurally related but softer and pliable medium chain length polyhydroxyalkanoate. Additives of oligomeric derivatives of this polymer improved ductility of poly(3-hydroxybutyrate), greatly widening the future applicability of this well-established biomaterial. In parallel, the binary polyhydroxyalkanoate materials also exhibited improved cell attachment and proliferation, a highly desirable outcome.

Sirtuin1promotes the proliferation of C2C12myoblast cells via the myostatin signaling pathway.

Accumulating evidence suggests that Sirtuin (Sirt)1 serves a significant role in proliferation and differentiation of myoblast cells; however the signaling mechanisms involved remain to be established. Myostatin (MSTN), a member of transforming growth factor‑β family, is an vital regulator of myoblast, fibroblast growth and differentiation. To determine if MSTN is involved in the regulation of myoblast cell proliferation by Sirt1, the present study administrated the Sirt1activator resveratrol, inhibitor nicotinamide (NAM) and MSTN inhibitor SB431542to C2C12myoblast cells. It was demonstrated that the Sirt1activator, resveratrol, repressed, whereas the Sirt1inhibitor, NAM, enhanced C2C12myoblast cells proliferation in a Sirt1‑dependent manner. SB431542promoted the proliferation of C2C12myoblast cells and reversed the inhibition effect of NAM on C2C12myoblast cell proliferation. Additionally, resveratrol upregulated the mRNA expression of MyoD, but inhibited the expression of MSTN. Additionally, NAM significantly repressed the expression of MyoD and the phosphorylation of P107 (p‑P107), but enhanced the expression of MSTN and the protein expression of P107. SB431542significantly mitigated the effect of NAM on the expression of MyoD, P107 and p‑P107. Taken together, these results indicated that Sirt1promotes the proliferation of C2C12myoblast cells via the MSTN signaling pathway.

Electroactive nanofibrous biomimetic scaffolds by thermally induced phase separation.

Fabrication of functional nanofibrous biomimetic scaffolds for tissue regeneration remains a challenge. This work demonstrates that functional nanofibrous scaffolds were fabricated from blends of polylactide with other functional polymers by a thermally induced phase separation (TIPS) technique, here exemplified by the fabrication of electroactive nanofibrous scaffolds from the blends of polylactide and an electroactive degradable tetraaniline-polylactide-tetraaniline (TPT) block copolymer by TIPS. The TPT copolymer was synthesized by coupling reaction between the carboxyl-capped tetraaniline and polylactide. The chemical structure, electroactivity, thermal properties and mechanical properties of TPT and polylactide/TPT blend films were characterized. The copolymer blends were fabricated into electroactive nanofibrous scaffolds by TIPS. The effect of aniline tetramer content, polymer concentration and phase separation temperature on the diameter of nanofibers was investigated. The adhesion and proliferation of C2C12 myoblast cells and protein adsorption on the electroactive biodegradable substrates were evaluated, and the results show that the electroactive materials are nontoxic and could enhance the C2C12 cell proliferation without electrical stimulation, and adsorbed more proteins compared to polylactide. The electrical stimulation on the electroactive substrates significantly increased the cell proliferation of C2C12 myoblasts. This work opens the way to fabricate functional nanofibrous scaffolds from the blends of polylactide and other functional polymers by TIPS.

Retardation of C2C12 myoblast cell proliferation by exposure to low-temperature atmospheric plasma.

As the first step in evaluating the possibility of low-temperature atmospheric plasma for clinical applications in the treatment of rhabdomyosarcoma (RMS), we determined the effects of plasma exposure on C2C12 myoblasts. The low-temperature atmospheric plasma was generated through an electrical discharge in argon gas. One minute of plasma exposure every 24h inhibited the cell proliferation, whereas myoblast differentiation was not affected. Plasma exposure increased the phosphorylation of ERK and JNK at 30min after the exposure, but the phosphorylation of both was decreased to less than control levels at 1 and 4h after the exposure. Plasma exposure increased the percentage of cells in the G2/M phase at 8h after the exposure. In conclusion, plasma exposure retarded the proliferation of C2C12 myoblasts by G2/M arrest. Therefore, plasma exposure can be a possible treatment for the anti-proliferative effects of malignant tumors, such as RMS, without affecting differentiated skeletal muscle cells.

In situ forming biodegradable electroactive hydrogels

In situ forming biodegradable electroactive hydrogels based on gelatin-graft-polyaniline enhanced the adhesion and proliferation of C2C12 myoblast cells.

Abstract 17550: PPAR-δ Activation Induces Angiomyogenesis Through the Potentiation of Endothelial Progenitor Cell Paracrine Network

Background Endothelial progenitor cells (EPCs) paracrine network has been known to play a key role in neovascularization. We focused on skeletal muscle regeneration as well as new vessel formation by EPCs and their modulation by PPAR-δ. Methods and Results GW501516 , a PPAR-δ agonist enhanced MMP-9 mRNA expression, protein excretion and enzyme activity among several paracrine factors in EPCs. MMP-9 in conditioned media (CM) from GW501516 treated EPCs degraded IGFBP-3 in Western blot. The CM treatment phosphorylated IGF-1 receptor (R) in HUVECs, enhancing BrdU incorporation, Matrigel tube formation and migration. It also induced proliferation of C2C12 myoblast cells and protected them from hypoxia. Systemic treatment of GW501516 in mice increased MMP-9 expression in EPCs and degraded IGFBP-3 in serum Western. In mice which received bone marrow transplantation (BMT) from WT mice, continuous infusion of GW501516 phosphorylated IGF-1R in ECs and skeletal muscle. It increased capillary density and regenerating myotubes with central nuclei, and decreased muscle fibrosis in MT stain. IF for Ki67 and TUNEL stain demonstrated the in vivo proliferation and protection from apoptosis of ECs and skeletal muscle cells by GW501516. Limb perfusion recovery was also enhanced in LDPI. BMT from MMP-9 KO mice or IGFBP-3 systemic infusion abolished these effects. Intramuscular injection of ex vivo GW501516 treated EPCs to ischemic limbs showed the same effects. But EPCs from MMP-9 KO mice reversed the results. In skin punch wound model, GW501516 systemic administration enhanced wound healing with increased angiogenesis in WT mice, not in MMP-9 KO mice. Conclusion EPC paracrine network through MMP-9 mediated IGF-1 signaling pathway is important in angiomyogenesis, and is potentiated by PPAR-δ activation. Our results suggest PPAR-δ agonist as a good candidate drug for patients with peripheral vascular diseases who need angiomyogenesis for recovery.