Differentiation Of C2C12 Myoblast Cells Research Articles

The ensured proliferative capacity of myoblast in serum-reduced conditions with Methyl-β-cyclodextrin.

To produce muscle fibers for cultured meat on a large scale, it is important to expand myoblasts in a serum-reduced or serum-free medium to avoid cost, ethical, and environmental issues. Myoblasts such as C2C12 cells differentiate quickly into myotubes and lose their ability to proliferate when the serum-rich medium is replaced with a serum-reduced medium. This study demonstrates that Methyl-β-cyclodextrin (MβCD), a starch-derived agent that depletes cholesterol, can inhibit further differentiation of myoblasts at the MyoD-positive stage by reducing plasma membrane cholesterol on C2C12 cells and primary cultured chick muscle cells. Furthermore, MβCD efficiently blocks cholesterol-dependent apoptotic cell death of myoblasts, which is one of the mechanisms by which it inhibits the differentiation of C2C12 myoblast cells, as dead cells of myoblast are necessary for the fusion of adjacent myoblasts during the differentiation process into myotubes. Importantly, MβCD maintains the proliferative capacity of myoblasts only under differentiation conditions with a serum-reduced medium, suggesting that its mitogenic effect is due to its inhibitory effect on myoblast differentiation into myotube. In conclusion, this study provides significant insights into ensuring the proliferative capacity of myoblasts in a future serum-free condition for cultured meat production.

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.

Potential Roles of Silicon/Silica‐Based Nanoparticles in 3D Printed Hydrogels for Skeletal Muscle Regeneration

Introduction : Skeletal muscle constitutes about 40% of the total body mass and plays a significant role in the movement of the human body. Although skeletal muscles have remarkable endogenous regenerative capacity, this capacity is overwhelmed following acute severe traumatic injuries. Furthermore, the associated oxidative damage can delay regeneration process and prolong recovery. These traumatic injuries result from combat- and/or trauma-induced muscle injuries and often lead to irreversible tissue damage and impaired vascularization. Volumetric muscle loss (VML) is a severe traumatic injury that results in a critical loss (≥ 20%) of the native muscle mass leading to permanent disability. Our hypothesis is that using 3D bioprinted hydrogel modified with silica-based nanoparticles (NPs) laden human skeletal muscle cells will provide the required architecture and enhance muscle regeneration in VML defects where high levels of reactive oxygen species (ROS) is predominant. Materials and Methods Sodium metasilicate powder was used to adjust and optimize the effective concentration of ionic silicon, while hydrogen peroxide was used as sources of ROS to simulate the oxidative damage conditions of VML injuries. Silica based nanoparticles were embedded into GelMA-based hydrogels for 3D printing of scaffolds that mimic the skeletal muscle architecture. Results Our preliminary data using ionic silicon indicated that Si-ions are not cytotoxic to myoblast cells under tested concentrations (0.1-2.0 mM) (Figure 1). Furthermore, Si-ions significantly enhanced myoblast cell viability, proliferation, and differentiation into myotubes as indicated by a higher fusion index compared to the control. In-vitro studies indicated that0.4 mM of H2O2 into the growth media significantly decreases the cell viability after 6 and 24 hr. compared to the control (**p < 0.01, n=4 per group). Addition of 0.5-1.0 mM of Si into the growth media significantly enhances the cell viability under conditions that mimic high ROS (i.e., H202 treated group). The 3D printed scaffolds of GelMA + NPs indicated a higher myoblast cell viability significantly reducing cell death compared to the bare GelMA scaffolds as shown at Figure 2. Conclusion Our preliminary findings conclude that 0.1 mM Si-ions enhance myoblast viability, proliferation, and differentiation of C2C12 myoblast cells. Using 0.4 mM of H2O2 can simulate the oxidative damage condition in myoblast cells in-vitro. Using 0.1-0.5 mM silicon ions can attenuate the oxidative damage (0.4 mM H2O2) on C2C12 myoblast cells. 3D printed hydrogels loaded with silica-based nanoparticles are promising materials for 3D printing of muscle constructs.

Circular RNA circ-FoxO3 Inhibits Myoblast Cells Differentiation.

CircRNA is a type of closed circular non-coding RNA formed by reverse splicing and plays an important role in regulating the growth and development of plants and animals. To investigate the function of circ-FoxO3 in mouse myoblast cells’ (C2C12) differentiation and proliferation, we used RT-qPCR to detect the expression level of circ-FoxO3 in mouse myoblast cells at different densities and different differentiation stages, and the specific interference fragment was used to inhibit the expression level of circ-FoxO3 in myoblast cells to observe its effect on myoblast cells proliferation and differentiation. We found that the expression level of circ-FoxO3 in myoblast cells increased with the prolongation of myoblast cells differentiation time, and its expression level decreased with the proliferation of myoblast cells. At the same time, we found that the differentiation ability of the cells was significantly increased (p < 0.05), but the cell proliferation was unchanged (p > 0.05) after inhibiting the expression of circ-FoxO3 in myoblast cells. Combining the results of bioinformatics analysis and the dual luciferase reporter experiment, we found that circ-FoxO3 is a sponge of miR-138-5p, which regulates muscle differentiation. Our study shows that circ-FoxO3 can inhibit the differentiation of C2C12 myoblast cells and lay a scientific foundation for further study of skeletal muscle development at circRNA levels.

Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Effect of Beta‐hydroxybutyrate on Myoblast Proliferation and Differentiation

Ketogenic diets (KDs) and fasting have been promoted for weight loss and health. KDs cause an increase in circulating levels of ketone bodies, including beta‐hydroxybutyrate (βHB). The effects of amplified levels of βHB on skeletal muscle proliferation and differentiation are largely unknown. Therefore, we tested whether βHB has an effect on proliferation and differentiation of C2C12 myoblast cells in vitro. Experiment 1: to assess proliferation, myoblasts were plated sparsely and incubated in growth media for 6 hours. They were then incubated overnight (~12 hours) with 0 – 10 mM bHB followed by the addition of BrDU to the media for an additional 2 hours to label proliferating cells. The cells were fixed and stained for BrDU and DAPI and proliferation index was calculated as the percentage of BrDU+ nuclei/total nuclei. We found that myoblast proliferation did not increase significantly with βHB. Experiment 2: to assess differentiation, myoblasts were grown to 60% confluence in growth media, followed by differentiation media for 4 days with 0–10 mM bHB. The myotubes were then fixed and stained with DAPI and antibodies against myosin heavy chain. Differentiation index was calculated as the percentage of nuclei associated with MHC+ myotubes. We found that myoblast differentiation increased 22% with 0.6125 mM βHB, and tended to increase (p = 0.12) at 1.25 mM bHB. This suggests that moderate levels of bHB (similar to those achieved in those on KDs or fasting) may be beneficial to muscle repair and/or regeneration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80–90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3’UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3’UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.

Open Fluidics: A Cell Culture Flow System Developed Over Wettability Contrast-Based Chips.

Biological tissues are recurrently exposed to several dynamic mechanical forces that influence cell behavior. On this work, the focus is on the shear stress forces induced by fluid flow. The study of flow-induced effects on cells leads to important advances in cardiovascular, cancer, stem cell, and bone biology understanding. These studies are performed using cell culture flow (CCF) systems, mainly parallel plate flow chambers (PPFC), and microfluidic systems. Here, it is proposed an original CCF system based on the open fluidics concept. The system is developed using a planar superhydrophobic platform with hydrophilic paths. The paths work as channels to drive cell culture medium flows without using walls for liquid confinement. The liquid streams are controlled just based on the wettability contrast. To validate the concept, the effect of the shear stress stimulus in the osteogenic differentiation of C2C12 myoblast cells is studied. Combining bone morphogenic protein (specifically BMP-2) stimulation with this mechanical stimulus, a synergistic effect is found on osteoblast differentiation. This effect is confirmed by the enhancement of alkaline phosphatase activity, a well-known early marker of osteogenic differentiation. The suggested CCF system combines characteristics and advantages of both the PPFC and microfluidic systems.

In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model

Skeletal muscle tissue engineering holds great promise for pharmacological studies. Herein, we demonstrated an in vitro drug testing system using tissue-engineered skeletal muscle constructs. In response to epigenetic drugs, myotube differentiation of C2C12 myoblast cells was promoted in two-dimensional cell cultures, but the levels of contractile force generation of tissue-engineered skeletal muscle constructs prepared by three-dimensional cell cultures were not correlated with the levels of myotube differentiation in two-dimensional cell cultures. In contrast, sarcomere formation and contractile activity in two-dimensional cell cultures were highly correlated with contractile force generation of tissue-engineered skeletal muscle constructs. Among the epigenetic drugs tested, trichostatin A significantly improved contractile force generation of tissue-engineered skeletal muscle constructs. Follistatin expression was also enhanced by trichostatin A treatment, suggesting the importance of follistatin in sarcomere formation of muscular tissues. These observations indicate that contractility data are indispensable for in vitro drug screening.

Myostatin promotes tenogenic differentiation of C2C12 myoblast cells through Smad3

Myostatin, a member of the transforming growth factor‐β (TGF‐β) superfamily, is expressed in developing and adult skeletal muscle and negatively regulates skeletal muscle growth. Recently, myostatin has been found to be expressed in tendons and increases tendon fibroblast proliferation and the expression of tenocyte markers. C2C12 is a mouse myoblast cell line, which has the ability to transdifferentiate into osteoblast and adipocyte lineages. We hypothesized that myostatin is capable of inducing tenogenic differentiation of C2C12 cells. We found that the expression of scleraxis, a tendon progenitor cell marker, is much higher in C2C12 than in the multipotent mouse mesenchymal fibroblast cell line C3H10T1/2. In comparison with other growth factors, myostatin significantly up‐regulated the expression of the tenogenic marker in C2C12 cells under serum‐free culture conditions. Immunohistochemistry showed that myostatin inhibited myotube formation and promoted the formation of spindle‐shaped cells expressing tenomodulin. We examined signaling pathways essential for tenogenic differentiation to clarify the mechanism of myostatin‐induced differentiation of C2C12 into tenocytes. The expression of tenomodulin was significantly suppressed by treatment with the ALK inhibitor SB341542, in contrast to p38MAPK (SB203580) and MEK1 (PD98059) inhibitors. RNAi silencing of Smad3 significantly suppressed myostatin‐induced tenomodulin expression. These results indicate that myostatin has a potential role in the induction of tenogenic differentiation of C2C12 cells, which have tendon progenitor cell characteristics, through activation of Smad3‐mediated signaling.

Stretchable degradable and electroactive shape memory copolymers with tunable recovery temperature enhance myogenic differentiation.

Conducting polymers can regulate cell behavior such cell adhesion, proliferation, and differentiation with or without electrical stimulation. Therefore, they have great potential for electrical signal sensitive tissue regeneration. Although conducting biomaterials with degradability have been developed, highly stretchable and electroactive degradable copolymers for soft tissue engineering have been rarely reported. On the other hand, shape memory polymers (SMPs) have been widely used in biomedical fields. However, SMPs based on polyesters usually are biologically inert. This work reported the design of super stretchable electroactive degradable SMPs based on polycaprolactone and aniline trimer with tunable recovery temperature around body temperature. These flexible electroactive SMPs facilitated the proliferation and differentiation of C2C12 myoblast cells compared with polycaprolactone, indicating that they are excellent scaffolding biomaterials in tissue engineering to repair skeletal muscle and possibly other tissues.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

BackgroundSnakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.MethodologyC2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.ResultsIn non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.ConclusionLLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

Ethanol extract of Cyclolepis genistoides D. Don (palo azul) induces formation of myotubes, which involves differentiation of C2C12 myoblast cells

In this study, we examined the cell differentiation effect of an ethanol extract of Cyclolepis genistoides D. Don, a herbaceous perennial belonging to the family Asteraceae (vernacular name: palo azul). Palo azul has numerous physiological effects that contribute to the prevention of metabolic syndromes, although the mechanism remains unclear. We previously suggested that palo azul has antidiabetic activity via an adipose differentiation effect. Here, we focused on whether palo azul promoted the differentiation of myoblasts. The mouse muscle myoblast cell line C2C12 was cultured and differentiated using horse serum with or without an ethanol extract of palo azul (12.5-200 μg/mL). Quantitative real-time polymerase chain reaction was performed to evaluate differentiation markers, including insulin-like growth factor-1 and myogenin. To evaluate myotube formation, myosin heavy-chain (MHC) expression and localization were detected by immunohistochemistry. Palo azul increased the expression of the differentiation markers. Furthermore, immunohistochemistry analysis revealed increased formation of MHC myotubes after palo azul treatment along with increased diameter and fusion indices of the myotubes. The expression level of MHC was also increased. In conclusion, palo azul may increase muscle mass in the body and improve insulin resistance conditions by facilitating the formation of myotubes by promoting myocyte differentiation.

Strong electroactive biodegradable shape memory polymer networks based on star-shaped polylactide and aniline trimer for bone tissue engineering.

Preparation of functional shape memory polymer (SMP) for tissue engineering remains a challenge. Here the synthesis of strong electroactive shape memory polymer (ESMP) networks based on star-shaped polylactide (PLA) and aniline trimer (AT) is reported. Six-armed PLAs with various chain lengths were chemically cross-linked to synthesize SMP. After addition of an electroactive AT segment into the SMP, ESMP was obtained. The polymers were characterized by (1)H NMR, GPC, FT-IR, CV, DSC, DMA, tensile test, and degradation test. The SMP and ESMP exhibited strong mechanical properties (modulus higher than GPa) and excellent shape memory performance: short recovery time (several seconds), high recovery ratio (over 94%), and high fixity ratio (almost 100%). Moreover, cyclic voltammetry test confirmed the electroactivity of the ESMP. The ESMP significantly enhanced the proliferation of C2C12 cells compared to SMP and linear PLA (control). In addition, the ESMP greatly improved the osteogenic differentiation of C2C12 myoblast cells compared to PH10 and PLA in terms of ALP enzyme activity, immunofluorescence staining, and relative gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). These intelligent SMPs and electroactive SMP with strong mechanical properties, tunable degradability, good electroactivity, biocompatibility, and enhanced osteogenic differentiation of C2C12 cells show great potential for bone regeneration.

Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

The histone demethylase KDM4B interacts with MyoD to regulate myogenic differentiation in C2C12 myoblast cells

Enzymes that mediate posttranslational modifications of histone and nonhistone proteins have been implicated in regulation of skeletal muscle differentiation. However, functions of histone demethylases that could counter the actions of H3-K9 specific histone methyltransferases remain still obscure. Here we present evidences that KDM4B histone demethylase regulates expression of myogenic regulators such as MyoD and thereby controls myogenic differentiation of C2C12 myoblast cells. We demonstrate that expression of KDM4B gradually increases during myogenic differentiation and depletion of KDM4B using shRNA results in inhibition of differentiation in C2C12 myoblast cells, which is correlated with decreased expression of MyoD and myogenin. In addition, we find that KDM4B shRNA represses expression of MyoD promoter-driven luciferase reporter and exogenous expression of MyoD rescues myogenic potential in KDM4B-depleted myoblast cells. We further show that KDM4B interacts with MyoD, binds to MyoD and myogenin promoters in vivo, and finally, is involved in demethylation of tri-methylated H3-K9 on promoters of MyoD and myogenin. Taken together, our data suggest that KDM4B plays key roles in myogenic differentiation of C2C12 cells, presumably by its function as a H3-K9 specific histone demethylase.

MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix

Osterix (Osx) is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Osx-null mice, which exhibit a complete absence of bone formation and arrested osteoblast differentiation, die immediately after birth. However, our understanding of the regulatory mechanism of Osx expression remains poor. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play pivotal roles in diverse biological processes, including the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms. In this study, we aimed to investigate the impact of miRNAs on Osx expression. Bioinformatic analyses predicted that miR-214 would be a potential regulator of Osx. The direct binding of miR-214 to the Osx 3′ untranslated region (3′ UTR) was demonstrated by a luciferase reporter assay using a construct containing the Osx 3′ UTR. Deletion mutant construction revealed that the Osx 3′ UTR contained two miR-214 binding sites. MiR-214 expression was inversely correlated with Osx expression in Saos-2 and U2OS cells. The forced expression of miR-214 in Saos-2 cells led to a reduction in the level of Osx protein. Moreover, the role of miR-214 in the osteogenic differentiation of C2C12 cells was investigated. We found that the osteogenic differentiation of C2C12 cells was enhanced by the downregulation of miR-214 expression, as measured by increased alkaline phosphatase activity and matrix mineralization. Taken together, these results indicate that miR-214 is a novel regulator of Osx, and that it plays an important role in the osteogenic differentiation of C2C12 cells as a suppressor.

Selective Androgen Receptor Modulator, YK11, Regulates Myogenic Differentiation of C2C12 Myoblasts by Follistatin Expression

The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.