scholarly journals Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

2020 ◽  
Vol 72 (3) ◽  
pp. 379-391
Author(s):  
Ana Stancic ◽  
Ivana Drvenica ◽  
Branko Bugarski ◽  
Vesna Ilic ◽  
Diana Bugarski
Keyword(s):  
Cell Therapy ◽  
Experimental Model ◽  
Myogenic Differentiation ◽  
Calf Serum ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
Heme Oxygenase 1 ◽  
C2c12 Myoblast ◽  
Free Hemoglobin ◽  
Functional Characteristics

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 ?M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1?) at a concentration of 10 ?M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury.

scholarly journals Ganoderma microsporum immunomodulatory protein, GMI, promotes C2C12 myoblast differentiation in vitro via upregulation of Tid1 and STAT3 acetylation

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244791
Author(s):  
Wan-Huai Teo ◽  
Jeng-Fan Lo ◽  
Yu-Ning Fan ◽  
Chih-Yang Huang ◽  
Tung-Fu Huang
Keyword(s):  
Myogenic Differentiation ◽  
Atp Synthesis ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Muscle Loss ◽  
C2c12 Myoblasts ◽  
Immunomodulatory Protein ◽  
Pre Treatment

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it’s crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.

287 PORCINE ADIPOSE-DERIVED STEM CELLS IN CO-CULTURE FUSE ACTIVELY WITH MOUSE MYOTUBES AND EXPRESS MYOGENIC MARKERS

2013 ◽  
Vol 25 (1) ◽  
pp. 291
Author(s):  
S. Jain ◽  
D. J. Milner ◽  
M. Bionaz ◽  
J. A. Cameron ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Myogenic Differentiation ◽  
Primary Source ◽  
Cell Types ◽  
C2c12 Cells ◽  
Immunofluorescent Staining ◽  
C2c12 Myotubes ◽  
Adipose Derived Stem Cells ◽  
Myogenic Markers

Mesenchymal stem cells (MSC) have been shown to be useful in regenerative medicine with their capability to give rise to various different cell types, including osteoblasts, adipocytes, chondrocytes, muscle cells, and neurons. Among MSC, bone marrow-derived stem cells (BMSC) are considered the primary source. Recently, many studies have shown the regenerative capabilities of adipose-derived stem cells (ASC). The ASC, with their greater abundance and ease of harvest, provide clear advantages over BMSC. We have previously demonstrated the myogenic differentiation of porcine ASC when co-cultured with differentiating C2C12 myoblasts in a myogenic differentiation-promoting medium. In this study, we sought to examine the myogenic potential of porcine ASC when co-cultured with fully differentiated murine myotubes. For the present study, we used porcine ASC isolated from the back fat of a transgenic gree fluorescent protein (GFP)-expressing pig at passage 3. The ASC were added to mouse C2C12 myotube cultures that had been induced towards myogenesis for 72 h. As controls, we co-cultured, in the same conditions, GFP-expressing endothelial cells (ENDO) from the aorta of the same pig. Additionally, we cultured ASC, ENDO, and C2C12 cells alone in myogenic-differentiation medium. Cultures were harvested at 12, 24, and 48 h after addition of porcine cells to myotube cultures for measurement of mouse- or porcine-specific myogenic markers by quantitative RT-PCR and immunohistochemistry. We were able to observe fusion of ASC GFP-expressing cells with pre-formed mouse myotubes by detection of myotubes expressing GFP. Additionally, immunofluorescent staining of co-cultures with an antibody specific for porcine nuclear Lamin A demonstrated the presence of ASC nuclei incorporated into myotubes. We observed large increases in gene expression of porcine-specific myogenin (MYOG; >900-fold) and desmin (DES; 8-fold). Unexpectedly, ENDO in co-culture with myotubes also had increased expression of DES (4-fold) and MYOG (400-fold), possibly indicating their de-differentiation and adaptation to a myogenic phenotype. In addition, expression of mouse-specific DES and MYOG were boosted in C2C12 myotubes when co-cultured for 48 h compared with C2C12 alone, suggesting enhanced myogenesis or prolonged survival of myotubes in co-culture. Cultures of ASC and ENDO alone did not display increased expression of myogenic markers. These results provide support for the use of ASC for muscle regeneration strategies, as in the case of damaged muscles and muscular dystrophy. In addition, compared with our previous observations, where ASC were co-cultured with undifferentiated C2C12, the ASC co-cultured with myotubes appeared to have an enhanced fusion and expression of myogenic markers. Finally, the capacity of ENDO to fuse and actively expressed muscle-specific genes deserves further investigation.

Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

2011 ◽  
Vol 300 (5) ◽  
pp. C1122-C1138 ◽  
Author(s):  
Henri Bernardi ◽  
Stephanie Gay ◽  
Yann Fedon ◽  
Barbara Vernus ◽  
Anne Bonnieu ◽  
...  
Keyword(s):  
Cell Fate ◽  
Satellite Cells ◽  
Myogenic Differentiation ◽  
Expression Profiles ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Strong Increase ◽  
C2c12 Myoblast ◽  
Wnt Proteins ◽  
Myogenic Activity

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

scholarly journals Hes6 regulates myogenic differentiation

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2195-2207
Author(s):  
Judy Cossins ◽  
Ann E. Vernon ◽  
Yun Zhang ◽  
Anna Philpott ◽  
Philip H. Jones
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Xenopus Embryos ◽  
Enhancer Of Split

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21Cip1, and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

scholarly journals Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

1996 ◽  
Vol 7 (10) ◽  
pp. 1587-1599 ◽  
Author(s):  
D S Franklin ◽  
Y Xiong
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Cell Cycle Arrest ◽  
Kinase Activity ◽  
Myogenic Differentiation ◽  
Critical Role ◽  
C2c12 Cells ◽  
C2c12 Myoblast ◽  
Mouse Muscle ◽  
Cycle Arrest

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.

scholarly journals Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion

2019 ◽  
Vol 317 (5) ◽  
pp. C1025-C1033 ◽  
Author(s):  
Val A. Fajardo ◽  
Colton J. F. Watson ◽  
Kirsten N. Bott ◽  
Fereshteh Moradi ◽  
Lucas A. Maddalena ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myogenic Differentiation ◽  
Vastus Lateralis ◽  
Critical Role ◽  
Myoblast Fusion ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
Mammalian Skeletal Muscle ◽  
Mrna Levels ◽  
Calcineurin Signaling

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

scholarly journals Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Kumiko Terada ◽  
Satomi Misao ◽  
Naoki Katase ◽  
Shin-ichiro Nishimatsu ◽  
Tsutomu Nohno
Keyword(s):  
Cell Proliferation ◽  
Wnt Signaling ◽  
Cellular Localization ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Muscle Differentiation ◽  
C2c12 Cells ◽  
C2c12 Myoblast ◽  
Smad Signaling ◽  
Myoblast Cell

Background.Wnt signaling is involved in muscle formation throughβ-catenin-dependent or -independent pathways, but interactions with other signaling pathways including transforming growth factorβ/Smad have not been precisely elucidated.Results.As Wnt4 stimulates myogenic differentiation by antagonizing myostatin (GDF8) activity, we examined the role of Wnt4 signaling during muscle differentiation in the C2C12 myoblast cell line. Among several extrinsic signaling molecules examined in a microarray analysis of C2C12 cells during the transition from cell proliferation to differentiation after mitogen deprivation,bone morphogenetic protein 4 (BMP4)expression was prominently increased.Wnt4overexpression had similar effects onBMP4expression. BMP4 was able to inhibit muscle differentiation when added to the culture medium. BMP4 and noggin had no effects on the cellular localization ofβ-catenin induced by Wnt3a; however, the BMP4-induced phosphorylation of Smad1/5/8 was enhanced by Wnt4, but not by Wnt3a. The BMP antagonist noggin effectively stimulated muscle differentiation through binding to endogenous BMPs, and the effect of noggin was enhanced by the presence of Wnt3a and Wnt4.Conclusion.These results suggest that BMP/Smad pathways are modified through Wnt signaling during the transition from progenitor cell proliferation to myogenic differentiation, although Wnt/β-catenin signaling is not modified with BMP/Smad signaling.

scholarly journals Human Resistin Inhibits Myogenic Differentiation and Induces Insulin Resistance in Myocytes

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Chun Hua Sheng ◽  
Zhen Wu Du ◽  
Yang Song ◽  
Xiao Dong Wu ◽  
Yu Cheng Zhang ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Cell Proliferation ◽  
Insulin Sensitivity ◽  
Glucose Uptake ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
Mrna Levels ◽  
Uptake Ratio ◽  
Human Resistin

This study is aimed to investigate the effect of human resistin on myocyte differentiation and insulin resistance. The human resistin eukaryotic expression vector was stable transfected into C2C12 myocyte cells and was transiently transfected into COS7 cells. The effects of human resistin on cell proliferation, cell cycle, and myogenic differentiation of C2C12 cells were examined. Glucose uptake assays was performed on C2C12 myotubes by using [3H] 2-deoxy-D-glucose. The mRNA levels of insulin receptor (IR) and glucose transporter 4 (GLUT4) were evaluated by semiquantitative RT-PCR. Results showed by the C2C12 cells transfected with human resistin gene compared with that without transfecting gene are as follows: (1) cell proliferation was significantly promoted, (2) after inducing differentiation, the myotube’s diameters and expression of desmin and myoglobin decreased, and (3) glucose uptake ratio was lowered and expression of IR and GLUT4 decreased. However, there was no significant difference in the glucose uptake ratio between C2C12 myotubes treated with a human resistin conditioned medium of COS7 cells and treated with control medium. These results suggest that maybe human resistin has not a direct role on insulin sensitivity of myocytes. However, maybe it impaired the insulin sensitivity of myocytes through suppressing myogenesis and stimulating proliferation of myoblasts.

scholarly journals Platelet-poor plasma of athletes is a potent inducer of myogenic differentiation of C2C12 myoblasts

2020 ◽  
Vol 74 (1) ◽  
pp. 18-33
Author(s):  
Irina Maslovaric ◽  
Vesna Ilic ◽  
Ana Stancic ◽  
Juan Santibanez ◽  
Drenka Trivanovic ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Platelet Rich Plasma ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Football Players ◽  
C2c12 Myoblast ◽  
Potent Inducer ◽  
Gelatinase Activity ◽  
Platelet Poor Plasma ◽  
Myoblast Cells

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months? frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20?C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters > dancers > football players; mean migratory capacity was: weightlifters = dancers > football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players > dancers > weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, long-term, frozen-stored PPP preserves its potential to modify myoblast morphology and function.

scholarly journals 2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways

2021 ◽  
Author(s):  
Hyunju Liu ◽  
Su-Mi Lee ◽  
Hosouk Joung
Keyword(s):  
Myogenic Differentiation ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Covalent Attachment ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Long Term Effects ◽  
Proliferation And Differentiation ◽  
Myoblast Proliferation ◽  
Myoblast Cells

AbstractSUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.

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