scholarly journals MYOC Promotes the Differentiation of C2C12 Cells by Regulation of the TGF-β Signaling Pathways via CAV1

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 686
Author(s):  
Yuhan Zhang ◽  
Shuang Li ◽  
Xin Wen ◽  
Huili Tong ◽  
Shufeng Li ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Confocal Microscopy ◽  
Gastrocnemius Muscle ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Laser Confocal Microscopy ◽  
Caveolin 1 ◽  
The Common ◽  
Sites Of Action ◽  
Cav1 Expression

Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-β pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-β pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-β by influencing CAV1 to promote the differentiation of C2C12 cells.

scholarly journals Diverse Action of Selected Statins on Skeletal Muscle Cells—An Attempt to Explain the Protective Effect of Geranylgeraniol (GGOH) in Statin-Associated Myopathy (SAM)

2019 ◽  
Vol 8 (5) ◽  
pp. 694 ◽  
Author(s):  
Anna Jaśkiewicz ◽  
Beata Pająk ◽  
Magdalena Łabieniec-Watała ◽  
Clara De Palma ◽  
Arkadiusz Orzechowski
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Cell Viability ◽  
Mitochondrial Respiration ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
C2c12 Cell ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Calpain Inhibitor

The present study is centered on molecular mechanisms of the cytoprotective effect of geranylgeraniol (GGOH) in skeletal muscle harmed by statin-associated myopathy (SAM). GGOH via autophagy induction was purportedly assumed to prevent skeletal muscle viability impaired by statins, atorvastatin (ATR) or simvastatin (SIM). The C2C12 cell line was used as the ‘in vitro’ model of muscle cells at different stages of muscle formation, and the effect of ATR or SIM on the cell viability, protein expression and mitochondrial respiration were tested. Autophagy seems to be important for the differentiation of muscle cells; however, it did not participate in the observed GGOH cytoprotective effects. We showed that ATR- and SIM-dependent loss in cell viability was reversed by GGOH co-treatment, although GGOH did not reverse the ATR-induced drop in the cytochrome c oxidase protein expression level. It has been unambiguously revealed that the mitochondria of C2C12 cells are not sensitive to SIM, although ATR effectively inhibits mitochondrial respiration. GGOH restored proper mitochondria functioning. Apoptosis might, to some extent, explain the lower viability of statin-treated myotubes as the pan-caspase inhibitor, N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK), partly reversed ATR- or SIM-induced cytotoxic effects; however, it does not do so in conjunction with caspase-3. It appears that the calpain inhibitor, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLM), restored the viability that was reduced by ATR and SIM (p < 0.001). GGOH prevents SAM, in part, as a consequence of a caspase-3 independent pathway, probably by calpain system inactivation.

scholarly journals Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development

2011 ◽  
Vol 43 (20) ◽  
pp. 1135-1143 ◽  
Author(s):  
Ryan L. Sheppard ◽  
Espen E. Spangenburg ◽  
Eva R. Chin ◽  
Stephen M. Roth
Keyword(s):  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Androgen Receptor ◽  
Transcriptional Activity ◽  
C2c12 Cell ◽  
Repeat Length ◽  
C2c12 Cells ◽  
Expression Vectors ◽  
Polyglutamine Repeat ◽  
Fusion Index

Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) ( P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.

scholarly journals Identification of CXCL11 as part of chemokine network controlling skeletal muscle development

2021 ◽  
Author(s):  
Malte Puchert ◽  
Christian Koch ◽  
Konstanze Zieger ◽  
Jürgen Engele
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Muscle Fibres ◽  
Skeletal Muscle Development ◽  
Functional Studies ◽  
Adult Rat ◽  
Limb Muscles ◽  
Chemokine Cxcl12

AbstractThe chemokine, CXCL12, and its receptors, CXCR4 and CXCR7, play pivotal roles during development and maintenance of limb muscles. CXCR7 additionally binds CXCL11, which uses CXCR3 as its prime receptor. Based on this cross-talk, we investigate whether CXCL11 would likewise affect development and/or function of skeletal muscles. Western blotting and immunolabelling demonstrated the developmentally restricted expression of CXCL11 in rat limb muscles, which was contrasted by the continuous expression of its receptors in proliferating and differentiating C2C12 cells as well as in late embryonic to adult rat limb muscle fibres. Consistent with a prime role in muscle formation, functional studies identified CXCL11 as a potent chemoattractant for undifferentiated C2C12 cells and further showed that CXCL11 does neither affect myoblast proliferation and differentiation nor metabolic/catabolic pathways in formed myotubes. The use of selective receptor antagonists unravelled complementary effects of CXCL11 and CXCL12 on C2C12 cell migration, which either require CXCR3/CXCR7 or CXCR4, respectively. Our findings provide new insights into the chemokine network controlling skeletal muscle development and function and, thus, might provide a base for future therapies of muscular diseases.

scholarly journals Syncytin-1 Expression is Increased in Skeletal Muscle of Humans with Obesity and is Inversely Correlated to Muscle Protein Synthesis

2021 ◽  
Author(s):  
Jayachandran Ravichandran ◽  
Lori R Roust ◽  
Christos Katsanos
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Protein Metabolism ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Anabolic Hormone ◽  
Lower Protein ◽  
Potential Link

Abstract Background: Various pathophysiological conditions alter protein metabolism in skeletal muscle, with obesity being one of them. Obesity impairs regeneration of skeletal muscle, and the same biological mechanism(s) may adversely affect protein metabolism in the muscle of these individuals. Methods: We used C2C12 cell line to evaluate the effects of the anabolic hormone insulin on the expression of protein syncytin-1, which regulates regeneration of muscle, and in the presence of fatty acids whose metabolism is altered in obesity. We used muscle biopsy samples from obese humans with lower muscle protein synthesis and lean controls to evaluate expression of syncytin-1 in obesity and its correlation with protein synthesis in muscle. Results: Insulin upregulated syncytin-1 expression in C2C12 cells and this response was impaired in the presence of the fatty acid palmitate, but not oleate. Expression of the protein 4E-BP1, which signals increase in protein synthesis in muscle, showed response similar to that of syncytin-1. Humans with obesity characterized by lower muscle protein synthesis had higher expression of syncytin-1 in muscle compared to lean humans (P < 0.01). The rate of synthesis of protein in skeletal muscle across humans subjects correlated inversely (r = -0.51; P = 0.03) with the expression of syncytin-1 in muscle. Conclusions: Our studies provide novel insights in the regulation of syncytin-1 in skeletal muscle, and describe potential link between syncytin-1 expression and protein metabolism in skeletal muscle of humans. Altered syncytin-1 expression in muscle may mediate lower protein turnover in muscle of humans with obesity.

MicroRNA-129-5p Inhibited C2C12 Myogenesis and Repressed Slow Fiber Gene Expression in vitro

2021 ◽  
Author(s):  
Ying Peng ◽  
Meixue Xu ◽  
Mingle Dou ◽  
Xin'E Shi ◽  
Gongshe Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Myogenic Differentiation ◽  
C2c12 Cell ◽  
Negative Regulator ◽  
C2c12 Cells ◽  
Loss Of Function ◽  
Mouse Tissues ◽  
Slow Fiber ◽  
Fiber Gene

The miR-129 family is widely reported as tumor repressors, while, their roles in skeletal muscle have not been fully investigated. Here, the function and mechanism of miR-129-5p in skeletal muscle, a member of the miR-129 family, were explored using C2C12 cell line. Our study shown that miR-129-5p was widely detected in mouse tissues, with the highest expression in skeletal muscle. Gain- and loss-of-function study shown that miR-129-5p could negatively regulate myogenic differentiation, indicated by reduced ratio of MyHC-positive myofibers and repressed expression of myogenic genes, such as MyoD, MyoG and MyHC. Furthermore, miR-129-5p was more enriched in fast extensor digitalis lateralis (EDL) than in slow soleus (SOL). Enhanced miR-129-5p could significantly reduce the expression of mitochondrial cox family, together with that of MyHC I, and knockdown of miR-129-5p conversely increased the expression of cox genes and MyHC I. Mechanistically, miR-129-5p directly targeted the 3'-UTR of Mef2a, which was suppressed by miR-129-5p agomir at both mRNA and protein levels in C2C12 cells. Moreover, overexpression of Mef2a could rescue the inhibitory effects of miR-129-5p on the expression of myogenic factors and MyHC I. Collectively, our data revealed that miR-129-5p as a negative regulator of myogenic differentiation and slow fiber gene expression, thus affecting body metabolic homeostasis.

scholarly journals Theophylline inhibits cigarette smoke-induced inflammation in skeletal muscle by upregulating HDAC2 expression and decreasing NF-κB activation

2019 ◽  
Vol 316 (1) ◽  
pp. L197-L205 ◽  
Author(s):  
Yanfei Bin ◽  
Ying Xiao ◽  
Dongmei Huang ◽  
Zhiying Ma ◽  
Yi Liang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mouse Model ◽  
Cigarette Smoke ◽  
Gastrocnemius Muscle ◽  
C2c12 Cells ◽  
Histone Deacetylase 2 ◽  
Protein Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Inflammatory Effect

Inflammation is associated with skeletal muscle dysfunction and atrophy in patients with chronic obstructive pulmonary disease (COPD). Theophylline has an anti-inflammatory role in COPD. However, the effects of theophylline on inflammation in skeletal muscle in COPD have rarely been reported. The aims of this study were to explore whether theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema and to investigate the molecular mechanism underlying this effect. In mice, cigarette smoke (CS) exposure for 28 wk resulted in atrophy of the gastrocnemius muscle. Histone deacetylase 2 (HDAC2) and nuclear factor-κBp65 (NF-κBp65) mRNA and protein levels were significantly decreased and increased, respectively, in gastrocnemius muscle. This effect was revered by aminophylline. The exposure of murine skeletal muscle C2C12 cells to CS extract (CSE) significantly increased IL-8 and TNF-α levels as well as NF-κBp65 mRNA and protein levels and NF-κBp65 activity. This effect was reversed by theophylline. HDAC2 knockdown enhanced the activity of NF-κBp65 and increased IL-8 and TNF-α levels in C2C12 cells. CSE significantly increased the interaction of HDAC2 with NF-κBp65 in C2C12 cells. These data suggest that theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema by upregulating HDAC2 expression and decreasing NF-κBp65 activation.

scholarly journals Septin-7 is indispensable for proper skeletal muscle architecture and function

2021 ◽  
Author(s):  
Monika Gonczi ◽  
Zsolt Raduly ◽  
Laszlo Szabo ◽  
Janos Fodor ◽  
Andrea Telek ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Critical Role ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Knock Out ◽  
Fourth Component ◽  
And Function ◽  
Mitochondrial Networks

Today septins are considered as the fourth component of the cytoskeleton with the Septin-7 isoform playing a critical role in the formation of higher order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin-7 conditional knock-down mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers the organization and localization of septin filaments is revealed, and an ontogenesis-dependent expression of Septin-7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knock-out of Septin-7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin-7 is essential in proper myotube differentiation. These and the transient increase in Septin-7 expression following muscle injury demonstrate its vital contribution to muscle regeneration and development.

scholarly journals Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1680
Author(s):  
Marius Drysch ◽  
Sonja Verena Schmidt ◽  
Mustafa Becerikli ◽  
Felix Reinkemeier ◽  
Stephanie Dittfeld ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Ischemia Reperfusion ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Ros Generation ◽  
Protective Effects ◽  
Activity Increase ◽  
Mitogen Activated Protein ◽  
Myostatin Inhibition ◽  
Hypoxia Reoxygenation

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a Myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn−/− cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn−/− cells and could serve as basis for further research.

Confocal microscopy study of membrane organelles of the skeletal muscle fiber in the process of Zenker’s (spreading) necrosis

2007 ◽  
Vol 1 (2) ◽  
pp. 183-190 ◽  
Author(s):  
S. A. Krolenko ◽  
S. Ya. Adamyan ◽  
T. N. Belyaeva ◽  
T. P. Mozhenok ◽  
A. V. Salova
Keyword(s):  
Skeletal Muscle ◽  
Confocal Microscopy ◽  
Muscle Fiber ◽  
Microscopy Study ◽  
Skeletal Muscle Fiber

Expression of avian Ca2+-ATPase in cultured mouse myogenic cells

1989 ◽  
Vol 9 (5) ◽  
pp. 1978-1986
Author(s):  
N J Karin ◽  
Z Kaprielian ◽  
D M Fambrough
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
C2c12 Cells ◽  
Cyanine Dye ◽  
Adult Rabbit ◽  
Base Pairs ◽  
Reading Frame ◽  
Cytoplasmic Vesicles ◽  
Fast Twitch ◽  
Chicken Skeletal Muscle

cDNA encoding Ca2+-ATPase was cloned from a chicken skeletal muscle library. The cDNA (termed FCa) comprised 3,239 base pairs, including an open reading frame encoding 994 amino acids which showed the highest degree of homology with the adult rabbit fast-twitch Ca2+-ATPase isoform (C. J. Brandl, S. de Leon, D. R. Martin, and D. H. MacLennan, J. Biol. Chem. 262:3768-3774, 1987). Radiolabeled FCa hybridized to a 3.2-kilobase transcript in chicken skeletal muscle RNA but not to cardiac muscle RNA, which confirmed its identity as encoding the fast Ca2+-ATPase isoenzyme. FCa was transfected into the mouse myogenic line C2C12, from which a protein of 100 kilodaltons was immunopurified by using a monoclonal antibody specific for the avian fast Ca2+-ATPase. Immunofluorescence microscopy of a line (designated C2FCa2) stably expressing the avian Ca2+-ATPase localized the protein to the nuclear envelope and a population of cytoplasmic vesicles. A similar pattern was observed when C2FCa2 cells were stained with DiOC6(3), a cyanine dye that labels endoplasmic reticulum and mitochondria (M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, and L. B. Chen, Cell 38:101-108, 1984). We conclude that the avian Ca2+-ATPase fast isoform is expressed and correctly targeted to the endoplasmic reticulum in mouse C2C12 cells.

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