Targeted Down-regulation of Caveolin-3 Is Sufficient to Inhibit Myotube Formation in Differentiating C2C12 Myoblasts

Whole body vibration training is widely used in rehabilitation and sports activities to improve muscle strength, balance, and flexibility. However, the molecular mechanisms of vertical vibration (VV) training and their effect on the myogenesis of myoblasts remain undefined. This study was undertaken to address the hypothesis that VV can enhance the expression of ECM proteins and myogenic regulatory factors (MRFs) in myoblasts and, in turn, increase myotube formation. Using real-time PCR, Western blot analysis, and immunofluorescence studies, we examined the effect of VV treatment with frequencies of 5, 8, or 10 Hz on the expression of ECM proteins and MRFs as well as myotube formation in C2C12 myoblasts. We showed that VV stimulation is safe and effective at stimulating myogenesis in C2C12 myoblasts. The levels of expression of the ECM proteins type I collagen and decorin were the highest after VV treatment at frequencies of 8 and 10 Hz. Expression of the MRFs MyoD and myogenin increased after VV stimulation in a time- and dose-dependent manner. The total number of myotubes formed, as well as the length and the average area of myotubes, were substantially increased following VV treatment at frequencies of 8 to 10 Hz. In conclusion, VV treatment at frequencies of 8 to 10 Hz can stimulate the expression of ECM proteins and MRFs in myoblasts and, in turn, increase myotube formation.

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Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.04.002 ◽

2011 ◽

Vol 408 (1) ◽

pp. 167-173 ◽

Cited By ~ 25

Author(s):

Young Woo Eom ◽

Jong Eun Lee ◽

Mal Sook Yang ◽

In Keun Jang ◽

Hyo Eun Kim ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Human Adipose Tissue ◽

C2c12 Myoblasts ◽

Myotube Formation ◽

Cellular Fusion

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Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Brazilian Journal of Medical and Biological Research ◽

10.1590/s0100-879x2005000700005 ◽

2005 ◽

Vol 38 (7) ◽

pp. 1025-1032 ◽

Cited By ~ 3

Author(s):

C.S. Mermelstein ◽

L.M. Amaral ◽

M.I.L. Rebello ◽

J.S.N. Reis ◽

R. Borojevic ◽

...

Keyword(s):

Intermediate Filament ◽

Cell Shape ◽

Down Regulation ◽

C2c12 Myoblasts

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C2C12 myoblasts are more sensitive to the toxic effects of simvastatin than myotubes and show impaired proliferation and myotube formation

Biochemical Pharmacology ◽

10.1016/j.bcp.2021.114649 ◽

2021 ◽

pp. 114649

Author(s):

Gerda M. Sanvee ◽

Jamal Bouitbir ◽

Stephan Krähenbühl

Keyword(s):

Toxic Effects ◽

C2c12 Myoblasts ◽

Myotube Formation

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Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

International Journal of Molecular Sciences ◽

10.3390/ijms21030745 ◽

2020 ◽

Vol 21 (3) ◽

pp. 745

Author(s):

Orly Lacham-Kaplan ◽

Donny M. Camera ◽

John A. Hawley

Keyword(s):

Synergistic Effect ◽

Striated Muscle ◽

Morphological Changes ◽

Estrogen Receptor Α ◽

Mitogen Activated Protein Kinase ◽

Myoblast Differentiation ◽

C2c12 Myoblasts ◽

Myotube Formation

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

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Cyclic Stretch Facilitates Myogenesis in C2C12 Myoblasts and Rescues Thiazolidinedione-Inhibited Myotube Formation

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2016.00027 ◽

2016 ◽

Vol 4 ◽

Cited By ~ 14

Author(s):

Ya-Ju Chang ◽

Yun-Ju Chen ◽

Chia-Wei Huang ◽

Shih-Chen Fan ◽

Bu-Miin Huang ◽

...

Keyword(s):

Cyclic Stretch ◽

C2c12 Myoblasts ◽

Myotube Formation

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Simvastatin impairs myoblast proliferation and myotube formation of C2C12 myoblasts and in mouse skeletal muscle

10.21203/rs.3.rs-348598/v1 ◽

2021 ◽

Author(s):

Gerda M Sanvee ◽

Jamal Bouitbir ◽

Stephan Kraehenbuehl

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Skeletal Muscle Regeneration ◽

Plasma Membrane Permeability ◽

Ki 67 ◽

C2c12 Myoblasts ◽

Mouse Skeletal Muscle ◽

Myotube Formation ◽

Myoblast Proliferation ◽

Adam 12

Abstract Statins reduce cardiovascular complications in patients with high LDL-cholesterol but are associated with myopathy. We investigated the possibility that statins impair skeletal muscle regeneration by assessing simvastatin toxicity on C2C12 myoblasts and myotubes and mouse skeletal muscle. Simvastatin increased plasma membrane permeability and decreased the cellular ATP content in both myoblasts and myotubes, but with a stronger effect on myoblasts. While insulin prevented cytotoxicity up to 8 hours after addition of simvastatin to myotubes, prevention in myoblasts required simultaneous addition. Mevalonate and geranylgeraniol also prevented simvastatin-associated cytotoxicity on myoblasts and myotubes. Simvastatin impaired the phosphorylation of the insulin receptor (IR β), Akt ser473 and S6rp, and increased phosphorylation of AMPK thr172 in both myotubes and myoblasts, which was prevented by insulin and mevalonate. Simvastatin impaired oxygen consumption and increased superoxide production by myoblasts and myotubes and induced apoptosis via cytochromc c release. In addition, simvastatin impaired proliferation and fusion of myoblasts to myotubes by inhibiting the expression of the nuclear transcription factor MyoD and of the metalloprotease ADAM-12. Decreased expression of the proliferation factor Ki-67 and of ADAM-12 were also observed in gastrocnemius of mice treated with simvastatin. In conclusion, myoblasts were more susceptible to the toxic effects of simvastatin and simvastatin impaired myoblast proliferation and myotube formation. Impaired muscle regeneration represents a new mechanism of statin myotoxicity and may be important in statin-associated myopathy.

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Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/7640272 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Stefano Testa ◽

Pietro D’Addabbo ◽

Ersilia Fornetti ◽

Roberta Belli ◽

Claudia Fuoco ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Heavy Chain ◽

Promoter Region ◽

Myogenic Differentiation ◽

Control Cell ◽

Gene Expressions ◽

C2c12 Myoblasts ◽

Myotube Formation ◽

First Time

The presence of tetraplex structures in the promoter region of the myogenic differentiation 1 gene (MyoD1) was investigated with a specific tetraplex-binding porphyrin (TMPyP4), to test its influence on the expression of MyoD1 itself and downstream-regulated genes during myogenic differentiation. TMPyP4-exposed C2C12 myoblasts, blocking MyoD1 transcription, proliferated reaching confluence and fused forming elongated structures, resembling myotubes, devoid of myosin heavy chain 3 (MHC) expression. Besides lack of MHC, upon MyoD1 inhibition, other myogenic gene expressions were also affected in treated cells, while untreated control cell culture showed normal myotube formation expressing MyoD1, Myog, MRF4, Myf5, and MHC. Unexpectedly, the myomaker (Mymk) gene expression was not affected upon TMPyP4 exposure during C2C12 myogenic differentiation. At the genomic level, the bioinformatic comparison of putative tetraplex sites found that three tetraplexes in MyoD1 and Myog are highly conserved in mammals, while Mymk and MHC did not show any conserved tetraplexes in the analysed regions. Thus, here, we report for the first time that the inhibition of the MyoD1 promoter function, stabilizing the tetraplex region, affects downstream myogenic genes by blocking their expression, while leaving the expression of Mymk unaltered. These results reveal the existence of two distinct pathways: one leading to cell fusion and one guaranteeing correct myotube differentiation.

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