• Journal of the Korean Society of Physical Medicine
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• v.14 no.2
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• pp.63-70
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• 2019

PURPOSE: While electrical stimulation (ES) is known to be a safe and flexible tool in rehabilitation therapy, it has had limited adoption in muscle regeneration. This study was performed to investigate whether ES can induce myogenic differentiation and to clarify the mechanism underlying the effects of ES on myogenic differentiation. METHODS: This study used rat L6 cell lines as myoblasts for myogenic differentiation. Electric stimulation was applied to the cells using a C-Pace EP culture pacer (IonOptix, Westwood, Ma, USA). The gene expressions of myogenic markers were examined using qPCR and immunochemistry. RESULTS: Our study showed that ES increased the thickness and length of myotubes during myogenic differentiation. It was found that ES increased the expression of myogenic markers, such as MyoD and Myogenin, and also activated the fusion of the myoblast cells. In addition, ES suppressed the expression of small GTPases, which can explain why ES promotes myogenic differentiation. CONCLUSION: We found that ES induced myogenic differentiation by suppressing small GTPases, inhibiting cell division. We suggest that ES-based therapies can contribute to the development of safe and efficient muscle regeneration.

• BMB Reports
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• v.53 no.11
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• pp.605-610
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• 2020

Skeletal myogenesis is a complex process that is finely regulated by myogenic transcription factors. Recent studies have shown that saturated fatty acids (SFA) can suppress the activation of myogenic transcription factors and impair the myogenic differentiation of progenitor cells. Despite the increasing evidence of the roles of miRNAs in myogenesis, the targets and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study examined the implications of SFA-induced miR-183-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) drastically reduced myogenic transcription factors, such as myoblast determination protein (MyoD), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C), and inhibited FHL1 expression and myogenic differentiation of C2C12 myoblasts, accompanied by the induction of miR-183-5p. The knockdown of FHL1 by siRNA inhibited myogenic differentiation of myoblasts. Interestingly, miR-183-5p inversely regulated the expression of FHL1, a crucial regulator of skeletal myogenesis, by targeting the 3'UTR of FHL1 mRNA. Furthermore, the transfection of miR-183-5p mimic suppressed the expression of MyoD, MyoG, MEF2C, and MyHC, and impaired the differentiation and myotube formation of myoblasts. Overall, this study highlights the role of miR-183-5p in myogenic differentiation through FHL1 repression and suggests a novel miRNA-mediated mechanism for myogenesis in a background of obesity.

• BMB Reports
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• v.55 no.2
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• pp.104-109
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• 2022

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.

• Molecules and Cells
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• v.38 no.4
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• pp.362-372
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• 2015

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 microarray 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 luciferase 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 Korean Journal of Physiology and Pharmacology
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• v.27 no.3
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• pp.277-287
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• 2023

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3'UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

• Biomedical Science Letters
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• v.29 no.3
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• pp.190-200
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• 2023

Skeletal muscle, essential for metabolism, thermoregulation, and immunity, undergoes myogenic differentiation that results in myotube formation. Trans-anethole (TA), the major constituent in essential oil produced by anise, star anise, and fennel, whose function in skeletal muscle has not yet been elucidated. Therefore, we investigated whether TA influenced muscle differentiation in mouse C2C12 myoblasts. Cells were induced to differentiate using a differentiation medium with or without TA (50 or 200 mg/mL) daily for 5 days. We measured myotube length and diameter after differentiation days 1, 3, and 5 and analyzed the expression of myogenic markers (myoblast determination protein 1, myogenin, myocyte enhancer factor 2, muscle creatine kinase, and myosin heavy chain) and atrophy-related genes (atrogin-1 and muscle ring finger-1 [MuRF-1]) using quantitative real-time PCR. Additionally, we observed the expression of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) using western blotting. Our data showed that TA significantly induced the formation of smaller and thinner myotubes and reduced the myogenic factor expression. Furthermore, the atrogin-1 and MuRF-1 expression markedly increased by TA. Consistent with these findings, TA significantly decreased the expression of total Akt and p-Akt. Taken together, these results indicate that TA inhibits myogenic differentiation of C2C12 cells via reduction of both total Akt and p-Akt. Our findings may provide valuable insights into the impact of PAA on individuals at risk of muscle atrophy.

• Journal of Animal Science and Technology
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• v.49 no.1
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• pp.25-32
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• 2007

The current study was undertaken to determine the effect of conjugated linoleic acid(CLA) isomers, cis-9, cis-11(c9c11), cis-9, trans-11(c9t11), trans-9, trans-11(t9t11), trans-10, cis-12(t10c12) on differentiation of pig preadipocytes and myogenic satellite cells during culture. Cells were isolated from new born pigs. The t10c12 isomer decreased differentiation of pig preadipocytes(92%), but not that of myogenic cells. The t9t11 isomer decreased differentiation of preadipocytes(14%) and increased that of myogenic cells (26%). No other CLA isomers affected differentiation of preadipocytes or myogenic cells. The effects of CLA on proliferation of preadipocytes and myogenic cells were small, compared to the effects on differentiation. These results suggest that CLA isomers have different effects on differentiaton of pig preadipocytes and myogenic cells.

• Biomedical Science Letters
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• v.29 no.1
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• pp.26-33
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• 2023

Ziziphus jujuba Mill. (ZJM), a traditional folk medicine and functional food in South Korea and China, has been reported to having pharmacological activities against anti-cancer, anti-oxidative, and anti-obesity. However, the effect of ZJM related to myoblast differentiation has not been known. In this study, we investigated the effects and mechanism of ZJM on myogenic differentiation of C2C12 cells. ZJM promotes myogenic differentiation and elevates the formation of multinucleated myotube compared to the control group. ZJM significantly increased the mRNA and protein expression of MyHC1, myogenin and MyoD in dose- and time-dependent manner. Interestingly, ZJM significantly inhibited the mRNA and protein expression of protein degradation markers, atrogin-1 and MuRF-1, in dose- and time-dependent manner. Taken together, our data suggest that ZJM is a potential functional candidate for muscle growth and strength by promoting myogenic differentiation.

• International Journal of Stem Cells
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• v.16 no.1
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• pp.78-92
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• 2023

Background and Objectives: This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen combined with 5-Aza in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs). Methods and Results: In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen combined with 5-Aza could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Myod, Myogenin and Mhc mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and 𝛽-actin protein expression). Finally, to verify the mechanism of myogenic differentiation of hydrogen-bound 5-Aza, we performed bioinformatics analysis and Western blot to detect the expression of p-P38 protein. Hydrogen combined with 5-Aza significantly enhanced the proliferation and myogenic differentiation of ADSCs in vitro by increasing the number of single-cell mitochondria and upregulating the expression of myogenic biomarkers such as Myod, Mhc and myotube formation. The expressions of p-P38 was up-regulated by hydrogen combined with 5-Aza. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor). Conclusions: Hydrogen alleviates the cytotoxicity of 5-Aza and synergistically promotes the myogenic differentiation capacity of adipose stem cells via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

• Biomedical Science Letters
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• v.10 no.3
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• pp.179-186
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• 2004

We examined a role of proteasome, the non-lysosomal multicatalytic protease complex,on the differentiation of chick embryonic myoblasts in culture. The peptide hydrolyzing activities of proteasome were found to change; the hydrolyzing activity against N-succinyl-Leu-Leu- Val- Tyr-7 -amido-4-methy1coumarin (SLLVY-AMC) was prominent and increased with myogenic differentiation. Proteasome inhibitors, N-carbobenzoxy-Leu-Leu-norvalinal (MG115) and N-carbobenzoxy-Ile-Glu (O-t-butyl)-Ala-Leucinal (PSI), blocked membrane fusion of myoblasts as well as the SLLVY-AMC hydrolyzing activity. Those inhibitory activities of the agents occurred in parallel, but were reversible and both cell fusion and the peptidase activity were restored when the agents were withdrawn from the culture medium. On the other hand, the agents caused accumulation of the ubiquitinylated proteins in the cytoskeletal proteins. These results suggest that each of the peptide hydrolyzing activities of proteasome is independently regulated during the myogenic differentiation and the chymotrypsin-like activity may play an important role in that process.