• The Korean Journal of Physiology and Pharmacology
• /
• 제27권3호
• /
• pp.277-287
• /
• 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.

• BMB Reports
• /
• 제55권2호
• /
• pp.104-109
• /
• 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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권5호
• /
• pp.649-658
• /
• 2010

Myogenic satellite cells (MSCs) are adult stem cells that activate and differentiate into myotubes. These stem cells are multipotent as they transdifferentiate into adipocyte-like cells, nerve cells and osteocytes. The effects of steroid hormones ($E_2$ and testosterone) were studied as a further step toward understanding the mechanism of MSCs proliferation and differentiation. In this study, MSCs were grown continuously for 87 days, implying that there may be a group of MSCs that continue to proliferate rather than undergoing differentiation. Isolated MSCs were cultured in Dulbecco's Modified Eagle's Medium supplemented with adult male, female or castrated bovine serum to observe the effect of steroid hormones on MSC proliferation. Cell proliferation was the highest in cultures supplemented with male serum followed by female and castrated serum. The positive effect of male hormone on MSC proliferation was confirmed by the observation of testosterone-mediated increased proliferation of cells cultured in medium supplemented with castrated serum. Furthermore, steroid hormone treatment of MSCs increased lipid accumulation in myotubes. Oil-Red-O staining showed that 17${\beta}$-estradiol ($E_2$) treatment avidly increased lipid accumulation, followed by $E_2$+testosterone and testosterone alone. To our knowledge, this is the first report of lipid accumulation in myotubes due to steroids in the absence of an adipogenic environment, and the effect of steroid hormones on cell proliferation using different types of adult bovine serum, a natural hormonal system. In conclusion, we found that sex steroids affect MSCs proliferation and differentiation, and lipid accumulation in myotubes.

• Journal of Animal Science and Technology
• /
• 제49권1호
• /
• pp.25-32
• /
• 2007

본 연구는 여러 CLA 이성체, 즉 cis-9, cis- 11(c9c11), cis-9, trans-11(c9t11), trans-9, trans-11 (t9t11) 및 trans-10, cis-12(t10c12)가 배양 중인 돼지 지방세포와 근육세포의 분화와 증식에 미치는 영향을 구명하고자 실시하였다. 세포는 신생자돈으로부터 분리했다. t10c12 이성체는 지방세포의 분화를 억제했는데(92%) 근육세포의 분화는 억제시키지 않았다. t9t11 이성체는 지방세포의 분화를 억제했는데(14%), 근육세포의 분화는 촉진시켰다(26%). 다른 CLA 이성체는 지방세포나 근육세포의 분화에 영향을 미치지 않았다. 그리고 CLA가 지방세포와 근육세포의 증식에 미치는 영향은 분화에 미치는 영향에 비해서 작았다. 위의 결과는 여러 CLA 이성체는 돼지 지방세포와 근육세포의 분화에 다른 영향을 미침을 나타낸다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제31권3호
• /
• pp.439-448
• /
• 2018

Objective: Myogenic satellite cells were isolated from semitendinosus muscle of prepubertal Korean black goat to observe the differential effect of linolenic and retinoic acid in thepresence of thiazolidinediones (TZD) and also to observe the production insulin sensitive preadipocyte. Methods: Cells were characterized for their stemness with cluster of differentiation 34 (CD34), CD13, CD106, CD44, Vimentin surface markers using flow cytometry. Cells characterized themselves as possessing significant (p<0.05) levels of CD13, CD34, CD106, Vimentin revealing their stemness potential. Goat myogenic satellite cells also exhibited CD44, indicating that they possessed a % of stemness factors of adipose lineage apart from their inherent stemness of paxillin factors 3/7. Results: Cells during proliferation stayed absolutely and firmly within the myogenic fate without any external cues and continued to show a significant (p<0.05) fusion index % to express myogenic differentiation, myosin heavy chain, and smooth muscle actin in 2% horse serum. However, confluent myogenic satellite cells were the ones easily turning into adipogenic lineage. Intriguingly, upregulation in adipose specific genetic markers such as peroxisome proliferation-activated receptor ${\gamma}$, adiponectin, lipoprotein lipase, and CCAAT/enhancer binding protein ${\alpha}$ were observed and confirmed in all given treatments. However, the amount of adipogenesis was found to be statistically significant (p<0.01) with linolenic acid as compared to retinoic acid in combination with TZD's. Conclusion: Retinoic acid was found to produce smaller preadipocytes which have been assumed to have insulin sensitization and hence retinoic acid could be used as a potential agent to sensitize tissues to insulin in combination with TZD's to treat diabetic conditions in humans and animals in future.

• International Journal of Stem Cells
• /
• 제16권1호
• /
• pp.78-92
• /
• 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.

• Animal Bioscience
• /
• 제36권2호
• /
• pp.295-306
• /
• 2023

Objective: Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Methods: Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. Results: The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. Conclusion: p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.

• Asian-Australasian Journal of Animal Sciences
• /
• 제22권10호
• /
• pp.1447-1460
• /
• 2009

Myogenic satellite cells have been isolated and identified by several recently elucidated molecular markers. Furthermore, knowledge about the precise function of these markers has provided insight into the early and terminal events of satellite cells during proliferation, differentiation, transdifferentiation, specification and activation. Recently, quiescent myogenic satellite cells have been associated with possession of Pax 3 and 7 that represent pluripotent stem cells capable of differentiating into other lineages. However, the mechanism by which myogenic satellite cells attain pluripotent potential remain elusive. Later, transdifferentiating ability of these cells to another lineage in the absence or presence of certain growth factor/ or agents has revolutionized the scope of these pluripotent myogenic satellite cells for manipulation of animal production (in terms of quality and quantity of muscle protein) and health (in terms of repair of skeletal muscle, cartilage or bone).

• 대한한방내과학회지
• /
• 제29권1호
• /
• pp.243-257
• /
• 2008

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. To determine MyoD expression by Dodamtang treatment, we compared the expression pattern of $C_{2}C_{12}$ mouse myoblasts that constitutively express myostatin with control cells. In vitro, increasing concentrations of Dodamtang reversibly prevented the myogenic blockage of myoblasts by myostatin expression. ELISA assay, Western and confocal analysis indicated that treatment of Dodamtang to the low serum culture media increased the levels of MyoD leading to the inhibition of myogenic differentiation by myostatin. The stable transfection of $C_{2}C_{12}$ myoblasts with myostatin expressing constructs did rescue MyoD-induced myogenic differentiation. Consistent with this, the treatment of Dodamtang rescued the expression of a MyoD in $C_{2}C_{12}$ myoblasts treated with myostatin. Taken together, these results suggest that induction of MyoD by Dodamtang inhibits myostatin activity and expression via SMAD3 resulting in the rescue of the myoblasts to differentiate into myotubes. Thus we propose that myostatin action by Dodamtang plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that blockage of functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

• Molecules and Cells
• /
• 제25권4호
• /
• pp.479-486
• /
• 2008

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with $10{\mu}M$ PD98059 prevented myogenin expression in response to a low dose of SB203580 ($3{\mu}M$) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.