myoblast proliferation
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MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs and play critical roles in the regulation of post-transcriptional gene expression. Our previous study uncovered that chi-miR-487b-3p is widespread in different goat tissues, which is significantly higher in muscle, especially in lamb. Here, we demonstrate the role of chi-miR-487b-3p as a myogenic miRNA that regulates skeletal muscle development. chi-miR-487b-3p overexpression was demonstrated to significantly inhibit goat myoblast proliferation and differentiation, whereas chi-miR-487b-3p inhibition resulted in the opposite effects. Next, chi-miR-487b-3p was predicted to target the 3′UTR of insulin receptor substrate 1 (IRS1) gene by Target-Scan and miRDB. The results of dual-luciferase assay, RT-qPCR, and western blot all confirmed that IRS1 might be a direct target of chi-miR-487b-3p as its expression was negatively regulated by chi-miR-487b-3p. siRNA silencing of IRS1 further demonstrated significant inhibition on goat myoblast proliferation and differentiation, confirming the effect of IRS1 downregulation by chi-miR-487b-3p in myogenesis. In addition, chi-miR-487b-3p knockout goat myoblast clones were generated using CRISPR/Cas9 technology, and we further illustrated that chi-miR-487b-3p regulates goat myoblast growth through the PI3K/Akt signaling pathway by targeting IRS1. Collectively, our work demonstrated that chi-miR-487b-3p is a potent inhibitor of skeletal myogenesis and provided new insights into the mechanisms of miRNA on the regulation of goat growth.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.

Intrauterine growth restricted (IUGR) fetuses are born with lower skeletal muscle mass, fewer proliferating myoblasts, and fewer myofibers compared to normally growing fetuses. Plasma concentrations of insulin, a myogenic growth factor, are lower in IUGR fetuses. We hypothesized that a two-week insulin infusion at 75% gestation would increase myoblast proliferation and fiber number in IUGR fetal sheep. Catheterized control fetuses received saline (CON-S, n=6), and the IUGR fetuses received either saline (IUGR-S, n=7) or insulin (IUGR-I, 0.014 ± 0.001 units/kg/hr, n=11) for 14 days. Fetal arterial blood gases and plasma amino acid levels were measured. Fetal skeletal muscles (biceps femoris, BF; and flexor digitorum superficialis, FDS) and pancreases were collected at necropsy (126 ± 2 dGA) for immunochemistry analysis, real-time qPCR, or flow cytometry. Insulin concentrations in IUGR-I and IUGR-S were lower vs. CON-S (P ≤ 0.05, group). Fetal arterial PaO2, O2 content, and glucose concentrations were lower in IUGR-I vs. CON-S (P ≤ 0.01) throughout the infusion period. IGF-1 concentrations tended to be higher in IUGR-I vs. IUGR-S (P=0.06), but both were lower vs. CON-S (P ≤ 0.0001, group). More myoblasts were in S/G2 cell cycle stage in IUGR-I vs. both IUGR-S and CON-S (145% and 113%, respectively, P ≤ 0.01). IUGR-I FDS muscle weighed 40% less and had 40% lower fiber number vs. CON-S (P ≤ 0.05) but were not different from IUGR-S. Myonuclear number per fiber and the mRNA expression levels of muscle regulatory factors were not different between groups. While the pancreatic β-cell mass was lower in both IUGR-I and IUGR-S compared to CON-S, the IUGR groups were not different from each other indicating that feedback inhibition by endogenous insulin did not reduce β-cell mass. A two-week insulin infusion at 75% gestation promoted myoblast proliferation in the IUGR fetus but did not increase fiber or myonuclear number. Myoblasts in the IUGR fetus retain the capacity to proliferate in response to mitogenic stimuli, but intrinsic defects in the fetal myoblast by 75% gestation may limit the capacity to restore fiber number.

The evolutionarily conserved signaling pathway Notch is unequivocally essential for embryogenesis. Notch’s contribution to the muscle repair process in adult tissue is complex and obscure but necessary. Notch integrates with other signals in a functional antagonist manner to direct myoblast activity and ultimately complete muscle repair. There is profound recent evidence describing plausible mechanisms of Notch in muscle repair. However, the story is not definitive as evidence is slowly emerging that negates Notch’s importance in myoblast proliferation. The purpose of this review article is to examine the prominent evidence and associated mechanisms of Notch’s contribution to the myogenic repair phases. In addition, we discuss the emerging roles of Notch in diseases associated with muscle atrophy. Understanding the mechanisms of Notch’s orchestration is useful for developing therapeutic targets for disease.

MicroRNAs are known to play a critical role in skeletal myogenesis and maintenance, and cofilin-2 (CFL2) is necessary for actin cytoskeleton dynamics and myogenic differentiation. Nonetheless, target molecules and the modes of action of miRNAs, especially those responsible for the inhibitory mechanism on the myogenesis by saturated fatty acids (SFA) or obesity, still remain unclear. Here, we reported the role played by miR-429-3p on CFL2 expression, actin filament dynamics, myoblast proliferation, and myogenic differentiation in C2C12 cells. Palmitic acid (PA), the most abundant SFA in diet, inhibited the myogenic differentiation of myoblasts, accompanied by CFL2 reduction and miR-429-3p induction. Interestingly, miR-429-3p suppressed the expression of CFL2 by targeting the 3′UTR of CFL2 mRNA directly. Transfection of miR-429-3p mimic in myoblasts increased F-actin formation and augmented nuclear YAP level, thereby promoting cell cycle progression and myoblast proliferation. Moreover, miR-429-3p mimic drastically suppressed the expressions of myogenic factors, such as MyoD, MyoG, and MyHC, and impaired myogenic differentiation of C2C12 cells. Therefore, this study unveiled the crucial role of miR-429-3p in myogenic differentiation through the suppression of CFL2 and provided implications of SFA-induced miRNA in the regulation of actin dynamics and skeletal myogenesis.

Previous studies have shown that gga-miR-2954 was highly expressed in the gonads and other tissues of male chickens, including muscle tissue. Yin Yang1 (YY1), which has functions in mammalian skeletal muscle development, was predicted to be a target gene of gga-miR-2954. The purpose of this study was to investigate whether gga-miR-2954 plays a role in skeletal muscle development by targeting YY1, and evaluate its function in the sexual dimorphism development of chicken muscle. Here, all the temporal and spatial expression profiles in chicken embryonic muscles showed that gga-miR-2954 is highly expressed in males and mainly localized in cytoplasm. Gga-miR-2954 exhibited upregulated expression of in vitro myoblast differentiation stages. Next, through the overexpression and loss-of-function experiments performed in chicken primary myoblasts, we found that gga-miR-2954 inhibited myoblast proliferation but promoted differentiation. During myogenesis, gga-miR-2954 could suppress the expression of YY1, which promoted myoblast proliferation and inhibited the process of myoblast cell differentiation into multinucleated myotubes. Overall, these findings reveal a novel role of gga-miR-2954 in skeletal muscle development through its function of the myoblast proliferation and differentiation by suppressing the expression of YY1. Moreover, gga-miR-2954 may contribute to the sex difference in chicken muscle development.

Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. The present research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.