Transcriptome-wide N6-Methyladenosine Methylome Profiling Reveals m6A Regulation of Skeletal Myoblast Differentiation in Cattle (Bos taurus)

N6-methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m6A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m6A-seq and RNA-seq to explore the m6A methylation modification pattern during bovine skeletal myogenesis. m6A-seq analysis revealed that m6A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691–8,094 m6A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m6A and mRNA levels, suggesting that m6A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m6A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m6A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m6A regulating skeletal muscle development.

Palmitic Acid-Induced miR-429-3p Impairs Myoblast Differentiation by Downregulating CFL2

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.

Requirement for PINCH in Skeletal Myoblast Differentiation

Background: Skeletal muscle is composed of bundles of myofibers ensheathed by extracellular matrix networks. Malformation of skeletal muscle during embryonic development results in congenital myopathies. Disease mechanisms of congenital myopathies remain unclear. PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Elucidation of the molecular mechanisms underlying skeletal myogenesis will offer new insights into pathogenesis of myopathies.Methods: We generated muscle-specific PINCH knock-out mice to study the functional role of PINCH in skeletal myogenesis. Histologic and Transmission Electron Microscopy analysis demonstrated that Impaired myogenic differentiation and maturation in mice with PINCH1 being ablated in skeletal muscle progenitors, and Ablation of PINCH1 and PINCH2 resulted in reduced size of muscle fibers and impaired multinucleation; Cell culture and immunostaining showed that defects in myoblast fusion and cytoskeleton assembly in PINCH double mutant mice; Western blotting showed that defects in expression of cytoskeleton proteins and proteins involved in myogenesis in DMUT skeletal muscles.Results: Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. Myofibers of PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes, but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin β1 and some cytoskeleton proteins, and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants, that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation, indicating a critical role of PINCH proteins in myogenic differentiation.Conclusion: Our results demonstrated an essential role of PINCH in skeletal myogenic differentiation.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

MicroRNA-494-3p inhibits formation of fast oxidative muscle fibres by targeting E1A-binding protein p300 in human-induced pluripotent stem cells

MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3′-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 (MYOD1) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3′-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.