Myoblast Differentiation Research Articles

Mitochondrial transplantation as a novel therapeutic approach in idiopathic inflammatory myopathy.

This study aimed to investigate the efficacy of mitochondrial transplantation as a therapeutic intervention for idiopathic inflammatory myopathy (IIM). This study used a comprehensive approach, incorporating both in vitro and in vivo IIM models, and conducted a first-in-human clinical trial to assess the effectiveness and safety of mitochondria isolated from human umbilical cord mesenchymal stem cells (PN-101). Mitochondria isolated from umbilical cord mesenchymal stem cells were designated as PN-101. The efficacy of PN-101 was assessed using myoblasts derived from patients with IIM and C2C12 mouse perforin/granzyme B-treated myoblasts as an in vitro IIM model. PN-101's effect on IIM was examined using C protein-induced myositis (CIM) mice as an in vivo model. The efficacy and safety of PN-101 were evaluated in a phase 1/2a clinical trial involving 9 adult patients with refractory polymyositis or dermatomyositis. The myoblasts derived from patients with IIM exhibited defects in mitochondrial function and myogenesis. PN-101 transplantation enhances muscle differentiation and mitochondrial function in IIM myoblasts. PN-101 also enhanced intracellular adenosine triphosphate content, cell viability, and myogenesis in C2C12 perforin/granzyme B-treated myoblasts. In an in vivo model, PN-101 reduced myositis severity by exhibiting anti-inflammatory effects and restoring the CIM-induced metabolic shift. In a phase 1/2a prospective clinical trial involving adult patients with refractory IIM, PN-101 demonstrated no severe adverse drug reactions and showed at least minimal improvement in the International Myositis Assessment and Clinical Studies Group (IMACS)-Total Improvement Scores (TISs) compared with baseline. PN-101 transplantation could serve as a novel treatment for IIM by enhancing mitochondrial repair and reducing inflammation in muscle tissues.

MMP2 regulates proliferation and differentiation in chicken primary myoblasts, and RNA-seq screens for key genes.

The growth and development of chicken skeletal muscle directly affects chicken meat production, which is very important for broiler industry. Matrix metallopeptidase 2 (MMP2) exists in skeletal muscle. However, the underlying regulating of MMP2 remain unknown. In this study, MMP2 promoted cell proliferation and inhibited cell differentiation after overexpression in chicken primary myoblasts cells (CPMs). When MMP2 was knocked down, it inhibited CPMs proliferation and promoted cell differentiation. Subsequently, RNA sequencing (RNA-seq) and bioinformatics analysis were performed on overexpressing MMP2. We identified 265 up-regulated genes and 229 down-regulated genes. Based on the fragments per kilobase million (FPKM) ≥ 10, the retained data were analyzed by Pearson correlation analysis. MMP2 was positively correlated with carboxypeptidase M (CPM), MSTRG.14120 and aldehyde dehydrogenase 1 family member A3 (ALDH1A3), and the correlation coefficient was the highest (0.998). MMP2 was negatively correlated with hes family bHLH transcription factor 1 (HES1), and the correlation coefficient was the highest (0.998). Go term was enriched in cellular components or biogenesis, cellular processes, and cell aggregation. KEGG was significantly enriched to the cancer pathway. qRT-PCR analysis validated the transcriptomic results of RNA-seq. In conclusion, these results provided new insights into the molecular mechanisms by which MMP2 affected the proliferation and differentiation of chicken myoblasts.

Puerarin Promotes the Migration and Differentiation of Myoblasts by Activating the FAK and PI3K/AKT Signaling Pathways.

Puerarin, a flavonoid compound present in the roots of radix puerariae, contributes to the development of tissues such as bone and nerve, but its role in skeletal muscle regeneration remains unclear. In this study, we employed C2C12 myoblasts and barium chloride (BaCl2)-based muscle injury models to investigate the effects of puerarin on myogenesis. Our study showed that puerarin stimulated the migration and differentiation of myoblasts in vitro. For the mechanism study, we found that puerarin's influence on cell migration was associated with the activation of FAK signaling; additionally, puerarin induced myoblast differentiation by upregulating the PI3K/AKT pathway. We also found that puerarin treatment could improve muscle regeneration following muscle injury. Taken together, our data indicate that puerarin facilitated myogenesis by promoting migration and differentiation, which suggests puerarin as a new candidate drug for the treatment of muscle loss diseases.

MUSTN1 Interaction With SMPX Regulates Muscle Development and Regeneration.

Pigs are important agricultural animals whose growth rate and meat production performance are related to muscle development. Musculoskeletal embryonic nuclear protein 1 (MUSTN1) participates in various biological processes, including myogenesis and growth in animals, but the physiological functions and mechanisms of porcine MUSTN1 on muscle development are unclear; thus, we aimed to elucidate them. We found that MUSTN1 was highly expressed in the muscles of fast-growing pigs. Functionally, MUSTN1 promoted myoblast proliferation and differentiation. MUSTN1 knockout mice exhibited reduced muscle mass and fibre cross-sectional area, decreased exercise endurance, and delayed muscle regeneration. Small muscle protein X-linked (SMPX) was identified as an interacting protein of MUSTN1, and its promotion of myogenic differentiation depended on MUSTN1. Furthermore, MUSTN1 stabilised SMPX and maintained myofiber morphology. This study suggests that MUSTN1 is a critical regulator in the control of muscle development and regeneration and is a potential target for animal genetic improvement and the treatment of human muscle disease.

Calponin 3 Regulates Myoblast Proliferation and Differentiation Through Actin Cytoskeleton Remodeling and YAP1-Mediated Signaling in Myoblasts.

An actin-binding protein, known as Calponin 3 (CNN3), modulates the remodeling of the actin cytoskeleton, a fundamental process for the maintenance of skeletal muscle homeostasis. Although the roles of CNN3 in actin remodeling have been established, its biological significance in myoblast differentiation remains largely unknown. This study investigated the functional significance of CNN3 in myogenic differentiation, along with its effects on actin remodeling and mechanosensitive signaling in C2C12 myoblasts. CNN3 knockdown led to a marked increase in filamentous actin, which promoted the nuclear localization of Yes-associated protein 1 (YAP1), a mechanosensitive transcriptional coactivator required for response to the mechanical cues that drive cell proliferation. Subsequently, CNN3 depletion enhanced myoblast proliferation by upregulating the expression of the YAP1 target genes related to cell cycle progression, such as cyclin B1, cyclin D1, and PCNA. According to a flow cytometry analysis, CNN3-deficient cells displayed higher S and G2/M phase fractions, which concurred with elevated proliferation rates. Furthermore, CNN3 knockdown impaired myogenic differentiation, as evidenced by reduced levels of MyoD, MyoG, and MyHC, key markers of myogenic commitment and maturation, and immunocytochemistry showed that myotube formation was diminished in CNN3-suppressed cells, which was supported by lower differentiation and fusion indices. These findings reveal that CNN3 is essential for myogenic differentiation, playing a key role in regulating actin remodeling and cellular localization of YAP1 to orchestrate the proliferation and differentiation in myogenic progenitor cells. This study highlights CNN3 as a critical regulator of skeletal myogenesis and suggests its therapeutic potential as a target for muscle atrophy and related disorders.

Ficus lindsayana Leaf Extract Protects C2C12 Mouse Myoblasts Against the Suppressive Effects of Bisphenol-A on Myogenic Differentiation.

Recently, toxicological and epidemiological research has provided strong support for the unfavorable effects of bisphenol-A (BPA, 2,2'-bis(4-hydroxyphenyl) propane) on myogenesis and its underlying mechanisms. Researchers have therefore been looking for new strategies to prevent or mitigate these injurious effects of BPA on the human body. It has been found that plant extracts may act as potential therapeutic agents or functional foods, preventing human diseases caused by BPA. We previously reported that Ficus lindsayana (FL) extract exhibits anti-inflammation activity in macrophages via suppressing the expression of inflammation-related molecules and anti-insulin resistance in inflammation-treated adipocytes. In this study, we investigated whether Ficus lindsayana leaf extract (FLLE) protects C2C12 mouse myoblasts against the suppressive effects of BPA on myogenic differentiation. The viability of BPA-stimulated C2C12 myoblasts was significantly increased when co-treated with FLLE (200 µg/mL), suggesting that the extract may lessen the inhibitory effects of BPA on cell division. We also found that FLLE significantly increased neo-myotube formation by inducing the fusion of myoblasts into multinucleated myotubes when compared to the BPA-treated control cells, without impacting cell viability. In addition, the levels of myogenin and myocyte enhancer factor 2A (MEF2A), which are crucial markers and regulators of myogenesis, were markedly increased by the addition of FLLE (50 µg/mL) to the BPA-treated C2C12 cells. This finding suggests that FLLE effectively improved myogenic differentiation in BPA-exposed myoblasts. FLLE treatment (50 µg/mL) significantly raised total Akt protein levels in the BPA-treated C2C12 cells, enhancing protein phosphorylation. In addition, FLLE (50 µg/mL) obviously increased the phosphorylation levels of p70S6K and 4E-BP1, key downstream targets of the Akt/mTOR signaling cascade, by elevating total p70S6K and 4E-BP1 levels. These results suggest that FLLE diminishes the decline in myogenic differentiation induced by BPA via the regulation of the myocyte differentiation-related signaling pathway. The information obtained from this study demonstrates the health benefits of this plant, which warrants further investigation as an alternative medicine, functional ingredient, or food supplement that can prevent the negative health effects of BPA or other toxicants.

Pervasive RNA-binding protein enrichment on TAD boundaries regulates TAD organization.

Mammalian genome is hierarchically organized by CTCF and cohesin through loop extrusion mechanism to facilitate the organization of topologically associating domains (TADs). Mounting evidence suggests additional factors/mechanisms exist to orchestrate TAD formation and maintenance. In this study, we investigate the potential role of RNA-binding proteins (RBPs) in TAD organization. By integrated analyses of global RBP binding and 3D genome mapping profiles from both K562 and HepG2 cells, our study unveils the prevalent enrichment of RBPs on TAD boundaries and define boundary-associated RBPs (baRBPs). We found that baRBP binding is correlated with enhanced TAD insulation strength and in a CTCF-independent manner. Moreover, baRBP binding is associated with nascent promoter transcription. Additional experimental testing was performed using RBFox2 as a paradigm. Knockdown of RBFox2 in K562 cells causes mild TAD reorganization. Moreover, RBFox2 enrichment on TAD boundaries is a conserved phenomenon in C2C12 myoblast (MB) cells. RBFox2 is downregulated and its bound boundaries are remodeled during MB differentiation into myotubes. Finally, transcriptional inhibition indeed decreases RBFox2 binding and disrupts TAD boundary insulation. Altogether, our findings demonstrate that RBPs can play an active role in modulating TAD organization through co-transcriptional association and synergistic actions with nascent promoter transcripts.

Impact of Magneto-Mechanical Actuation on Cell Differentiation: A Study Using Wireless, 3D-Printed Device and a Porous Ferrogel.

Cells perceive external and internally generated forces of different kinds, significantly impacting their cellular biology. In the relatively nascent field of mechanobiology, the impact of such forces is studied and further utilized to broaden the knowledge of cellular developmental pathways, disease progression, tissue engineering, and developing novel regenerative strategies. However, extensive considerations of mechanotransduction pathways for biomedical applications are still broadly limited due to a lack of affordable technologies in terms of devices and simple magnetic actuatable materials. Herein, synthesizing a monophasic, macroporous, in situ-fabricated gelatin-based ferrogel is reported using polyethylene glycol (PEG) coated-iron oxide (magnetite) particles with high magnetization. Developing a 3D printed, compact, and wireless device capable of providing a wide range of magneto-mechanical actuation using magnetic field susceptible materials in a noncontact manner is reported. Using the device and ferrogel, C2C12 myoblast differentiation is studied under magnetic field actuation, and significant differences in the myogenin, a differentiation marker, expression behavior are observed. Due to careful design considerations, robust component selection, and easy availability oflow-cost precursor for magnetic responsive material fabrication, the device-ferrogel combination can be easily adapted to routine biological studies, thereby helping mechanobiology to be utilized for developing exciting new biomedical strategies.

Polygonatum sibiricum polysaccharide ameliorates skeletal muscle aging and mitochondrial dysfunction via PI3K/Akt/mTOR signaling pathway.

Sarcopenia is currently a life-threatening disease for the elderly. Polygonatum sibiricum polysaccharide (PSP) has anti-oxidative stress and anti-inflammatory effects. However, the effects of PSP on skeletal muscle aging, myoblast differentiation and mitochondrial dysfunction through PI3K/Akt/mTOR signaling pathway has not been explored. To explore the effects and related mechanisms of PSP on muscle aging, myoblast differentiation and mitochondrial dysfunction. The chemical components of Polygonatum sibiricum were determined using the UHPLC-MS/MS method. The common targets and biological pathways between PSP and sarcopenia were investigated by network pharmacology analysis. In vitro C2C12 cells experiments were performed to reveal the effects of PSP on muscle aging, myotube differentiation, and mitochondrial damage. In addition, in vivo experiments were designed with the mouse model of D-gal-induced aging to evaluate the ameliorative impact of PSP on the skeletal muscle mass and function. Polygonatum sibiricum mainly included 466 bioactive components. Polygonatum sibiricum and sarcopenia had 278 common targets by network pharmacology analysis, which were associated with mitochondrial function and PI3K/Akt/mTOR pathway. In vitro experiment indicated that PSP significantly enhanced the viability of C2C12 cells and myotube differentiation by down-regulating p21, p53, p16, MuRF1 and Atrogin-1and up-regulating MyoD, Myogenin, and MyHC. However, the addition of LY294002, PI3K/Akt/mTOR pathway inhibitor, partially reversed the anti-aging and anti-oxidative stress effects of PSP. PSP also significantly improved mitochondrial membrane potential and decreased mitochondrial ROS levels by upregulating the phosphorylation of the PI3K/Akt/mTOR pathway. In vivo experimental data indicated that PSP significantly enhanced muscle strength, endurance, mass of skeletal muscle (quadriceps and gastrocnemius) and cross-sectional area (CSA) of skeletal muscle in D-gal induced aging mice. PSP exhibits significant ameliorative effects on skeletal muscle aging and atrophy, as well as mitochondrial dysfunction by activating the PI3K/Akt/mTOR signaling pathway. Our study uniquely investigates the effects of PSP on skeletal muscle aging and mitochondrial dysfunction with a specific focus on the PI3K/Akt/mTOR signaling pathway, which highlights the potential of PSP as a novel therapeutic agent for sarcopenia, offering an alternative to current treatment strategies.

STIMulating IRE1: how store-operated Ca2+ entry intersects with ER proteostasis

The endoplasmic reticulum (ER) controls intracellular Ca2+ dynamics. Depletion of ER Ca2+ stores results in short-term activation of store-operated Ca2+ entry (SOCE) via STIM1/Orai1 at ER-plasma membrane (ER-PM) contact sites (MCSs) and the long-term activation of the unfolded protein response (UPR), securing ER proteostasis. Recent work by Carreras-Sureda and colleagues describes a bidirectional control between IRE1 and STIM1 within the ER lumen that regulates ER-PM contact assembly and SOCE to sustain T-cell activation and myoblast differentiation.

Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder that significantly impairs muscle regeneration following injuries, contributing to numerous complications and reduced quality of life. There is an urgent need for therapeutic strategies that can enhance muscle regeneration and alleviate these pathological mechanisms. In this study, we evaluate the therapeutic efficacy of W-GA nanodots, which are composed of gallic acid (GA) and tungstate (W6+), on muscle regeneration in type 2 diabetes mellitus (T2D)-induced muscle injury, with a focus on their anti-inflammatory and antioxidative effects. This study synthesized ultrasmall W-GA nanodots that were optimized for improved stability and bioactivity under physiological conditions. In vitro assessments included cell viability, apoptosis, reactive oxygen species (ROS) generation, and myotube differentiation in C2C12 myoblasts under hyperglycemic conditions. In vivo, T2D was induced in C57BL/6 mice, followed by muscle injury and treatment with W-GA. Muscle repair, fibrosis, and functional recovery were assessed through histological analysis and gait analysis using the CatWalk system. The W-GA nanodots significantly enhanced muscle cell proliferation, decreased ROS, and reduced apoptosis in vitro. In vivo, compared with the control group, the W-GA-treated group exhibited notably improved muscle regeneration, decreased fibrosis, and enhanced functional recovery. The treatment notably modulated the inflammatory response and oxidative stress in diabetic muscle tissues, facilitating improved regenerative dynamics and muscle function. W-GA nanodots effectively counter the pathological mechanisms of diabetic myopathy by enhancing regenerative capacity and reducing oxidative stress and inflammation. This nanomedicine approach offers a promising therapeutic avenue for improving muscle health and overall quality of life in individuals suffering from T2D. However, further studies are needed to explore the clinical applications and long-term efficacy of these nanodots in preventing diabetic complications.

The Role of Primary Cilia in Myoblast Proliferation and Cell Cycle Regulation during Myogenesis

The process of mammalian myogenesis is fundamental to understanding muscle development and holds broad relevance across multiple fields, from developmental biology to regenerative medicine. This review highlights two key aspects: myoblast proliferation and the role of cilia in this process. Myoblasts, as muscle precursor cells, must undergo tightly regulated cycles of proliferation and differentiation to ensure proper muscle growth and function. Recent research has uncovered an essential role for primary cilia, hair-like sensory organelles on the cell surface, in modulating signaling pathways crucial to myogenesis. Cilium-mediated signaling appears to regulate various stages of myogenesis, including the control of myoblast differentiation. Furthermore, primary cilia undergo multiple cycles of formation and disassembly during myogenesis, presumably enabling detailed, context-dependent regulation of their functions. In particular, the regulation of myoblast proliferation through cell cycle control by primary cilia is an important topic that requires further investigation. By examining the interactions between primary cilia and myoblasts, this review aims to provide new insights into the molecular and cellular mechanisms driving muscle development, with potential applications for understanding muscle-related diseases and advancing therapeutic strategies. Additionally, advancements in imaging and image analysis technologies have become indispensable for studying these processes at the cellular level. This review also addresses these technological advancements and current challenges.Key words: myogenesis, myoblast, proliferation, cilia, imaging.

꾸지뽕나무 뿌리 추출물이 C2C12 세포의 mTOR 매개 근육 분화에 미치는 영향

Skeletal muscle differentiation is essential to maintain proper posture and long-term functional stability. Understanding the mechanisms regulating myogenic differentiation can facilitate the development of effective therapeutic approaches for muscle degeneration. Therefore, this study aimed to explore the effect of the Cudrania tricuspidataroot extract (CRE) on the myogenic differentiation of C2C12 myoblasts, focusing on the mechanistic target of rapamycin kinase (mTOR) pathway. CRE enhanced the insulin-like growth factor 2-muscle-specific enhancer activity,which was suppressed by rapamycin, thereby confirming mTOR pathwayinvolvement. Additionally, CRE significantly promoted myotube formation, fusion, and differentiation and modulated the levels of key transcription factors, myogenic differentiation factor (upregulated) and paired box-7 (downregulated), indicative of early-stage myogenesis. Overall, CRE promoted mTOR-mediated differentiation of C2C12 myoblasts, acting as a novel therapeutic candidate for muscle degeneration and associated disorders.

Oar‐miR‐411a‐5p Promotes Proliferation and Differentiation in Hu Sheep Myoblasts Under Heat Stress by Targeting SMAD2

ABSTRACTMicroRNAs (miRNAs) are endogenous noncoding RNAs that produce a remarked effect on regulating posttranscriptional gene expression. Our previous study identified a decrease in the expression of oar‐miR‐411a‐5p from umbilical plasma in intrauterine growth restriction (IUGR) Hu lambs subjected to maternal heat stress. In this study, we demonstrated that oar‐miR‐411a‐5p could modulate skeletal muscle development. Overexpression of oar‐miR‐411a‐5p significantly enhanced proliferation and differentiation in heat‐stressed Hu sheep myoblasts while suppressing apoptosis. Conversely, inhibition of oar‐miR‐411a‐5p resulted in opposing effects. Subsequently, RNAhybrid analysis revealed targeted sites between oar‐miR‐411a‐5p and the 3′ untranslated region (UTR) of SMAD2. This suggested that SMAD2 is a direct target gene of oar‐miR‐411a‐5p, as its expression was negatively modulated by oar‐miR‐411a‐5p, a finding corroborated by dual‐luciferase assay and RT‐qPCR. Furthermore, co‐transfection of oar‐miR‐411a‐5p and SMAD2 into Hu sheep myoblasts indicated that oar‐miR‐411a‐5p modulated heat‐stressed myoblast growth by targeting SMAD2. In conclusion, these findings elucidate the function of oar‐miR‐411a‐5p in promoting the development of heat‐stressed Hu sheep myoblasts, thereby enhancing our understanding of how miRNAs influence skeletal muscle growth in heat‐stressed Hu sheep.

Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of body weight occurring in about 80% of cancer patients, frequently representing the leading cause of death. Dietary intervention is emerging as a promising therapeutic strategy to counteract cancer-induced wasting. Serine is the second most-consumed amino acid (AA) by cancer cells and has emerged to be strictly necessary to preserve skeletal muscle structure and functionality. Here, we demonstrate that decreased serine availability during tumor progression promotes myotubes diameter reduction in vitro and induces muscle wasting in in vivo mice models. By investigating the metabolic crosstalk between colorectal cancer cells and muscle cells, we found that incubating myotubes with conditioned media from tumor cells relying on exogenous serine consumption triggers pronounced myotubes diameter reduction. Accordingly, culturing myotubes in a serine-free medium induces fibers width reduction and suppresses the activation of the AKT-mTORC1 pathway with consequent impairment in protein synthesis, increased protein degradation, and enhanced expression of the muscle atrophy-related genes Atrogin1 and MuRF1. In addition, serine-starved conditions affect myoblast differentiation and mitochondrial oxidative metabolism, finally inducing oxidative stress in myotubes. Consistently, serine dietary deprivation strongly strengthens cancer-associated weight loss and muscle atrophy in mice models. These findings uncover serine consumption by tumor cells as a previously undisclosed driver in cancer cachexia, opening new routes for possible therapeutic approaches.

Myoblast-derived ADAMTS-like 2 promotes skeletal muscle regeneration after injury

Skeletal muscle regeneration and functional recovery after minor injuries requires the activation of muscle-resident myogenic muscle stem cells (i.e. satellite cells) and their subsequent differentiation into myoblasts, myocytes, and ultimately myofibers. We recently identified secreted ADAMTS-like 2 (ADAMTSL2) as a pro-myogenic regulator of muscle development, where it promoted myoblast differentiation. Since myoblast differentiation is a key process in skeletal muscle regeneration, we here examined the role of ADAMTSL2 during muscle regeneration after BaCl2 injury. Specifically, we found that muscle regeneration was delayed after ablation of ADAMTSL2 in myogenic precursor cells and accelerated following injection of pro-myogenic ADAMTSL2 protein domains. Mechanistically, ADAMTSL2 regulated the number of committed myoblasts, which are the precursors for myocytes and regenerating myofibers. Collectively, our data support a role for myoblast-derived ADAMTSL2 as a positive regulator of muscle regeneration and provide a proof-of-concept for potential therapeutic applications.

Essential Role of Cortactin in Myogenic Differentiation: Regulating Actin Dynamics and Myocardin-Related Transcription Factor A-Serum Response Factor (MRTFA-SRF) Signaling.

Cortactin (CTTN) is an actin-binding protein regulating actin polymerization and stabilization, which are vital processes for maintaining skeletal muscle homeostasis. Despite the established function of CTTN in actin cytoskeletal dynamics, its role in the myogenic differentiation of progenitor cells remains largely unexplored. In this study, we investigated the role of CTTN in the myogenic differentiation of C2C12 myoblasts by analyzing its effects on actin cytoskeletal remodeling, myocardin-related transcription factor A (MRTFA) nuclear translocation, serum response factor (SRF) activation, expression of myogenic transcription factors, and myotube formation. CTTN expression declined during myogenic differentiation, paralleling the reduction in MyoD, suggesting a potential role in the early stages of myogenesis. We also found that CTTN knockdown in C2C12 myoblasts reduced filamentous actin, enhanced globular actin levels, and inhibited the nuclear translocation of MRTFA, resulting in suppressed SRF activity. This led to the subsequent downregulation of myogenic regulatory factors, such as MyoD and MyoG. Furthermore, CTTN knockdown reduced the nuclear localization of YAP1, a mechanosensitive transcription factor, further supporting its regulatory roles in cell cycle and proliferation. Consequently, CTTN depletion impeded proliferation, differentiation, and myotube formation in C2C12 myoblasts, highlighting its dual role in the coordination of cell cycle regulation and myogenic differentiation of progenitor cells during myogenesis. This study identifies CTTN as an essential regulator of myogenic differentiation via affecting the actin remodeling-MRTFA-SRF signaling axis and cell proliferation.

Enhancer Enh483 regulates myoblast proliferation and differentiation of buffalo myoblasts by targeting FAXC.

A detailed understanding of the precise regulatory mechanisms governing buffalo skeletal muscle is crucial for improving meat quality and yield. Proper skeletal muscle fate decisions necessitate the accurate regulation of key enhancers. This study screened nine potential enhancers linked to muscle development by analysing ATAC-seq data from buffalo myoblasts during the proliferative and differentiative phases. The enhancer activity of these candidates was confirmed in buffalo myoblasts, C2C12, and human skeletal muscle myoblasts using a dual-luciferase reporter system. The CRISPRi system and RT-qPCR were used to test the effects of 9 candidate enhancers on buffalo myoblasts. The active enhancer, Enh483, was selected based on its significant impact. Upon successful inhibition of Enh483 using CRISPRi, decreases in the expression of buffalo myogenic proliferation marker genes (PCNA, CyclinD1, and CDK2) were observed via RT-qPCR and Western blot. Subsequent proliferation assays using CCK-8 and EdU confirmed the promotive effect of Enh483 on buffalo myogenic cell proliferation. Following a 5-day differentiation induction period, changes in the expression of differentiation marker genes (MyoD1, MyoG, and MyHC) were analysed using RT-qPCR and Western blot. Additionally, fused myotube numbers were quantified, and the impact of Enh483 on buffalo myogenic cell differentiation was assessed through immunofluorescence. Our findings indicate that Enh483 facilitates buffalo myogenic cell differentiation. Further interaction analysis utilising 3C-PCR revealed a direct association between Enh483 and the FAXC promoter. In summary, the results from this study lay a foundational framework for deciphering the intricate regulatory mechanisms underpinning buffalo muscle development.

Melatonin supplementation to sows in mid to late gestation affects offspring circadian, myogenic, and growth factors in pre and postnatal skeletal muscle.

The neuroendocrine hormone melatonin is associated with circadian rhythms and has antioxidant and vasodilative properties. In cattle, melatonin rescues fetal growth during maternal nutrient restriction in a seasonally dependent manner, but melatonin research in swine is limited. The objective of this study was to evaluate effects of dietary melatonin supplementation during mid to late gestation on circadian rhythm and muscle growth and development of the longissimus dorsi in utero and postnatally. Sows received 20mg dietary melatonin daily (MEL) or no melatonin supplement (CON). Experiment 1 supplemented sows from gestational age (dGA) 38 ± 1 to 99 ± 1, experiment 2 supplemented sows from 41 to 106 ± 1 dGA, and experiment 3 supplemented sows from 60 dGA to farrowing. At harvest, morphometric measurements of all fetuses were taken, while the small (SM), medium (MED), and large (LG) piglet from each litter were used for further analysis. Prenatal data were analyzed using the MIXED procedure of SAS, and postnatal data were analyzed using the GLIMMIX procedure. Fetal morphometrics were analyzed for fixed effect of treatment, and transcript abundance was analyzed for treatment, time, and size. Postnatal parameters were analyzed for fixed effects of treatment, size, and production stage. In Exp. 1, MEL increased (P = 0.016) Period 1 (PER1) transcript abundance in the evening (PM) compared to the morning (AM). In Exp. 1, myogenin (MYOG) transcript abundance was increased (P = 0.033) in MEL fetuses in the AM compared to MEL in the PM. Myogenic factor 5 (MYF5) and paired box 7 (PAX7) were increased (P = 0.016) in the PM. Fetuses from MEL treated sows had increased (P < 0.05) BW, curve crown rump length, and head circumference in Exp. 2. In Exp. 2, MEL increased (P = 0.012) PER1 and Period 2 (PER2) transcript abundance in the PM. In Exp. 2, myoblast differentiation 1 (MYOD) was increased (P = 0.016) in SM and MED fetuses, while MYF5 and PAX7 were increased (P = 0.019) in SM fetuses. Postnatal BW was increased (P = 0.025) in MED and LG MEL treated offspring compared to CON. Insulin-like growth factor 1 (IGF1) was downregulated (P = 0.050) in MEL treated offspring, while insulin-like growth factor 1 receptor (IGF1R) was upregulated (P = 0.009) in MEL offspring. These results indicate that maternal melatonin supplementation during gestation modulates fetal circadian regulatory genes and alters myogenic genes during growth.

The miR-6240 target gene Igf2bp3 promotes myoblast fusion by enhancing myomaker mRNA stability

BackgroundMyoblast fusion plays a crucial role in myogenesis. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) functions as an RNA N6-methyladenosine reader and exerts important roles in various biological processes. While our prior study suggested Igf2bp3 contributes to myogenesis, its molecular regulatory mechanism is largely unclear.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and western blot were used for gene expression analysis. siRNA and CRISPRi technologies were conducted to knockdown the expression of Igf2bp3. CRISPR/Cas9 technology was performed to knockout Igf2bp3. The Igf2bp3 overexpression vector was designed using the pcDNA3.1(+) vector. Immunofluorescence detection was employed for subcellular localization and cell differentiation analysis. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays were conducted for cell proliferation and fusion detection. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized for regulatory mechanism analysis of Igf2bp3.ResultsThe overexpression of Igf2bp3 enhances myoblast fusion while knockdown of Igf2bp3 blocks the formation of myotubes. miR-6240 promotes myoblast proliferation while preventing myoblast differentiation and fusion by targeting the 3′ untranslated rgion (UTR) of Igf2bp3. Notably, the impacts of miR-6240 mimics on myoblast proliferation, differentiation, and fusion can be effectively counteracted by the overexpression of Igf2bp3. Moreover, our findings elucidate a direct interaction between Igf2bp3 and the myoblast fusion factor myomaker (Mymk). Igf2bp3 binds to Mymk to enhance its mRNA stability. This interaction results in increased expression of Mymk and heightened myoblast fusion.ConclusionsOur study unveils Igf2bp3 as a novel post-transcriptional regulator of myoblast fusion through the miR-6240/Mymk axis, significantly contributing to our understanding of skeletal muscle development.Graphical