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Communications Biology May 2024Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding...
Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.
Topics: Animals; MicroRNAs; Muscle Development; Chickens; Gene Expression Profiling; RNA, Long Noncoding; Cell Differentiation; Cell Proliferation; Myoblasts; Chick Embryo
PubMed: 38698103
DOI: 10.1038/s42003-024-06226-1 -
NPJ Science of Food Apr 2024Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture...
Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.
PubMed: 38693150
DOI: 10.1038/s41538-024-00263-0 -
Genomics May 2024Skeletal muscle satellite cells (SMSCs) play an important role in regulating muscle growth and regeneration. Chromatin accessibility allows physical interactions that...
Skeletal muscle satellite cells (SMSCs) play an important role in regulating muscle growth and regeneration. Chromatin accessibility allows physical interactions that synergistically regulate gene expression through enhancers, promoters, insulators, and chromatin binding factors. However, the chromatin accessibility altas and its regulatory role in ovine myoblast differentiation is still unclear. Therefore, ATAC-seq and RNA-seq analysis were performed on ovine SMSCs at the proliferation stage (SCG) and differentiation stage (SCD). 17,460 DARs (differential accessibility regions) and 3732 DEGs (differentially expressed genes) were identified. Based on joint analysis of ATAC-seq and RNA-seq, we revealed that PI3K-Akt, TGF-β and other signaling pathways regulated SMSCs differentiation. We identified two novel candidate genes, FZD5 and MAP2K6, which may affect the proliferation and differentiation of SMSCs. Our data identify potential cis regulatory elements of ovine SMSCs. This study can provide a reference for exploring the mechanisms of the differentiation and regeneration of SMSCs in the future.
Topics: Animals; Satellite Cells, Skeletal Muscle; Cell Differentiation; Sheep; Muscle Development; Frizzled Receptors; RNA-Seq; Signal Transduction; Cells, Cultured; Chromatin Immunoprecipitation Sequencing; Transforming Growth Factor beta; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Cell Proliferation
PubMed: 38692440
DOI: 10.1016/j.ygeno.2024.110851 -
Science Advances May 2024Overactivation of the transforming growth factor-β (TGFβ) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to...
Overactivation of the transforming growth factor-β (TGFβ) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFβ induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFβ signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFβ target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFβ-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.
Topics: Muscular Dystrophy, Duchenne; Histone-Lysine N-Methyltransferase; Muscle Fibers, Skeletal; Transforming Growth Factor beta; Humans; Signal Transduction; Animals; Cell Differentiation; Mice; Myoblasts; Fibrosis; Gene Expression Regulation
PubMed: 38691608
DOI: 10.1126/sciadv.adj8042 -
Science Advances May 2024The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of...
The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.
Topics: Humans; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 4; CRISPR-Cas Systems; Genotype; Homeodomain Proteins; Muscular Dystrophy, Facioscapulohumeral; Myoblasts; PAX7 Transcription Factor; Phenotype; Telomere; Translocation, Genetic
PubMed: 38691604
DOI: 10.1126/sciadv.adl1922 -
American Journal of Physiology. Cell... May 2024Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal...
Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following . Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level. Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.
Topics: Humans; Muscle Fibers, Skeletal; Insulin-Like Growth Factor I; Cell Differentiation; Muscle Contraction; Phenotype; Cells, Cultured; Glucose Transporter Type 4; Myosin Heavy Chains; Glucose; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Muscle, Skeletal
PubMed: 38690930
DOI: 10.1152/ajpcell.00654.2023 -
Acta Biochimica Et Biophysica Sinica Apr 2024Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that...
Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of promotes C2C12 cell differentiation. Additionally, knockdown of , specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration . Mechanistically, we show that knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.
PubMed: 38690615
DOI: 10.3724/abbs.2024062 -
Communications Biology Apr 2024Adaptation to hypothermia is important for skeletal muscle cells under physiological stress and is used for therapeutic hypothermia (mild hypothermia at 32 °C). We...
Adaptation to hypothermia is important for skeletal muscle cells under physiological stress and is used for therapeutic hypothermia (mild hypothermia at 32 °C). We show that hypothermic preconditioning at 32 °C for 72 hours improves the differentiation of skeletal muscle myoblasts using both C2C12 and primary myoblasts isolated from 3 month and 18-month-old mice. We analyzed the cold-shock proteome of myoblasts exposed to hypothermia (32 °C for 6 and 48 h) and identified significant changes in pathways related to RNA processing and central carbon, fatty acid, and redox metabolism. The analysis revealed that levels of the cold-shock protein RBM3, an RNA-binding protein, increases with both acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation and maintenance of mitochondrial metabolism. We also show that overexpression of RBM3 at 37 °C is sufficient to promote mitochondrial metabolism, cellular proliferation, and differentiation of C2C12 and primary myoblasts. Proteomic analysis of C2C12 myoblasts overexpressing RBM3 show significant enrichment of pathways involved in fatty acid metabolism, RNA metabolism and the electron transport chain. Overall, we show that the cold-shock protein RBM3 is a critical factor that can be used for controlling the metabolic network of myoblasts.
Topics: Animals; RNA-Binding Proteins; Mice; Cell Differentiation; Myoblasts; Mitochondria; Proteome; Cold-Shock Response; Cell Line
PubMed: 38688991
DOI: 10.1038/s42003-024-06196-4 -
Frontiers in Cell and Developmental... 2024Regeneration and repair are prerequisites for maintaining effective function of skeletal muscle under high energy demands, and myogenic differentiation is one of the key... (Review)
Review
Regeneration and repair are prerequisites for maintaining effective function of skeletal muscle under high energy demands, and myogenic differentiation is one of the key steps in the regeneration and repair process. A striking feature of the process of myogenic differentiation is the alteration of mitochondria in number and function. Mitochondrial dysfunction can activate a number of transcriptional, translational and post-translational programmes and pathways to maintain cellular homeostasis under different types and degrees of stress, either through its own signaling or through constant signaling interactions with the nucleus and cytoplasm, a process known as the mitochondrial stress responses (MSRs). It is now believed that mitochondrial dysfunction is closely associated with a variety of muscle diseases caused by reduced levels of myogenic differentiation, suggesting the possibility that MSRs are involved in messaging during myogenic differentiation. Also, MSRs may be involved in myogenesis by promoting bioenergetic remodeling and assisting myoblast survival during myogenic differentiation. In this review, we will take MSRs as an entry point to explore its concrete regulatory mechanisms during myogenic differentiation, with a perspective to provide a theoretical basis for the treatment and repair of related muscle diseases.
PubMed: 38681520
DOI: 10.3389/fcell.2024.1381417 -
International Journal of Molecular... Apr 2024During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for...
During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from , which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of , , and but strongly repressed that of , suggesting the maintenance of myogenic stemness amid repressed expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with (C2C12-tTA-MyoD), implying its independence of the down-regulation of . In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of . Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.
Topics: Muscle Development; Animals; Mice; MyoD Protein; Cell Proliferation; Fibroblast Growth Factor 2; Myoblasts; Cell Line; PAX7 Transcription Factor; PAX3 Transcription Factor; Myogenic Regulatory Factor 5; Cyclin D1; Satellite Cells, Skeletal Muscle; Cell Differentiation; Proto-Oncogene Proteins c-akt; Muscle, Skeletal
PubMed: 38673893
DOI: 10.3390/ijms25084308