RESUMEN
Insulin-like growth factor (IGF)-I mediates long-term activities that determine cell fate, including cell proliferation and differentiation. This study aimed to characterize the mechanisms by which IGF-I determines cell fate from the aspect of IGF-I signaling dynamics. In L6 myoblasts, myogenic differentiation proceeded under low IGF-I levels, whereas proliferation was enhanced under high levels. Mathematical and experimental analyses revealed that IGF-I signaling oscillated at low IGF-I levels but remained constant at high levels, suggesting that differences in IGF-I signaling dynamics determine cell fate. We previously reported that differential insulin receptor substrate (IRS)-1 levels generate a driving force for cell competition. Computational simulations and immunofluorescence analyses revealed that asynchronous IRS-1 protein oscillations were synchronized during myogenic processes through cell competition. Disturbances of cell competition impaired signaling synchronization and cell fusion, indicating that synchronization of IGF-I signaling oscillation is critical for myoblast cell fusion to form multinucleate myotubes.
Asunto(s)
Diferenciación Celular , Factor I del Crecimiento Similar a la Insulina , Mioblastos , Transducción de Señal , Factor I del Crecimiento Similar a la Insulina/metabolismo , Mioblastos/metabolismo , Mioblastos/citología , Animales , Línea Celular , Proliferación Celular , Desarrollo de Músculos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratas , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/citología , Fusión CelularRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína Forkhead Box O1 , Músculo Esquelético , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Humanos , Animales , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Masculino , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Femenino , Drosophila , Desarrollo de Músculos/fisiología , Persona de Mediana Edad , Anciano , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mioblastos/metabolismoRESUMEN
BACKGROUND: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP+ pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG)n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation. METHODS: DMPK (CTG)n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated. RESULTS: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1. CONCLUSIONS: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden.
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Diferenciación Celular , Fibras Musculares Esqueléticas , Distrofia Miotónica , Proteína Quinasa de Distrofia Miotónica , Pericitos , Humanos , Distrofia Miotónica/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/terapia , Distrofia Miotónica/patología , Fibras Musculares Esqueléticas/metabolismo , Pericitos/metabolismo , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismo , Mioblastos/metabolismo , Mioblastos/citología , Desarrollo de Músculos , Células Cultivadas , Masculino , Adulto , Femenino , Técnicas de Cocultivo , Persona de Mediana Edad , Fusión CelularRESUMEN
BACKGROUND: DNA G-quadruplexes (G4s) represent a distinctive class of non-canonical DNA secondary structures. Despite their recognition as potential therapeutic targets in some cancers, the developmental role of G4 structures remains enigmatic. Mammalian embryonic myogenesis studies are hindered by limitations, prompting the use of chicken embryo-derived myoblasts as a model to explore G4 dynamics. This study aims to reveal the embryonic G4s landscape and elucidate the underlying mechanisms for candidate G4s that influence embryonic myogenesis. RESULTS: This investigation unveils a significant reduction in G4s abundance during myogenesis. G4s stabilizer pyridostatin impedes embryonic myogenesis, emphasizing the regulatory role of G4s in this process. G4 Cut&Tag sequencing and RNA-seq analyses identify potential G4s and DEGs influencing embryonic myogenesis. Integration of G4 and DEG candidates identifies 32 G4s located in promoter regions capable of modulating gene transcription. WGBS elucidates DNA methylation dynamics during embryonic myogenesis. Coordinating transcriptome data with DNA G4s and DNA methylation profiles constructs a G4-DMR-DEG network, revealing nine interaction pairs. Notably, the NFATC2 promoter region sequence is confirmed to form a G4 structure, reducing promoter mCpG content and upregulating NFATC2 transcriptional activity. CONCLUSIONS: This comprehensive study unravels the first embryonic genomic G4s landscape, highlighting the regulatory role of NFATC2 G4 in orchestrating transcriptional activity through promoter DNA methylation during myogenesis.
Asunto(s)
G-Cuádruplex , Desarrollo de Músculos , Desarrollo de Músculos/genética , Animales , Embrión de Pollo , Mioblastos/metabolismo , Metilación de ADNRESUMEN
SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.
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Diferenciación Celular , Movimiento Celular , Desarrollo de Músculos , Músculo Esquelético , Proteína MioD , Factores de Empalme Serina-Arginina , Animales , Ratones , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Proteína MioD/metabolismo , Proteína MioD/genética , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Mioblastos/metabolismo , Empalme AlternativoRESUMEN
Senescent cells are characterized by multiple features such as increased expression of senescence-associated ß-galactosidase activity (SA ß-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase-Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.
Asunto(s)
Diferenciación Celular , Senescencia Celular , Mioblastos , Prostaglandina D2 , Proteínas Proto-Oncogénicas p21(ras) , Animales , Ratones , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Senescencia Celular/efectos de los fármacos , Mioblastos/metabolismo , Mioblastos/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Diferenciación Celular/efectos de los fármacos , Fenotipo Secretor Asociado a la Senescencia , Línea Celular , Doxorrubicina/farmacologíaRESUMEN
CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family that is closely related to tumor occurrence and progression, plays crucial roles in the immune system, cardiovascular system, and male reproductive system. Recently, CMTM3 has emerged as a potential target for treating diseases related to bone formation. However, additional studies are needed to understand the mechanisms by which CMTM3 regulates the process of osteogenic differentiation. In this study, we observed a significant downregulation of Cmtm3 expression during the transdifferentiation of C2C12 myoblasts into osteoblasts induced by BMP4. Cmtm3 overexpression suppressed proliferation and osteogenic differentiation in BMP4-induced C2C12 cells, whereas its knockdown conversely facilitated the process. Mechanistically, Cmtm3 overexpression upregulated both the protein and mRNA levels of p53 and p21. Conversely, Cmtm3 knockdown exerted the opposite effects. Additionally, we found that Cmtm3 interacts with p53 and increases protein stability by inhibiting proteasome-mediated ubiquitination and degradation. Notably, Trp53 downregulation abrogated the inhibitory effect of Cmtm3 on BMP4-induced proliferation and osteogenic differentiation of C2C12 myoblasts. Collectively, our findings provide key insights into the role of CMTM3 in regulating myoblast proliferation and transdifferentiation into osteoblasts, highlighting its significance in osteogenesis research.
Asunto(s)
Proliferación Celular , Transdiferenciación Celular , Quimiocinas , Proteínas con Dominio MARVEL , Mioblastos , Osteogénesis , Proteína p53 Supresora de Tumor , Animales , Humanos , Ratones , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Quimiocinas/metabolismo , Proteínas con Dominio MARVEL/metabolismo , Proteínas con Dominio MARVEL/genética , Mioblastos/metabolismo , Mioblastos/citología , Osteoblastos/metabolismo , Osteoblastos/citología , Osteogénesis/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
AIMS: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. MAIN METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. KEY FINDINGS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SIGNIFICANCE: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.
Asunto(s)
Diferenciación Celular , Peroxidación de Lípido , Mitocondrias , Mioblastos , Especies Reactivas de Oxígeno , Humanos , Especies Reactivas de Oxígeno/metabolismo , Diferenciación Celular/efectos de los fármacos , Mioblastos/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Desarrollo de Músculos/fisiología , Desarrollo de Músculos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Dinaminas/metabolismo , Fosforilación , Línea CelularRESUMEN
Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.
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Diferenciación Celular , Proliferación Celular , Pollos , Mioblastos , Telomerasa , Animales , Mioblastos/citología , Mioblastos/metabolismo , Línea Celular , Telomerasa/metabolismo , Telomerasa/genética , Desarrollo de Músculos/genética , Técnicas de Cultivo de Célula/métodos , Transfección , Embrión de PolloRESUMEN
Myogenesis is a highly orchestrated process whereby muscle precursor cells, myoblasts, develop into muscle fibers to form skeletal muscle during embryogenesis and regenerate adult muscle. Here, we studied the RNA-binding protein FUS (fused in sarcoma), which has been implicated in muscular and neuromuscular pathologies but is poorly characterized in myogenesis. Given that FUS levels declined in human and mouse models of skeletal myogenesis, and that silencing FUS enhanced myogenesis, we hypothesized that FUS might be a repressor of myogenic differentiation. Interestingly, overexpression of FUS delayed myogenesis, accompanied by slower production of muscle differentiation markers. To identify the mechanisms through which FUS inhibits myogenesis, we uncovered RNA targets of FUS by ribonucleoprotein immunoprecipitation (RIP) followed by RNA-sequencing (RNA-seq) analysis. Stringent selection of the bound transcripts uncovered Tnnt1 mRNA, encoding troponin T1 (TNNT1), as a major effector of FUS influence on myogenesis. We found that in myoblasts, FUS retained Tnnt1 mRNA in the nucleus, preventing TNNT1 expression; however, reduction of FUS during myogenesis or by silencing FUS released Tnnt1 mRNA for export to the cytoplasm, enabling TNNT1 translation and promoting myogenesis. We propose that FUS inhibits myogenesis by suppressing TNNT1 expression through a mechanism of nuclear Tnnt1 mRNA retention.
Asunto(s)
Diferenciación Celular , Desarrollo de Músculos , Mioblastos , Proteína FUS de Unión a ARN , Troponina T , Desarrollo de Músculos/genética , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Animales , Ratones , Humanos , Troponina T/metabolismo , Troponina T/genética , Mioblastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Músculo Esquelético/metabolismo , Línea CelularRESUMEN
Starch was mixed with a gel to produce a starch-based gel ink, which exhibited favorable printing characteristics. Through the optimization of infill density, 3D-printed scaffolds with 50% infill density and a highly ordered microstructure were successfully fabricated. The addition of calcium carbonate nanoparticles-glucono delta lactone (CaCO3 NPs-GDL) had notable effects on the swelling degree, in vitro digestion, water stability, and pore distribution of the scaffolds. When the amount of CaCO3 NPs in the starch-based gel was 0.075 g, the resulting 3D-printed gel scaffold with a 50% infill density proved to be the most suitable for cultivating cell-based meat. It featured pore sizes ranging from 80 to 120 µm and a compression modulus of 246.76 Pa. After 7 days of proliferation, the C2C12 mouse skeletal myoblasts exhibited an approximately 2.81-fold increase in cell numbers. The fusion index and maturation index of C2C12 cells on the scaffolds were 57.00 ± 0.45% and 34.56 ± 0.56%, respectively. The starch-based gel scaffolds demonstrated excellent water stability and in vitro degradability. Moreover, C2C12 cells exhibited successful proliferation and differentiation on the starch-based scaffolds, ultimately leading to the production of cell-based meat. This study developed a starch-based composite gel scaffold for the manufacture of cell-based meat.
Asunto(s)
Geles , Carne , Impresión Tridimensional , Almidón , Andamios del Tejido , Almidón/química , Animales , Ratones , Andamios del Tejido/química , Geles/química , Carne/análisis , Línea Celular , Proliferación Celular/efectos de los fármacos , Ingeniería de Tejidos , Mioblastos/citología , Mioblastos/metabolismo , Carbonato de Calcio/química , Diferenciación Celular/efectos de los fármacos , Carne in VitroRESUMEN
The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales , Factores de Transcripción MEF2 , Desarrollo de Músculos , Mioblastos , Animales , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Desarrollo de Músculos/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Mioblastos/metabolismo , Discos Imaginales/metabolismo , Factores de Transcripción MEF2/metabolismo , Factores de Transcripción MEF2/genética , Alas de Animales/metabolismo , Alas de Animales/crecimiento & desarrollo , Diferenciación Celular , Factores Reguladores MiogénicosRESUMEN
Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts.
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Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Islas de CpG , Metilación de ADN , Mioblastos , Fosfoproteínas , Animales , Metilación de ADN/genética , Metilación de ADN/efectos de los fármacos , Ratones , Mioblastos/metabolismo , Mioblastos/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Línea Celular , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Islas de CpG/genética , Decitabina/farmacología , Ratas , Azacitidina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cellular agriculture, an emerging technology, aims to produce animal-based products such as meat through scalable tissue culture methods. Traditional techniques rely on chemically undefined media using fetal bovine serum (FBS) or chemically defined media utilizing specific growth factors. To be a viable alternative to conventional meat production, cellular agriculture requires cost-effective materials with established supply chains for growth media. Here, we investigate hydrolysates from Kikuyu grass, Alfalfa grass, and cattle rearing pellets. We identified conditions that promote C2C12 myoblast cell growth in media containing 0.1% and 0% serum. These effects are more pronounced in combination with existing growth promoters such as insulin, transferrin, and selenium. Overall, the rearing pellet hydrolysates were most effective in promoting growth particularly when in combination with the growth promoters. Our findings suggest that leveraging these materials, along with known growth factors, can facilitate the development of improved, scalable, and commercially viable media for cellular agriculture.
Asunto(s)
Agricultura , Hidrolisados de Proteína , Animales , Bovinos , Agricultura/métodos , Ratones , Hidrolisados de Proteína/química , Medicago sativa/química , Medicago sativa/crecimiento & desarrollo , Medicago sativa/metabolismo , Línea Celular , Mioblastos/citología , Mioblastos/metabolismo , Proliferación Celular/efectos de los fármacos , Medios de Cultivo/metabolismo , Medios de Cultivo/química , Poaceae/química , Poaceae/metabolismoRESUMEN
Myoblast is a kind of activated muscle stem cell. Its biological activities, such as proliferation, migration, differentiation, and fusion, play a crucial role in maintaining the integrity of the skeletal muscle system. These activities of myoblasts can be significantly influenced by the extracellular matrix. Collagen, being a principal constituent of the extracellular matrix, substantially influences these biological activities. In skeletal muscle, collagen I and III are two kinds of primary collagen types. Their influence on myoblasts and the difference between them remain ambiguous. The purpose of this study is to discover the influence of collagen I and III on biological function of myoblasts and compare their differences. We used C2C12 cell line and primary myoblasts to discover the effect of collagen I and III on proliferation, migration and differentiation of myoblasts and then performed the transcriptome sequencing and analysis. The results showed that both collagen I and III enhanced the proliferation of myoblasts, with no statistical difference between them. Similarly, collagen I and III enhanced the migration of myoblasts, with collagen I was more pronounced in Transwell assay. On the contrary, collagen I and III inhibited myoblasts differentiation, with collagen III was more pronounced at gene expression level. The transcriptome sequencing identified DEGs and enrichment analysis elucidated different terms between Type I and III collagen. Collectively, our research preliminarily elucidated the influence of collagen I and III on myoblasts and their difference and provided the preliminary experimental foundation for subsequent research.
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Diferenciación Celular , Movimiento Celular , Proliferación Celular , Colágeno Tipo I , Mioblastos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Mioblastos/citología , Mioblastos/metabolismo , Animales , Ratones , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Línea CelularRESUMEN
Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an L-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant L-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.
Asunto(s)
Técnicas de Cocultivo , Ácido Láctico , Carne , Animales , Ácido Láctico/metabolismo , Ratas , Técnicas de Cocultivo/métodos , Medio de Cultivo Libre de Suero , Proliferación Celular , Synechococcus/metabolismo , Synechococcus/genética , Synechococcus/crecimiento & desarrollo , Línea Celular , Mioblastos/metabolismo , Mioblastos/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Carne in VitroRESUMEN
Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.
Asunto(s)
Fibras Musculares Esqueléticas , Miositis por Cuerpos de Inclusión , Humanos , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/genética , Miositis por Cuerpos de Inclusión/patología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Diferenciación Celular , Anciano , Femenino , Masculino , Células Cultivadas , Transcriptoma , Mioblastos/metabolismo , Mioblastos/patología , Biopsia , Perfilación de la Expresión Génica , Persona de Mediana EdadRESUMEN
Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.
Asunto(s)
Diferenciación Celular , Ratones Endogámicos mdx , MicroARNs , Desarrollo de Músculos , Mioblastos , Miogenina , Animales , Mioblastos/metabolismo , Ratones , Desarrollo de Músculos/fisiología , Desarrollo de Músculos/genética , Línea Celular , Miogenina/genética , Miogenina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Silenciamiento del Gen , Factor de Transcripción PAX7/metabolismo , Factor de Transcripción PAX7/genética , Regulación de la Expresión Génica , SestrinasRESUMEN
Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.
Asunto(s)
Fusión Celular , Endorribonucleasas , Desarrollo de Músculos , Músculo Esquelético , Mioblastos , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Proteína 1 de Unión a la X-Box , Animales , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Ratones , Mioblastos/metabolismo , Mioblastos/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Desarrollo de Músculos/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Células Satélite del Músculo Esquelético/metabolismo , Regeneración/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica , Proteínas de la Membrana , Proteínas MuscularesRESUMEN
We have recently identified the uncharacterized ZNF555 protein as a component of a productive complex involved in the morbid function of the 4qA locus in facioscapulohumeral dystrophy. Subsequently named DiPRO1 (Death, Differentiation, and PROliferation related PROtein 1), our study provides substantial evidence of its role in the differentiation and proliferation of human myoblasts. DiPRO1 operates through the regulatory binding regions of SIX1, a master regulator of myogenesis. Its relevance extends to mesenchymal tumors, such as rhabdomyosarcoma (RMS) and Ewing sarcoma, where DiPRO1 acts as a repressor via the epigenetic regulators TIF1B and UHRF1, maintaining methylation of cis-regulatory elements and gene promoters. Loss of DiPRO1 mimics the host defense response to virus, awakening retrotransposable repeats and the ZNF/KZFP gene family. This enables the eradication of cancer cells, reprogramming the cellular decision balance towards inflammation and/or apoptosis by controlling TNF-α via NF-kappaB signaling. Finally, our results highlight the vulnerability of mesenchymal cancer tumors to si/shDiPRO1-based nanomedicines, positioning DiPRO1 as a potential therapeutic target.