RESUMEN
Skeletal muscles undergo robust regeneration upon injury, and infiltrating immune cells play a major role in not only clearing damaged tissues but also regulating the myogenic process through secreted cytokines. Chemokine C-C motif ligand 8 (Ccl8), along with Ccl2 and Ccl7, has been reported to mediate inflammatory responses to suppress muscle regeneration. Ccl8 is also expressed by muscle cells, but a role of the muscle cell-derived Ccl8 in myogenesis has not been reported. In this study, we found that knockdown of Ccl8, but not Ccl2 or Ccl7, led to increased differentiation of C2C12 myoblasts. Analysis of existing single-cell transcriptomic datasets revealed that both immune cells and muscle stem cells (MuSCs) in regenerating muscles express Ccl8, with the expression by MuSCs at a much lower level, and that the temporal patterns of Ccl8 expression were different in MuSCs and macrophages. To probe a function of muscle cell-derived Ccl8 in vivo, we utilized a mouse system in which Cas9 was expressed in Pax7+ myogenic progenitor cells (MPCs) and Ccl8 gene editing was induced by AAV9-delivered sgRNA. Depletion of Ccl8 in Pax7+ MPCs resulted in accelerated muscle regeneration after barium chloride-induced injury in both young and middle-aged mice, and intramuscular administration of a recombinant Ccl8 reversed the phenotype. Accelerated regeneration was also observed when Ccl8 was depleted in Myf5+ or MyoD+ MPCs by similar approaches. Our results suggest that muscle cell-derived Ccl8 plays a unique role in regulating the initiation of myogenic differentiation during injury-induced muscle regeneration.
Asunto(s)
Diferenciación Celular , Quimiocina CCL8 , Desarrollo de Músculos , Músculo Esquelético , Mioblastos , Regeneración , Animales , Ratones , Regeneración/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Músculo Esquelético/lesiones , Desarrollo de Músculos/fisiología , Quimiocina CCL8/metabolismo , Quimiocina CCL8/genética , Mioblastos/metabolismo , Mioblastos/fisiología , Ratones Endogámicos C57BL , Línea Celular , Masculino , Quimiocina CCL7/metabolismo , Quimiocina CCL7/genética , Macrófagos/metabolismoRESUMEN
Tumor necrosis factor α (TNFα) exhibits diverse biological functions; however, its regulatory roles in myogenesis are not fully understood. In the present study, we explored the function of TNFα in myoblast proliferation, differentiation, migration, and myotube fusion in primary myoblasts and C2C12 cells. To this end, we constructed TNFα muscle-conditional knockout ( TNFα-CKO) mice and compared them with flox mice to assess the effects of TNFα knockout on skeletal muscles. Results indicated that TNFα-CKO mice displayed phenotypes such as accelerated muscle development, enhanced regenerative capacity, and improved exercise endurance compared to flox mice, with no significant differences observed in major visceral organs or skeletal structure. Using label-free proteomic analysis, we found that TNFα-CKO altered the distribution of several muscle development-related proteins, such as Hira, Casz1, Casp7, Arhgap10, Gas1, Diaph1, Map3k20, Cfl2, and Igf2, in the nucleus and cytoplasm. Gene set enrichment analysis (GSEA) further revealed that TNFα deficiency resulted in positive enrichment in oxidative phosphorylation and MyoD targets and negative enrichment in JAK-STAT signaling. These findings suggest that TNFα-CKO positively regulates muscle growth and development, possibly via these newly identified targets and pathways.
Asunto(s)
Ratones Noqueados , Desarrollo de Músculos , Músculo Esquelético , Regeneración , Factor de Necrosis Tumoral alfa , Animales , Desarrollo de Músculos/fisiología , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Línea Celular , Diferenciación Celular , Mioblastos/metabolismo , Mioblastos/fisiologíaRESUMEN
Repeated rounds of fusion between apposing myoblasts allow muscles to become multinucleated. New research finds that myoblasts undergoing fusion in the Drosophila embryo respond to hormone signaling from a nearby tissue, resulting in the activation of a myoblast-specific gene necessary for the fusion process.
Asunto(s)
Fusión Celular , Mioblastos , Animales , Mioblastos/metabolismo , Mioblastos/fisiología , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transducción de Señal , Comunicación CelularRESUMEN
Muscle injuries are common among athletes and often treated with platelet-rich plasma (PRP). However, whether the leukocyte concentration affects the efficacy of PRP in treating muscle injuries remains unclear. This study investigated the effects of leukocyte-poor platelet-rich plasma (LP-PRP) and leukocyte-rich platelet-rich plasma (LR-PRP) on myoblast proliferation and the molecular mechanisms underlying these effects. Myoblasts were treated with 0.5% LP-PRP, 0.5% LR-PRP, 1% LP-PRP, or 1% LR-PRP for 24 h. The gene expression of the LP-PRP- and LR-PRP-treated myoblasts was determined using RNA sequencing analysis. Cell proliferation was evaluated using an bromodeoxyuridine (BrdU) assay, and cell cycle progression was assessed through flow cytometry. The expression of cyclin A, cyclin-dependent kinase 1 (cdk1), and cdk2 was examined using Western blotting. The expression of myoblast determination protein 1 (MyoD1) was examined through Western blotting and immunofluorescence staining. The LP-PRP and LR-PRP both promoted the proliferation of myoblasts and increased differential gene expression of myoblasts. Moreover, the LP-PRP and LR-PRP substantially upregulated the expression of cyclin A, cdk1, and cdk2. MyoD1 expression was induced in the LP-PRP and LR-PRP-treated myoblasts. Our results corroborate the finding that LP-PRP and LR-PRP have similar positive effects on myoblast proliferation and MyoD1 expression.
Asunto(s)
Ciclina A , Mioblastos , Plasma Rico en Plaquetas , Humanos , Proteína Quinasa CDC2/metabolismo , Proliferación Celular , Ciclina A/metabolismo , Leucocitos/fisiología , Mioblastos/fisiología , Plasma Rico en Plaquetas/metabolismo , Regulación hacia ArribaRESUMEN
Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.
Asunto(s)
Músculo Esquelético , Pez Cebra , Animales , Ratones , Pez Cebra/genética , Músculo Esquelético/fisiología , Miofibrillas/fisiología , Morfogénesis , Mioblastos/fisiologíaRESUMEN
Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.
Asunto(s)
Canales Iónicos , Regeneración , Células Satélite del Músculo Esquelético , Animales , Ratones , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Canales Iónicos/genética , Canales Iónicos/fisiología , Músculo Esquelético/fisiología , Mioblastos/fisiología , Transducción de Señal , Células Satélite del Músculo Esquelético/fisiología , Regeneración/genética , Regeneración/fisiologíaRESUMEN
MircoRNAs (miRNAs) play an important role in skeletal muscle development. Previous study had found that miR-495-3p was differentially expressed in fetal and adult goat skeletal muscle, but its function in myogenic proliferation and differentiation are unclear. Herein, we found the expression of miR-495-3p in C2C12 was downregulated during proliferation stage and upregulated during differentiation stage. Functionally, overexpression of miR-495-3p in C2C12 inhibited proliferation, and promoted myogenic differentiation. Mechanistically, the luciferase reporter assay demonstrated that cadherin 2 (CDH2) was a potential target gene of miR-495-3p. Importantly, overexpression of miR-495-3p inhibited CDH2 expression. Furthermore, knockdown of CDH2 in C2C12 inhibited proliferation and promoted myogenic differentiation. Together, the results showed that miR-495-3p inhibits C2C12 proliferation and promotes myogenic differentiation through targeting CDH2.
Asunto(s)
MicroARNs , Animales , MicroARNs/genética , MicroARNs/metabolismo , Mioblastos/fisiología , Proliferación Celular/genética , Cadherinas/genética , Cadherinas/metabolismo , Desarrollo de Músculos/genética , Diferenciación Celular/genéticaRESUMEN
1. Methyltransferase-like 21C (METTL21C) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) play important roles in the proliferation of chicken myoblasts. However, it remains unclear whether there is protein-protein interaction between METTL21C and IGF2BP1 to regulate proliferation of chicken myoblasts.2. In this study, the Igf2bp1 gene was amplified from cDNA of liver tissue of Lueyang black-bone chicken to construct the overexpression vector HA-Igf2bp1. The HA-Igf2bp1 and Flag-Mettl21c vectors were individually transfected and co-transfected into HEK293T, respectively. Co-immunoprecipitation (Co-IP) assay indicated a protein-protein interaction between METTL21C and IGF2BP1.3. Using the Western blotting and LC-MS/MS, it was found that METTL21C could mediate the lysine methylation modification of IGF2BP1. Furthermore, the His-tagged overexpression vector HA-Igf2bp1-His was constructed, transfected and co-transfected with Flag-Mettl21c into HEK293T. His-tagged IGF2BP1 was purified by nickel ion affinity chromatography. Western blotting revealed that IGF2BP1 was successfully purified, and the trimethylation modification level of co-transfection group was significantly elevated compared with the single-transfection Igf2bp1 group.4. Mettl21c and Igf2bp1 overexpression vectors were transfected and co-transfected into primary chicken myoblasts, respectively. The results of 5-ethynyl-2'-deoxyuridine assay and the expression level of Pax7 and MyoD indicated that overexpression of Igf2bp1 alone inhibited the chicken myoblast proliferation, whereas co-expression of Mettl21c and Igf2bp1 eliminated the inhibitory effects of Igf2bp1, thereby favouring cell proliferation and differentiation.5. The results, for the first time, revealed that METTL21C mediated the lysine trimethylation modification of IGF2BP1 to regulate the proliferation of chicken myoblasts, which provided a new insight into in-depth analysis of the molecular mechanism of METTL21C methylation involved in regulating the growth and development of skeletal muscle in Lueyang black-bone chicken.
Asunto(s)
Pollos , Lisina , Animales , Humanos , Pollos/genética , Lisina/metabolismo , Lisina/farmacología , Cromatografía Liquida/veterinaria , Células HEK293 , Espectrometría de Masas en Tándem/veterinaria , Mioblastos/fisiología , Proliferación Celular/genéticaRESUMEN
Cancer-associated cachexia (CAC) is a multifactorial wasting syndrome characterized by loss of skeletal muscle. Interleukin-11 (IL11), one of the IL6 family cytokines, is highly expressed in various types of cancer including cancers frequently associated with cachexia. However, the impact of IL11 on muscle metabolism remains to be determined. Since one of the mechanisms of muscle wasting in cachexia is defective muscle regeneration due to impaired myogenic differentiation, we examined the effect of IL11 on the differentiation of C2C12 mouse myoblasts. Treatment of C2C12 cells with recombinant mouse IL11 resulted in decreased myotube formation. In addition, IL11 treatment reduced the protein and mRNA levels of myosin heavy chain (MHC), a marker of myogenic differentiation. Moreover, the levels of myogenic regulatory factors including myogenin and Mrf4 were significantly reduced by IL11 treatment. IL11 treatment increased the number of BrdU-positive cells and the level of phosphorylated retinoblastoma (Rb) protein, while the levels of p21Waf1 and p27Kip1 were reduced by IL11 treatment in differentiating C2C12 cells, suggesting that IL11 interferes with cell cycle exit during the early stages of myogenic differentiation. Consistent with this, IL11 treatment at the late stage of differentiation did not affect myotube formation and MHC expression. IL11 treatment resulted in an activation of ERK, STAT3, and AKT in differentiating C2C12 cells. However, only ERK inhibitors including PD98059 and U0126 were able to ameliorate the suppressive effect of IL11 on the expression of MHC and myogenin. Additionally, pretreatment with PD98059 and U0126 resulted in improved myotube formation and reduced BrdU staining in IL11-treated cells. Together, our results suggest that IL11 inhibits myogenic differentiation through delayed cell cycle exit in an ERK-dependent manner. To our knowledge, this study is the first to demonstrate an inhibitory role of IL11 in myogenic differentiation and identifies the previously unrecognized role of IL11 as a possible mediator of CAC.
Asunto(s)
Diferenciación Celular , Interleucina-11 , Mioblastos , Animales , Ratones , Bromodesoxiuridina , Caquexia , Quinasas MAP Reguladas por Señal Extracelular , Interleucina-11/farmacología , Desarrollo de Músculos , Miogenina/genética , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Neoplasias , Mioblastos/efectos de los fármacos , Mioblastos/fisiologíaRESUMEN
This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (-159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region -159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.
Asunto(s)
Proteína MioD , Mioblastos , Bovinos , Animales , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/fisiología , Regiones Promotoras Genéticas , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Desarrollo de Músculos/genéticaRESUMEN
Muscle cell fusion is a multistep process where the final step of the reaction drives progression beyond early hemifusion events to complete fusion. This step requires activity of the muscle-specific fusogen Myomerger, a single-pass transmembrane protein containing 84 amino acids with an ectodomain that includes two α-helices. Previous studies have demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. An obvious question is how such destabilizing activity might be regulated to avoid membrane and cellular damage, and how the two juxtaposed helices cooperate in fusion. Using cellular fusion assays and in vitro liposome assays, we report that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We demonstrate that externalized phosphatidylserine (PS), a lipid previously implicated in myoblast fusion, has a determinant role in the regulation of Myomerger activity. The membrane-proximal, amphipathic Helix-1 is normally disordered and its α-helical structure is induced by PS, making membrane interactions more efficacious. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and augments the membrane-stressing effects of Helix-1. These data reveal that Myomerger fusogenic activity is an exquisitely orchestrated event involving its two ectodomain helices, which are controlled by membrane lipid composition, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final step of myoblast fusion.
Asunto(s)
Fusión Celular , Proteínas de la Membrana , Mioblastos , Fosfatidilserinas , Animales , Línea Celular , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mioblastos/fisiologíaRESUMEN
Uremic sarcopenia is a serious clinical problem associated with physical disability and increased morbidity and mortality. Methylglyoxal (MG) is a highly reactive, dicarbonyl uremic toxin that accumulates in the circulatory system in patients with chronic kidney disease (CKD) and is related to the pathology of uremic sarcopenia. The pathophysiology of uremic sarcopenia is multifactorial; however, the details remain unknown. We investigated the mechanisms of MG-induced muscle atrophy using mouse myoblast C2C12 cells, focusing on intracellular metabolism and mitochondrial injury. We found that one of the causative pathological mechanisms of uremic sarcopenia is metabolic flow change to fatty acid synthesis with MG-induced ATP shortage in myoblasts. Evaluation of cell viability revealed that MG showed toxic effects only in myoblast cells, but not in myotube cells. Expression of mRNA or protein analysis revealed that MG induces muscle atrophy, inflammation, fibrosis, and oxidative stress in myoblast cells. Target metabolomics revealed that MG induces metabolic alterations, such as a reduction in tricarboxylic acid cycle metabolites. In addition, MG induces mitochondrial morphological abnormalities in myoblasts. These changes resulted in the reduction of ATP derived from the mitochondria of myoblast cells. Our results indicate that MG is a pathogenic factor in sarcopenia in CKD.
Asunto(s)
Insuficiencia Renal Crónica , Sarcopenia , Adenosina Trifosfato/metabolismo , Animales , Femenino , Humanos , Indicán/farmacología , Inflamación/inducido químicamente , Inflamación/patología , Masculino , Ratones , Atrofia Muscular , Mioblastos/patología , Mioblastos/fisiología , Estrés Oxidativo , Piruvaldehído/toxicidad , Insuficiencia Renal Crónica/metabolismoRESUMEN
Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Mioblastos/citología , Proteínas Señalizadoras YAP/metabolismo , Actinas/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Proteínas de Microfilamentos/genética , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/fisiología , Proteínas Señalizadoras YAP/genéticaRESUMEN
In vitro skeletal muscle cell production is emerging in the field of artificial lab-grown meat as alternative future food. Currently, there is an urgent paradigm shift towards a serum replacement culture system. Surprisingly, little is known about the impact of serum-free culture on skeletal muscle cells to date. Therefore, we performed metabolic profiling of the C2C12 myoblasts and myotubes in serum-free mediums (B27, AIM-V) and compared it with conventional serum supplementation culture. Furthermore, cell morphology, viability, and myogenic differentiation were observed for 7 days of cultivation. Intriguingly, the metabolic difference is more dominant between the cell status than medium effects. In addition, proliferative myoblast showed more distinct metabolic differences than differentiated myotubes in different culture conditions. The intracellular levels of GL3P and UDP-GlcNAc were significantly increased in myotubes versus myoblast. Non-essential amino acids and pyruvate reduction and transamination showed significant differences among serum, B27, and AIM-V cultures. Intracellular metabolite profiles indicated that C2C12 myotubes cultured in serum and B27 had predominant glycolytic and oxidative metabolism, respectively, indicating fast and slow types of muscle confirmed by MHC immunostaining. This work might be helpful to understand the altered metabolism of skeletal muscle cells in serum-free culture and contribute to future artificial meat research work.
Asunto(s)
Medio de Cultivo Libre de Suero , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Mioblastos/metabolismo , Mioblastos/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Industria de Alimentos , Carne , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Fenotipo , Factores de TiempoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Tsantan Sumtang (TS), a traditional Tibetan medicine, has been used in the clinic for the treatment of myocardial ischemia (MI) for ages, however, the bioactive ingredients that are responsible for improving MI remain unknown. AIM OF THE STUDY: This study investigated the chemical components of TS and their medicinal efficacies at cell levels, in order to expound the bioactive ingredients in TS. MATERIALS AND METHODS: First, a response-surface methodology was employed to determine the optimum ethanol reflux extraction process of polyphenols in TS (PTS) due to their close correlation with MI improvement. Second, a serum pharmacochemistry technique was used to analyze the compounds of PTS absorbed into the blood of rats. Third, hypoxia-, H2O2-, and adriamycin (ADM)-induced H9c2 cell injury models were used to investigate the cardioprotective effects of these compounds in vitro. Fourth, protective effects of isovitexin, quercitrin, and isoeugenol on mitochondrial function were further tested. RESULTS: The optimum extraction conditions for obtaining PTS were an ethanol concentration of 78.22%, an extraction time of 67.4 min, and a material-liquid ratio of 1:72.60 mL/g. Serum pharmacochemistry analysis detected 21 compounds, of which 11 compounds were always present in the blood within 5 h. Cytotoxicity and the protective effect of 11 compounds in hypoxia-, H2O2-, and ADM-induced H9c2 cell injury models shown that isovitexin, quercitrin, and isoeugenol had almost no cytotoxicity, and they could elevate the survival rate in injured H9c2 cells. Furthermore, isovitexin, quercitrin, and isoeugenol could decrease mitochondrial reactive oxygen species (ROS) releasion, inhibite mitochondrial permeability transition pore (mPTP) opening, ameliorate the change of mitochondrial membrane potential (MMP) to exert mitochondrial protection effect. CONCLUSION: Isovitexin, quercitrin, and isoeugenol exhibited cardioprotective effect at cell levles, these three compounds might be the bioactive ingredients in TS. These findings elucidate the pharmacodynamic substances and mechanisms of TS, guiding its clinical use.
Asunto(s)
Medicina Tradicional Tibetana , Mioblastos/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Polifenoles/farmacología , Animales , Antibióticos Antineoplásicos/toxicidad , Apigenina/administración & dosificación , Apigenina/química , Apigenina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Doxorrubicina/toxicidad , Eugenol/administración & dosificación , Eugenol/análogos & derivados , Eugenol/química , Eugenol/farmacología , Peróxido de Hidrógeno/toxicidad , Mioblastos/fisiología , Fitoterapia , Polifenoles/sangre , Polifenoles/química , Polifenoles/farmacocinética , Quercetina/administración & dosificación , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Kirsten rat sarcoma 2 viral oncogene homolog (Kras) is a proto-oncogene that encodes the small GTPase transductor protein KRAS, which has previously been found to promote cytokine secretion, cell survival, and chemotaxis. However, its effects on preadipocyte differentiation and lipid accumulation are unclear. In this study, the effects of KRAS inhibition on proliferation, autophagy, and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines. The results showed that KRAS was localized mainly in the nuclei of 3T3-L1 and C2C12 cells. Inhibition of KRAS altered mammalian target of rapamycin (Mtor), proliferating cell nuclear antigen (Pcna), Myc, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein beta (C/ebp-ß), diacylglycerol O-acyltransferase 1 (Dgat1), and stearoyl-coenzyme A desaturase 1 (Scd1) expression, thereby reducing cell proliferation capacity while inducing autophagy, enhancing differentiation of 3T3-L1 and C2C12 cells into mature adipocytes, and increasing adipogenesis and the capacity to store lipids. Moreover, during differentiation, KRAS inhibition reduced the levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p38, and phosphatidylinositol 3 kinase (PI3K) activation. These results show that KRAS has unique regulatory effects on cell proliferation, autophagy, adipogenic differentiation, and lipid accumulation.
Asunto(s)
Adipogénesis , Autofagia , Proliferación Celular , Fibroblastos/metabolismo , Mioblastos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal , Células 3T3 , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Células Cultivadas , Diacilglicerol O-Acetiltransferasa/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/fisiología , Regulación de la Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metabolismo de los Lípidos , Ratones , Mioblastos/fisiología , PPAR gamma/genética , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Estearoil-CoA Desaturasa/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Manosiltransferasas/fisiología , Mioblastos/fisiología , Animales , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Glicosilación , Manosiltransferasas/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mioblastos/citología , Fosforilación/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.
Asunto(s)
Acetilcolina/metabolismo , Microtúbulos/fisiología , Mioblastos/fisiología , Unión Neuromuscular/fisiología , Neuropéptidos/farmacología , Receptores Colinérgicos/metabolismo , Sinapsis/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Ratones , Microtúbulos/efectos de los fármacos , Mioblastos/citología , Mioblastos/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Receptores Colinérgicos/genética , Transmisión SinápticaRESUMEN
Muscle stem cells (MuSCs) are essential for muscle growth, maintenance and repair. Over the past decade, experiments in Drosophila have been instrumental in understanding the molecular and cellular mechanisms regulating MuSCs (also known as adult muscle precursors, AMPs) during development. A large number of genetic tools available in fruit flies provides an ideal framework to address new questions which could not be addressed with other model organisms. This review reports the main findings revealed by the study of Drosophila AMPs, with a specific focus on how AMPs are specified and properly positioned, how they acquire their identity and which are the environmental cues controlling their behavior and fate. The review also describes the recent identification of the Drosophila adult MuSCs that have similar characteristics to vertebrates MuSCs. Integration of the different levels of MuSCs analysis in flies is likely to provide new fundamental knowledge in muscle stem cell biology largely applicable to other systems.
Asunto(s)
Desarrollo de Músculos/fisiología , Mioblastos/fisiología , Regeneración/fisiología , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Drosophila/citología , Drosophila/fisiología , Modelos Biológicos , Mioblastos/citología , Transducción de SeñalRESUMEN
Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.