RESUMEN
Adiponectin has previously been investigated for exerting its protective effect against myocardial injury through anti-apoptotic and anti-oxidative actions. Therefore, the present study aimed to investigate the nature and mechanism of adiponectin inhibition of H2O2-induced apoptosis in chicken skeletal myoblasts. Skeletal muscle satellite cells were differentiated and assigned into three groups. Group C was on the blank control group, group H was stimulated with the H2O2 (500 µmol/L, 4 h) alone group, group A + H was pre-treated with adiponectin (10 µg/mL, 24 h) and stimulated with the H2O2 (500 µmol/L, 4 h) group. Cytotoxicity inhibited by adiponectin was evaluated by the CCK-8 assay. The degree of apoptosis and oxidative damage was investigated by the TdT-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining assays. Oxidative stress was assessed by evaluating lipid peroxidation, superoxide dismutase, and reduced glutathione. Acridine orange (AO) staining detected lysosomal membrane permeability. The changes in mitochondrial membrane potential (MMP) were analyzed using 5,5,6,6'-tetrachloro-1,1,3,3-tetraethylimidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The lysosomal function, mitochondrial function, and apoptosis-related mRNA and protein expression levels were quantified by real-time quantitative PCR and western blot, respectively. The results suggested that adiponectin treatment attenuated H2O2-induced cytotoxicity and oxidative stress in skeletal myoblasts. Compared with H2O2 treatment, TUNEL and ROS staining demonstrated lower apoptosis upon adiponectin treatment. AO staining confirmed the amelioration of lysosomal membrane damage, and JC-1 staining revealed an increase in mitochondrial membrane potential after adiponectin treatment. At the molecular level, adiponectin treatment inhibited the expression of the lysosomal apoptotic factors cathepsin B, chymotrypsin B, and the mitochondrial apoptotic pathway cytochrome-c (cyt-c) and caspase-8; decreased the apoptotic marker gene Bax; and increased the expression of the anti-apoptotic marker gene Bcl-2. Adiponectin treatment attenuated H2O2-induced apoptosis in skeletal myoblasts, possibly by inhibiting oxidative stress and apoptosis through the lysosomal-mitochondrial axis.
Asunto(s)
Adiponectina , Apoptosis , Pollos , Peróxido de Hidrógeno , Lisosomas , Potencial de la Membrana Mitocondrial , Mitocondrias , Mioblastos Esqueléticos , Estrés Oxidativo , Animales , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/toxicidad , Apoptosis/efectos de los fármacos , Adiponectina/farmacología , Adiponectina/metabolismo , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/citología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.
Asunto(s)
Desarrollo de Músculos/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Diferenciación Celular/fisiología , Humanos , Modelos Biológicos , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/fisiología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/fisiologíaRESUMEN
The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.
Asunto(s)
Desarrollo de Músculos/fisiología , Regeneración/fisiología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiología , Animales , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Macrófagos/fisiología , Modelos Biológicos , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Regeneración/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.
Asunto(s)
Células Madre Mesenquimatosas/citología , Imagen Molecular/métodos , Mioblastos Esqueléticos/citología , Infarto del Miocardio/patología , Miocardio/patología , Animales , Modelos Animales de Enfermedad , Genes Reporteros , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mioblastos Esqueléticos/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismoRESUMEN
Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Humanos , Ratones , Fibras Musculares Esqueléticas/citología , Mioblastos Esqueléticos/citologíaRESUMEN
Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.
Asunto(s)
Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mioblastos Esqueléticos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Mioblastos Esqueléticos/citologíaAsunto(s)
Fusión de Membrana/genética , Proteínas de la Membrana/genética , Síndrome de Mobius/genética , Proteínas Musculares/genética , Enfermedades Musculares/genética , Mutación Missense , Mioblastos Esqueléticos/metabolismo , Síndrome de Pierre Robin/genética , Animales , Diferenciación Celular , Fusión Celular , Modelos Animales de Enfermedad , Humanos , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Síndrome de Mobius/metabolismo , Síndrome de Mobius/patología , Proteínas Musculares/deficiencia , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Mioblastos Esqueléticos/citología , Síndrome de Pierre Robin/metabolismo , Síndrome de Pierre Robin/patología , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Pez CebraRESUMEN
The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described. Here we show that depletion of TPR results in an aberrant morphology of murine proliferating C2C12 myoblasts (MBs) and differentiated C2C12 myotubes (MTs). The ChIP-Seq data revealed that TPR binds to genes linked to muscle formation and function, such as myosin heavy chain (Myh4), myocyte enhancer factor 2C (Mef2C) and a majority of olfactory receptor (Olfr) genes. We further show that TPR, possibly via lysine-specific demethylase 1 (LSD1), promotes the expression of Myh4 and Olfr376, but not Mef2C. This provides a novel insight into the mechanism of myogenesis; however, more evidence is needed to fully elucidate the mechanism by which TPR affects specific myogenic genes.
Asunto(s)
Fibras Musculares Esqueléticas , Mioblastos Esqueléticos , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Expresión Génica , Regulación de la Expresión Génica , Ratones , Desarrollo de Músculos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismoRESUMEN
SESN2 and JNK are emerging powerful stress-inducible proteins in regulating lipid metabolism. The aim of this study was to determine the underlying mechanism of SESN2/JNK signaling in exercise to improve lipid disorder induced by high-fat diet (HFD). Our data showed that HFD and SESN2 knockout resulted in abnormalities including elevated body weight, increased fat mass, serum total cholesterol, lipid biosynthesis related proteins, and a concomitant increase of pJNK-Thr183/Tyr185. The above changes were reversed by exercise training. SESN2 silencing or JNK inhibition in palmitate-treated C2C12 further confirmed that SESN2 and JNK play a vital role in lipid biosynthesis. Rescue experiment further demonstrated that SESN2 reduced lipid biosynthesis through inhibition of JNK. SESN2/JNK signaling axis regulates lipid biosynthesis in both animal and cell models with abnormalities of lipid metabolism induced by HFD or palmitate treatment. This study provided evidence that exercise ameliorated lipid metabolic disorder induced by HFD feeding or by SESN2 knockout. SESN2 may improve lipid metabolism through inhibition JNK expression in skeletal muscle cells, providing a molecular mechanism that may represent an attractive target for the treatment of lipid disorder. Novelty: Exercise improved lipid disorder induced by HFD feeding and SESN2 knockout. SESN2 and JNK play a vital role in lipid biosynthesis in vivo and in vitro. SESN2 suppressed JNK to improve lipid metabolism in skeletal muscle cells.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Trastornos del Metabolismo de los Lípidos/metabolismo , Trastornos del Metabolismo de los Lípidos/prevención & control , Peroxidasas/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Composición Corporal , Línea Celular , Trastornos del Metabolismo de los Lípidos/etiología , Lípidos/biosíntesis , Lípidos/sangre , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismoRESUMEN
Musculoskeletal stromal cells' (MSCs') metabolism impacts cell differentiation as well as immune function. During osteogenic and adipogenic differentiation, BM-MSCs show a preference for glycolysis during proliferation but shift to an oxidative phosphorylation (OxPhos)-dependent metabolism. The MSC immunoregulatory fate is achieved with cell polarization, and the result is sustained production of immunoregulatory molecules (including PGE2, HGF, IL1RA, IL6, IL8, IDO activity) in response to inflammatory stimuli. MSCs adapt their energetic metabolism when acquiring immunomodulatory property and shift to aerobic glycolysis. This can be achieved via hypoxia, pretreatment with small molecule-metabolic mediators such as oligomycin, or AKT/mTOR pathway modulation. The immunoregulatory effect of MSC on macrophages polarization and Th17 switch is related to the glycolytic status of the MSC. Indeed, MSCs pretreated with oligomycin decreased the M1/M2 ratio, inhibited T-CD4 proliferation, and prevented Th17 switch. Mitochondrial activity also impacts MSC metabolism. In the bone marrow, MSCs are present in a quiescent, low proliferation, but they keep their multi-progenitor function. In this stage, they appear to be glycolytic with active mitochondria (MT) status. During MSC expansion, we observed a metabolic shift toward OXPhos, coupled with an increased MT activity. An increased production of ROS and dysfunctional mitochondria is associated with the metabolic shift to glycolysis. In contrast, when MSC underwent chondro or osteoblast differentiation, they showed a decreased glycolysis and inhibition of the pentose phosphate pathway (PPP). In parallel the mitochondrial enzymatic activities increased associated with oxidative phosphorylation enhancement. MSCs respond to damaged or inflamed tissue through the transfer of MT to injured and immune cells, conveying a type of signaling that contributes to the restoration of cell homeostasis and immune function. The delivery of MT into injured cells increased ATP levels which in turn maintained cellular bioenergetics and recovered cell functions. MSC-derived MT may be transferred via tunneling nanotubes to undifferentiated cardiomyocytes and leading to their maturation. In this review, we will decipher the pathways and the mechanisms responsible for mitochondria transfer and activity. The eventual reversal of the metabolic and pro-inflammatory profile induced by the MT transfer will open new avenues for the control of inflammatory diseases.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Animales , Biomarcadores , Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Reprogramación Celular , Metabolismo Energético , Humanos , Inmunomodulación , Trasplante de Células Madre Mesenquimatosas/métodos , Mitocondrias/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Evidence is growing for a role of vitamin D in regulating skeletal muscle mass, strength and functional capacity. Given the role the kidneys play in activating total vitamin D, and the high prevalence of vitamin D deficiency in Chronic Kidney Disease (CKD), it is possible that deficiency contributes to the low levels of physical function and muscle mass in these patients. METHODS: This is a secondary cross-sectional analysis of previously published interventional study, with in vitro follow up work. 34 CKD patients at stages G3b-5 (eGFR 25.5 ± 8.3 mL/min/1.73m2; age 61 ± 12 years) were recruited, with a sub-group (n = 20) also donating a muscle biopsy. Vitamin D and associated metabolites were analysed in plasma by liquid chromatography tandem-mass spectroscopy and correlated to a range of physiological tests of muscle size, function, exercise capacity and body composition. The effects of 1α,25(OH)2D3 supplementation on myogenesis and myotube size was investigated in primary skeletal muscle cells from vitamin D deficient donors. RESULTS: In vivo, there was no association between total or active vitamin D and muscle size or strength, but a significant correlation with VÌO2Peak was seen with total vitamin D (25OHD). in vitro, 1α,25(OH)2D3 supplementation reduced IL-6 mRNA expression, but had no effect upon proliferation, differentiation or myotube diameter. CONCLUSIONS: Vitamin D deficiency is not a prominent factor driving the loss of muscle mass in CKD, but may play a role in reduced exercise capacity.
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Tolerancia al Ejercicio/fisiología , Insuficiencia Renal Crónica/fisiopatología , Deficiencia de Vitamina D/fisiopatología , Anciano , Calcitonina/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estudios Transversales , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Fuerza Muscular/fisiología , Músculo Esquelético/fisiopatología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Insuficiencia Renal Crónica/complicaciones , Vitamina D/sangre , Vitamina D/metabolismo , Deficiencia de Vitamina D/etiologíaRESUMEN
The demethylation of histone lysine residues, one of the most important modifications in transcriptional regulation, is associated with various physiological states. KDM2B is a demethylase of histones H3K4, H3K36, and H3K79 and is associated with the repression of transcription. Here, we present a novel mechanism by which KDM2B demethylates serum response factor (SRF) K165 to negatively regulate muscle differentiation, which is counteracted by the histone methyltransferase SET7. We show that KDM2B inhibited skeletal muscle differentiation by inhibiting the transcription of SRF-dependent genes. Both KDM2B and SET7 regulated the balance of SRF K165 methylation. SRF K165 methylation was required for the transcriptional activation of SRF and for the promoter occupancy of SRF-dependent genes. SET7 inhibitors blocked muscle cell differentiation. Taken together, these data indicate that SRF is a nonhistone target of KDM2B and that the methylation balance of SRF as maintained by KDM2B and SET7 plays an important role in muscle cell differentiation.
Asunto(s)
Diferenciación Celular , Proteínas F-Box/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Músculo Esquelético/metabolismo , Factor de Respuesta Sérica/metabolismo , Sitios de Unión , Biomarcadores , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Proteínas F-Box/genética , Regulación de la Expresión Génica , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Metilación , Modelos Biológicos , Músculo Esquelético/citología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Unión Proteica , Elementos de Respuesta , Transcripción GenéticaRESUMEN
The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(ε-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes' alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle, as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle's physiological functions.
Asunto(s)
Hidrogeles/química , Nanopartículas del Metal/química , Microtecnología , Músculo Esquelético/fisiología , Poliésteres/química , Polietilenglicoles/química , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Oro/química , Nanopartículas del Metal/ultraestructura , Ratones , Mioblastos Esqueléticos/citologíaRESUMEN
3-Monochloro-1,2-propanediol (3-MCPD) is a chemical compound that is unintentionally produced during food processing such as acid hydrolysis. There has been reports regarding the role of this chemical compound in reproductive toxicity, as well as genotoxicity, neurotoxicity, and kidney toxicity. In this study, the in vitro muscle toxicity of 3-MCPD was assessed using C2C12 myoblast cells. The reduction in muscle regulatory factors (MRFs), which is related to muscle differentiation, was identified as significant with the increase concentration of 3-MCPD. Also, significantly decreased protein expression in mTOR and p70S6 kinase, which are the downstream targets of the pathway associated with muscle synthesis, was also confirmed. Therefore, the inhibitory effect of 3-MCPD on muscle differentiation is considered to be the cause of suppressing mTOR and p70S6 kinase expression. In conclusion, it was confirmed that 3-MCPD inhibits muscle differentiation in C2C12 myoblasts through suppressing the expression of several genetic factors involving muscle differentiation.
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Diferenciación Celular/efectos de los fármacos , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , alfa-Clorhidrina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Relación Estructura-ActividadRESUMEN
Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-ß (TGF-ß)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-ß/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. SIGNIFICANCE OF THE STUDY: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte-selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF-ß activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte-selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.
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Regulación de la Expresión Génica , Mioblastos Esqueléticos/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Proteína Desacopladora 1/biosíntesis , Animales , Bovinos , Células Cultivadas , Mioblastos Esqueléticos/citologíaRESUMEN
Although less attention was paid to understanding physical localization changes in cell nuclei recently, depicting chromatin interaction maps is a topic of high interest. Here, we focused on defining extensive physical changes in chromatin organization in the process of skeletal myoblast differentiation. Based on RNA profiling data and 3D imaging of myogenic (NCAM1, DES, MYOG, ACTN3, MYF5, MYF6, ACTN2, and MYH2) and other selected genes (HPRT1, CDH15, DPP4 and VCAM1), we observed correlations between the following: (1) expression change and localization, (2) a gene and its genomic neighbourhood expression and (3) intra-chromosome and microscopical locus-centromere distances. In particular, we demonstrated the negative regulation of DPP4 mRNA (p < 0.001) and protein (p < 0.05) in differentiated myotubes, which coincided with a localization change of the DPP4 locus towards the nuclear lamina (p < 0.001) and chromosome 2 centromere (p < 0.001). Furthermore, we discuss the possible role of DPP4 in myoblasts (supported by an inhibition assay). We also provide positive regulation examples (VCAM1 and MYH2). Overall, we describe for the first time existing mechanisms of spatial gene expression regulation in myoblasts that might explain the issue of heterogenic responses observed during muscle regenerative therapies.
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Diferenciación Celular , Cromatina/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Expresión Génica , Mioblastos Esqueléticos/metabolismo , Humanos , Mioblastos Esqueléticos/citologíaRESUMEN
Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl- concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl- concentration by extracellular Cl- depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl- efflux early on during myogenic differentiation, and a moderate intracellular Cl- concentration is necessary for myoblast fusion.
Asunto(s)
Cloruros/metabolismo , Proteínas de la Membrana/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Animales , Carbenoxolona/farmacología , Diferenciación Celular/fisiología , Fusión Celular , Línea Celular , Citosol/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Transporte Iónico/efectos de los fármacos , Ratones , Desarrollo de Músculos/fisiología , Mioblastos Esqueléticos/efectos de los fármacosRESUMEN
The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3'UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3'UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3'UTR was considerably increased, whereas Dicer1 depletion or 3'UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3'UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-ß abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.
Asunto(s)
MicroARNs/metabolismo , Desarrollo de Músculos , Mioblastos Esqueléticos/citología , Transactivadores/fisiología , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Humanos , Ratones , Fibras Musculares Esqueléticas/metabolismoRESUMEN
HMGB2, a DNA-binding protein, highly expresses during embryogenesis and plays an important role in development of some organs and tissues. However, it remains to be further investigated weather HMGB2 influences muscle development. In this work, we identified HMGB2 as an essential factor in myogenesis. Compared to wild type (WT) mice, body weights of systemic hmgb2 homozygous knockout (hmgb2-/- ) mice especially males were reduced. Diameter and cross-section area of tibialis anterior (TA) muscle fibers as well as expression of Myogenin and MyHC were all decreased in hmgb2-/- mice. CTX injury model revealed that HMGB2 was required for satellite cell proliferation and muscle regeneration. Moreover, HMGB2 interacted with S6K1 and regulated the kinase activity of S6K1 during cell proliferation. Knockdown and inactivation of S6K1 in C2C12 cells both resulted in impaired proliferation and differentiation. Furthermore, expression of cyclin D1 and Myf5 were both decreased when HMGB2 or S6K1 were knocked down and kinase activity of S6K1 was inhibited. These results indicate that HMGB2 is required for skeletal muscle development and regeneration, and HMGB2 maintains proliferation of myoblasts through regulating kinase activity of S6K1.
Asunto(s)
Proteína HMGB2/fisiología , Desarrollo de Músculos/fisiología , Músculo Esquelético/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/fisiología , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/fisiología , Regeneración , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/fisiologíaRESUMEN
In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.