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.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• exprese genu MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• kosterní svalová vlákna * cytologie   metabolismus   MeSH
• myoblasty kosterní * cytologie   metabolismus   MeSH
• myši MeSH
• protoonkogenní proteiny metabolismus   MeSH
• regulace genové exprese MeSH
• těžké řetězce myosinu metabolismus   MeSH
• vývoj svalů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Glutamine has been considered essential for rapidly dividing cells, but its effect on mitochondrial function is unknown. MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle biopsy samples (n = 9) and exposed for 20 days to 6 different glutamine concentrations (0, 100, 200, 300, 500, and 5000 µM). Cells were trypsinized and manually counted every 5 days. Seven days before the end of exposure, half of these cells were allowed to differentiate to myotubes. Afterward, energy metabolism in both myotubes and myoblasts was assessed by extracellular flux analysis (Seahorse Biosciences, Billerica, MA). The protocol for myoblasts was optimized in preliminary experiments. To account for different mitochondrial density or cell count, data were normalized to citrate synthase activity. RESULTS: Fastest myoblast proliferation was observed at 300 µM glutamine, with a significant reduction at 0 and 100 µM. Glutamine did not influence basal oxygen consumption, anaerobic glycolysis or respiratory chain capacity. Glutamine significantly (P = .015) influenced the leak through the inner mitochondrial membrane. Efficiency of respiratory chain was highest at 200-300 µM glutamine (~90% of oxygen used for adenosine triphosphate synthesis). Increased glutamine concentration to 500 or 5000 µM caused mitochondrial uncoupling in myoblasts and myotubes, decreasing the efficiency of the respiratory chain to ~70%. CONCLUSION: Glutamine concentrations, consistent with moderate clinical hypoglutaminemia (300 µM), bring about an optimal condition of myoblast proliferation and for efficiency of aerobic phosphorylation in an in vitro model of human skeletal muscle. These data support the hypothesis of hypoglutaminemia as an adaptive phenomenon in conditions leading to bioenergetic failure (eg, critical illness).

• MeSH
• biopsie MeSH
• energetický metabolismus účinky léků   MeSH
• fosforylace účinky léků   MeSH
• glutamin metabolismus   farmakologie   MeSH
• kosterní svalová vlákna cytologie   účinky léků   MeSH
• kosterní svaly cytologie   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• myoblasty kosterní cytologie   účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• techniky in vitro MeSH
• transport elektronů účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Our present study aimed to investigate the effect of lentiviral-mediated RNAi using short hairpin RNA (shRNA) targeting Smad4 on TGF-beta1 induced fibrosis. shRNAs targeting Smad4 were designed and the most efficient shRNA was screened. This shRNA was introduced into a lentiviral vector which was used to infect C2C12 myoblasts, and then the Smad4 expression was detected. Cells were divided into: C2C12 cells group, TGF-beta1 induction group, transfection group, and transfection after TGF-beta1 induction group. C2C12 myoblasts were transfected with lentivirus carrying Smad4-shRNA and treated with TGF-beta1 to induce the differentiation into myofibroblasts. Fluorescence Real-time-PCR and the western blot assay were employed to detect the expressions of collagen I and alpha-SMA. The results showed that the protein and mRNA expression of Smad4 in the C2C12 cells transfected with Smad4-shRNA1 was significantly reduced when compared with C2C12 before transfection. In the TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly increased as compared to the C2C12 cells group. In the transfection after TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly increased compared to the transfection group, and the protein expressions significantly increased, respectively. In the transfection after TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly decreased compared to the TGF-beta1 induction group, and the protein expressions significantly reduced, respectively. The results indicate that suppression of Smad4 expression can efficiently inhibit the TGF-beta1 induced fibrosis in myoblasts. The findings suggest Smad4 may become a novel target for the treatment of skeletal muscle fibrosis.

• MeSH
• barvení a značení metody   využití   MeSH
• fibróza genetika   komplikace   terapie   MeSH
• financování organizované MeSH
• fluorescenční mikroskopie využití   MeSH
• kosterní svaly cytologie   zranění   MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• myoblasty kosterní cytologie   fyziologie   patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   využití   MeSH
• protein Smad4 genetika   metabolismus   MeSH
• satelitní buňky kosterního svalu fyziologie   metabolismus   MeSH
• statistika jako téma MeSH
• transformující růstový faktor beta1 genetika   metabolismus   MeSH
• western blotting využití   MeSH
• Check Tag
• lidé MeSH