BACKGROUND & AIM: Dysfunction of skeletal muscle satellite cells might impair muscle regeneration and prolong ICU-acquired weakness, a condition associated with disability and delayed death. This study aimed to elucidate the distinct metabolic effects of critical illness and β-OH-butyrate on satellite cells isolated from these patients. METHODS: Satellite cells were extracted from vastus lateralis muscle biopsies of patients with ICU-acquired weakness (n = 10) and control group of healthy volunteers or patients undergoing elective hip replacement surgery (n = 10). The cells were exposed to standard culture media supplemented with β-OH-butyrate to assess its influence on cell proliferation by ELISA, mitochondrial functions by extracellular flux analysis, electron transport chain complexes by high resolution respirometry, and ROS production by confocal microscopy. RESULTS: Critical illness led to a decline in maximal respiratory capacity, ATP production and glycolytic capacity and increased ROS production in ICU patients' cells. Notably, the function of complex II was impaired due to critical illness but restored to normal levels upon exposure to β-OH-butyrate. While β-OH-butyrate significantly reduced ROS production in both control and ICU groups, it had no significant impact on global mitochondrial functions. CONCLUSION: Critical illness induces measurable bioenergetic dysfunction of skeletal muscle satellite cells. β-OH-butyrate displayed a potential in rectifying complex II dysfunction caused by critical illness and this warrants further exploration.
- MeSH
- adenosintrifosfát metabolismus MeSH
- dospělí MeSH
- energetický metabolismus účinky léků MeSH
- kritický stav * MeSH
- kultivované buňky MeSH
- kyselina 3-hydroxymáselná * farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- proliferace buněk účinky léků MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- satelitní buňky kosterního svalu * účinky léků metabolismus MeSH
- senioři MeSH
- svalová slabost MeSH
- svalové mitochondrie účinky léků metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Myosatellite cells are myogenic stem cells that can transform to provide nuclei for existing muscles or generate new muscle fibers as documented after extended exercise programs. OBJECTIVES: The authors investigated whether the simultaneous application of High-Intensity Focused Electromagnetic (HIFEM) and Synchrode radiofrequency (RF) affects the levels of satellite cells similarly as the prolonged exercise does to achieve muscle growth. METHODS: Three 30-minute simultaneous HIFEM and Synchrode RF treatments (once a week) were administered over the abdominal area of 5 Large White swine aged approximately 6 months. All animals were anesthetized during the treatments and biopsy acquisition. Biopsies of muscle tissue were collected at baseline, 4 days, 2 weeks, and 1 month post-treatment. After binding the specific antibodies, the NCAM/CD56 levels, a marker of activated satellite cells, were quantified employing the immunofluorescence microscopy technique with a UV lamp. RESULTS: Examined slices showed a continuous increase in satellite cell levels throughout the study. Four days after the treatment, we observed a 26.1% increase in satellite cells, which increased to 30.2% at 2-week follow-up. Additional histological analysis revealed an increase in the cross-sectional area of muscle fibers and the signs of newly formed fibers of small diameters at 2 weeks after the treatment. No damage to muscle tissue and no adverse effects related to the treatment were observed. CONCLUSIONS: The findings indicate that the simultaneous application of HIFEM and novel Synchrode RF treatment can initiate differentiation of satellite cells to support the growth of existing muscles and, presumably, even the formation of new myofibers.
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
The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.
- MeSH
- buněčná adheze fyziologie MeSH
- buněčná diferenciace fyziologie MeSH
- epidermální růstový faktor metabolismus fyziologie MeSH
- extracelulární matrix MeSH
- kmenové buňky cytologie MeSH
- kosterní svalová vlákna cytologie MeSH
- kosterní svaly cytologie MeSH
- kultivované buňky MeSH
- myoblasty cytologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- pohyb buněk fyziologie MeSH
- primární buněčná kultura MeSH
- proliferace buněk fyziologie MeSH
- satelitní buňky kosterního svalu metabolismus fyziologie MeSH
- signální transdukce MeSH
- vývoj svalů fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The cellular components of the satellite cell niche participate in the regulation of skeletal muscle regeneration. Beside myogenic cells at different developmental stages, this niche is formed by cells of the immune system, the interstitial connective tissue and the vascular system. Unambiguous determination of the origin of these cell types could contribute to optimization of the cell-based therapy of skeletal muscle disorders. In our work, we intravenously transplanted mouse GFP+ unseparated bone marrow cells into whole-body lethally irradiated immunocompetent mice four weeks before cardiotoxin-induced injury of the recipients' skeletal muscles. Seven and 28 days after the toxin injection, the injured regenerating and contralateral intact muscles were examined for identification of GFP+ bone marrow-derived cells by direct fluorescence, protein immunohistochemistry and immunogold transmission electron microscopy. In both the intact and injured muscles, GFP positivity was determined in immune cells, mainly in macrophages, and in interstitial spindleshaped cells. Moreover, in the injured muscles, rare GFP+ endothelial cells of the blood vessels and newly formed myotubes and muscle fibres were present. Our results confirmed the ability of bone marrowderived cells to contribute to the cellular component of the satellite cell niche in the intact and regenerating skeletal muscle. These cells originated not only from haematopoietic stem cells, but obviously also from other stem or progenitor cells residing in the bone marrow, such as multipotent mesenchymal stromal cells and endothelial progenitors.
- MeSH
- buňky kostní dřeně cytologie MeSH
- fluorescence MeSH
- kosterní svaly zranění patologie ultrastruktura MeSH
- myši inbrední C57BL MeSH
- nika kmenových buněk * MeSH
- regenerace fyziologie MeSH
- satelitní buňky kosterního svalu cytologie MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články 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
- Klíčová slova
- metody stanovení množství a ukládání intramyocelulárních lipidů, diabetologický výzkum, perkutánní biopsie kosterního svalu,
- MeSH
- biomedicínský výzkum metody trendy MeSH
- diabetes mellitus 2. typu * diagnóza etiologie terapie MeSH
- financování organizované MeSH
- genomika metody trendy MeSH
- imunohistochemie metody trendy MeSH
- inzulinová rezistence genetika imunologie MeSH
- jehlová biopsie * metody trendy využití MeSH
- kosterní svaly * enzymologie metabolismus patofyziologie MeSH
- lidé MeSH
- proteomika metody trendy MeSH
- satelitní buňky kosterního svalu cytologie imunologie metabolismus MeSH
- statistika jako téma MeSH
- svalové mitochondrie metabolismus mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3' untranslated region (3' UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3' UTR of c-Myb was also important because the expression of c-Myb coding region with its 3' UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3' UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.
- MeSH
- 3' nepřekládaná oblast genetika MeSH
- buněčná diferenciace genetika MeSH
- buněčné linie MeSH
- fúze buněk MeSH
- imunohistochemie MeSH
- kardiotoxiny farmakologie MeSH
- kosterní svalová vlákna cytologie metabolismus MeSH
- kosterní svaly účinky léků patofyziologie MeSH
- kultivované buňky MeSH
- myoblasty cytologie metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protoonkogenní proteiny c-myb genetika metabolismus MeSH
- regenerace účinky léků genetika MeSH
- satelitní buňky kosterního svalu cytologie metabolismus MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes. METHODOLOGY/PRINCIPAL FINDINGS: We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels. CONCLUSIONS/SIGNIFICANCE: Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.
- MeSH
- buněčná diferenciace MeSH
- buněčné dýchání MeSH
- buněčné linie MeSH
- elektronový transportní řetězec metabolismus MeSH
- galaktosa metabolismus MeSH
- glukosa metabolismus MeSH
- glykolýza MeSH
- kosterní svalová vlákna metabolismus MeSH
- kultivační média MeSH
- myoblasty kosterní metabolismus MeSH
- myši MeSH
- oxidace-redukce MeSH
- svalové mitochondrie metabolismus MeSH
- transport elektronů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
