There is increasing pressure on meat producers worldwide due to the need for higher yields and improved meat quality. This is why anabolic androgenic steroids (AAS) have been widely used in most countries, due to their ability to accelerate animal muscle growth. However, out of concern for their side effects, EU states have banned their use and implemented control mechanisms. But they are reaching their limits, and therefore, it is necessary to look for new ways and investigate the mechanism of action of AAS on muscle tissue. This study replicated the administration of banned AAS (testosterone, nandrolone and their combination) and observed their effect on pig muscle. The pig model was purposely chosen for the study, as no such research has been carried out on this species. At the same time, pork is one of the most consumed meats in Europe. It focused on histological changes in muscle structure, specifically the size of muscle fibres and the number of satellite cells per muscle fibre. Furthermore, ultrastructural changes in muscle fibres, the diameter of myofibrils, the number of myofibrils per area, the distance between myofibrils and the size of sarcomeres were examined. The results using the techniques of histology, fluorescent labelling and transmission electron microscopy showed that, after the application of AAS, there is an increase in the diameter of muscle fibres, an increase in the diameter of myofibrils, a decrease in the number of myofibrils per surface area and, in the case of testosterone, an increase in the distance between myofibrils and an increase in the length of sarcomeres. There was also a significant increase in the number of satellite cells per muscle fibre. The detected statistically significant differences between control and experimental groups provide evidence that selected histological parameters could be additional mechanisms for detecting the presence of AAS in pork meat in the future.

• MeSH
• anabolika * farmakologie   MeSH
• kosterní svalová vlákna * účinky léků   ultrastruktura   MeSH
• kosterní svaly účinky léků   anatomie a histologie   ultrastruktura   MeSH
• myofibrily * účinky léků   ultrastruktura   MeSH
• nandrolon * farmakologie   MeSH
• prasata anatomie a histologie   MeSH
• sarkomery účinky léků   ultrastruktura   MeSH
• satelitní buňky kosterního svalu účinky léků   ultrastruktura   MeSH
• testosteron * farmakologie   MeSH
• transmisní elektronová mikroskopie veterinární   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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

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.

• MeSH
• dánio pruhované * genetika   MeSH
• kosterní svaly * fyziologie   MeSH
• morfogeneze MeSH
• myoblasty fyziologie   MeSH
• myofibrily fyziologie   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural 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.

• MeSH
• fluorescenční mikroskopie MeSH
• kosterní svaly MeSH
• molekuly buněčné adheze nervové MeSH
• prasata MeSH
• rádiové vlny škodlivé účinky   MeSH
• satelitní buňky kosterního svalu * MeSH
• technologie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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

While GASP-1 and GASP-2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the noncompetitive and competitive antitrypsin activities of the full-length GASP-1 and GASP-2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the antitrypsin activity of GASP-1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP-1 and GASP-2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP-2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly interacting glutamine residue.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné linie MeSH
• intracelulární signální peptidy a proteiny chemie   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• mezibuněčné signální peptidy a proteiny chemie   metabolismus   MeSH
• myoblasty cytologie   metabolismus   MeSH
• myši MeSH
• proliferace buněk fyziologie   MeSH
• sekundární struktura proteinů MeSH
• simulace molekulární dynamiky * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• 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 aim of this study was to evaluate cell diversity by considering how Ca(2+) signaling has been adapted in skeletal muscle cell function. We characterized single C2C12 myoblasts through intracellular Ca(2+) signaling kinetics after exposure to specific drugs and calcium blockers using fast fluorescence microspectrofluorimetry followed by ATP effect analysis, which confirmed the expression of functional purinergic adenosine and P2 receptors. Further, we found that glutamate sensitivity of C2C12 cells was mediated by ionotropic glutamate receptors; on the other hand, most cells were responsive to cyclopiazonic acid, which inhibits the sarco-endoplasmic reticulum Ca(2+)-ATPase pump. These results suggest that C2C12 cells possess functional L- and P/Q-type voltage-operated Ca(2+) channels, ryanodine receptors and functional sarcoplasmic reticulumCa(2+) stores (typical for muscle cells), adenosine and P2 purinergic receptors, as well as ionotropic glutamate receptors. The evaluation of intracellular Ca(2+) signaling is a promising approach towards a better understanding and control of the physiopathological properties of myogenic cells that could be used as a predictive factor in the selection of optimal cells for scaffold recellularization or for tissue engineered constructs used in stem cell therapy.

• MeSH
• buněčné linie MeSH
• intracelulární tekutina fyziologie   MeSH
• kosterní svalová vlákna fyziologie   MeSH
• kultivované buňky MeSH
• myoblasty fyziologie   MeSH
• myši MeSH
• vápníková signalizace fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Microcurrent electrical neuromuscular stimulation (MENS) is known as an extracellular stimulus for the regeneration of injured skeletal muscle in sports medicine. However, the effects of MENS-associated increase in muscle protein content are not fully clarified. The purpose of this study was to investigate the effects of MENS on the muscular protein content, intracellular signals, and the expression level of caveolin-3 (Cav-3), tripartite motif-containing 72 (TRIM72) and MM isoenzyme of creatine kinase (CK-MM) in skeletal muscle using cell culture system. C2C12 myotubes on the 7th day of differentiation phase were treated with MENS (intensity: 10-20 microA, frequency: 0.3 Hz, pulse width: 250 ms, stimulation time: 15-120 min). MENS-associated increase in the protein content of myotubes was observed, compared to the untreated control level. MENS upregulated the expression of Cav-3, TRIM72, and CK-MM in myotubes. A transient increase in phosphorylation level of Akt was also observed. However, MENS had no effect on the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 and 5'AMP-activated protein kinase. MENS may increase muscle protein content accompanied with a transient activation of Akt and the upregulation of Cav-3 and TRIM72.

• MeSH
• buněčné linie MeSH
• elektrická stimulace metody   MeSH
• kaveolin 3 biosyntéza   MeSH
• kosterní svalová vlákna metabolismus   MeSH
• myoblasty metabolismus   MeSH
• myši MeSH
• svalové proteiny biosyntéza   MeSH
• transportní proteiny biosyntéza   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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