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Sarcoglycanopathies are rare limb girdle muscular dystrophies, still incurable, even though symptomatic treatments may slow down the disease progression. Most of the disease-causing defects are missense mutations leading to a folding defective protein, promptly removed by the cell’s quality control, even if possibly functional. Recently, we repurposed small molecules screened for cystic fibrosis as potential therapeutics in sarcoglycanopathy. Indeed, cystic fibrosis transmembrane regulator (CFTR) correctors successfully recovered the defective sarcoglycan-complex in vitro. Our aim was to test the combined administration of some CFTR correctors with C17, the most effective on sarcoglycans identified so far, and evaluate the stability of the rescued sarcoglycan-complex. We treated differentiated myogenic cells from both sarcoglycanopathy and healthy donors, evaluating the global rescue and the sarcolemma localization of the mutated protein, by biotinylation assays and western blot analyses. We observed the additive/synergistic action of some compounds, gathering the first ideas on possible mechanism/s of action. Our data also suggest that a defective α-sarcoglycan is competent for assembly into the complex that, if helped in cell traffic, can successfully reach the sarcolemma. In conclusion, our results strengthen the idea that CFTR correctors, acting probably as proteostasis modulators, have the potential to progress as therapeutics for sarcoglycanopathies caused by missense mutations.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder with predominant myotonia and muscular dystrophy which is caused by CTG-repeat expansions in the DMPK gene. These repeat expansions are transcribed and the resulting mRNA accumulates RNA-binding proteins involved in splicing, resulting in a general splicing defect. We observed nuclear envelope (NE) alterations in DM1 primary myoblasts. These included invaginations of the NE as well as an altered composition of the nuclear lamina. Specifically, we investigated NE transmembrane proteins (NETs) in DM1 primary myoblasts, staining to determine if their distribution was altered compared to controls and if this could contribute to these structural defects. We also tested the expression of these NETs in muscle and how localization changes in the DM1 primary myoblasts undergoing differentiation in vitro to myotubes. We found no changes in the localization of the tested NETs, but most tended to exhibit reduced expression with increasing DMPK-repeat length. Nonetheless, the DM1 patient expression range was within the expression range of the controls. Additionally, we found a down-regulation of the possible nesprin 1 giant isoform in DM1 primary myoblasts which could contribute to the increased NE invaginations. Thus, nesprin 1 may be an interesting target for further investigation in DM1 disease pathology.

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) β/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.

There are several three-dimensional (3D) skeletal muscle (SkM) tissue engineered models reported in the literature. 3D SkM tissue engineering (TE) aims to recapitulate the structure and function of native (in vivo) tissue, within an in vitro environment. This requires the differentiation of myoblasts into aligned multinucleated myotubes surrounded by a biologically representative extracellular matrix (ECM). In the present work, a new commercially available 3D SkM TE culture chamber manufactured from polyether ether ketone (PEEK) that facilitates suitable development of these myotubes is presented. To assess the outcomes of the myotubes within these constructs, morphological, gene expression, and ECM remodeling parameters were compared against a previously published custom-built model. No significant differences were observed in the morphological and gene expression measures between the newly introduced and the established construct configuration, suggesting biological reproducibility irrespective of manufacturing process. However, TE SkM fabricated using the commercially available PEEK chambers displayed reduced variability in both construct attachment and matrix deformation, likely due to increased reproducibility within the manufacturing process. The mechanical differences between systems may also have contributed to such differences, however, investigation of these variables was beyond the scope of the investigation. Though more expensive than the custom-built models, these PEEK chambers are also suitable for multiple use after autoclaving. As such this would support its use over the previously published handmade culture chamber system, particularly when seeking to develop higher-throughput systems or when experimental cost is not a factor.

ContextMyotonic dystrophies (DM) are dominantly inherited muscle disorders characterized by myotonia, muscle weakness, and wasting. The reasons for sarcopenia in DMs are uncleared and multiple factors are involved. Irisin, a positive hormone regulator of muscle growth and bone, may play a role.ObjectivesTo investigate (1) circulating irisin in a series of DM1 and DM2 male patients compared with healthy controls and (2) the relationships between irisin and anthropometric, metabolic and hormonal parameters.Design and study participantsThis is a cross-sectional study. Fasting blood samples for glucometabolic, gonadic, bone markers, and irisin were collected from 28 ambulatory DM1, 10 DM2, and 23 age-matched healthy male subjects. Body composition and bone mineralization [bone mineral density (BMD)] were measured by DEXA. Echocardiographic assessment and visceral adiposity, namely, liver and epicardial fat, were investigated by ultrasound. Irisin released from cultured myotubes derived from 3 DM1, 3 DM2, and 3 healthy donors was assayed.ResultsPlasma irisin levels were definitely lower in both DM1 and DM2 patients than in controls with no difference between DM1 and DM2. Irisin released from DM1 and DM2 myotubes was similar to that released from myotubes of the non-DM donors, though diabetic DM2 myotubes released more irisin than DM1 myotubes. There was no correlation between irisin and muscle strength or lean mass in both DM1 and DM2 patients. In DM1 patients, plasma irisin levels correlated negatively with oxygen consumption and positively with insulin resistance, while in DM2 patients plasma irisin levels positively correlated with fat mass at arms and legs levels. No correlation with visceral fat, left ventricular mass, and gonadal hormones could be detected. In both DM1 and DM2 patients, legs BMD parameters positively correlated with plasma irisin levels.ConclusionPlasma irisin is reduced in both DM1 and DM2 male patients likely reflecting muscle mass reduction. Moreover, insulin resistance may contribute to modulation of plasma irisin in DM1 patients. The irisin-mediated cross talk muscle–adipose tissue–bone may be active also in the male myotonic dystrophies’ model.

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

McArdle disease, also termed ‘glycogen storage disease type V’, is a disorder of skeletal muscle carbohydrate metabolism caused by inherited deficiency of the muscle-specific isoform of glycogen phosphorylase (GP-MM). It is an autosomic recessive disorder that is caused by mutations in the PYGM gene and typically presents with exercise intolerance, i.e. episodes of early exertional fatigue frequently accompanied by rhabdomyolysis and myoglobinuria. Muscle biopsies from affected individuals contain subsarcolemmal deposits of glycogen. Besides GP-MM, two other GP isoforms have been described: the liver (GP-LL) and brain (GP-BB) isoforms, which are encoded by the PYGL and PYGB genes, respectively; GP-BB is the main GP isoform found in human and rat foetal tissues, including the muscle, although its postnatal expression is dramatically reduced in the vast majority of differentiated tissues with the exception of brain and heart, where it remains as the major isoform. We developed a cell culture model from knock-in McArdle mice that mimics the glycogen accumulation and GP-MM deficiency observed in skeletal muscle from individuals with McArdle disease. We treated mouse primary skeletal muscle cultures in vitro with sodium valproate (VPA), a histone deacetylase inhibitor. After VPA treatment, myotubes expressed GP-BB and a dose-dependent decrease in glycogen accumulation was also observed. Thus, this in vitro model could be useful for high-throughput screening of new drugs to treat this disease. The immortalization of these primary skeletal muscle cultures could provide a never-ending source of cells for this experimental model. Furthermore, VPA could be considered as a gene-expression modulator, allowing compensatory expression of GP-BB and decreased glycogen accumulation in skeletal muscle of individuals with McArdle disease.

Jatropha gossypifolia L. (Euphorbiaceae) is a plant widely used in the treatment of type 2 diabetes mellitus (T2DM), but there are few scientific reports validating its activity in this area. In this work and through a bioguided assay, a crude extract stimulated glucose uptake in C2C12 myotubes up to 30%, thereby reducing insulin resistance induced by fatty acids compared to the basal control. A chromatographic fraction applied intraperitoneally (IP) in mice reduced glucose by 42% in a mouse model of T2DM, after administration of 10 doses during 20 days. A flavanone was purified from this active fraction and its structure was assigned by 1H- and 13C-NMR (1D and 2D) and MS. This compound retains the previously reported activity, stimulating in vitro the glucose uptake in a concentration-dependent manner. This study indicates that Jatropha gossypifolia L. extracts enhance glucose uptake in cultured myotubes and adipocytes and also improving glucose tolerance in an in vivo model.

Microcantilever platforms are functional models for studying skeletal muscle force dynamics in vitro. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding the chronic investigations of skeletal muscles for drug efficacy and toxicity. To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) β/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA. [ABSTRACT FROM AUTHOR]

Grifola frondosa is a mushroom that has anti-obesity effects, but the detailed mechanism is poorly understood. In this study, we found that lipid soluble extracts derived from G. frondosa (GFE) had peroxisome proliferator-activated receptor δ (PPARδ) agonist activity, inducing the expression of PPARδ-target genes in vitro. Furthermore, administration of GFE to high-fat diet-induced obese mice lowered the total blood cholesterol levels, upregulated the expression of PPARδ-target genes in skeletal muscles and improved glucose intolerance. Additionally, analyses of C2C12 myotubes revealed that GFE restored glucose uptake, which was inhibited by sodium palmitate, to normal levels. Unexpectedly, such acceleration was not abolished by a PPARδ antagonist. These results suggest that G. frondosa is a novel functional food that may prevent life-style related diseases like obesity and diabetes, and that these beneficial effects are likely to be mediated through the activation of PPARδ and a PPARδ-independent insulin signaling pathway. [ABSTRACT FROM AUTHOR]

Botulin toxin (BTX) is widely used for treating skeletal muscle spasticity. Experimental reports on BTX treatment were mainly focused on the neuromuscular junction, while relatively little is known about toxin effects on the muscle cell itself. We investigated possible impact of BTX type A on skeletal muscle cell transcriptome by microarray analysis in muscle-derived cell cultures (fibroblasts, myoblasts and myotubes) from controls and spastic patients, and results were then validated at transcript and protein level. BTX-A treatment of control cells induced major changes in the myogenic component of the transcriptome, whereas the same treatment had a negligible effect in the fibrogenic component. BTX-A treatment of cell cultures from spastic patients induced an increased number of genes differentially expressed both in the fibrogenic and myogenic components. Specifically, BTX-A had a major effect on cell cycle-related genes in myoblasts, on muscle contraction-related genes in myotubes, and on extracellular matrix-related genes in fibroblasts from spastic patients. Our findings show that in vitro BTX-A treatment differentially affects transcript expression in muscle cells from spastic patients compared to those from controls suggesting a direct effect of BTX-A on muscle-specific functional pathways. [ABSTRACT FROM AUTHOR]

Enhanced oxidative stress plays an important role in the progression and onset of diabetes and its complications. Strategies or efforts meant to reduce the oxidative stress are needed which may mitigate these pathogenic processes. The present study aims to investigate the in vitro ameliorative potential of nine antioxidant molecules in L6 myotubes under oxidative stress condition induced by 4-hydroxy-2-nonenal and also to comprehend the gene expression patterns of oxidative stress genes upon the supplementation of different antioxidants in induced stress condition. The study results demonstrated a marked increase in the level of malondialdehyde and protein carbonyl content with a subsequent increase in the free radicals that was reversed by the pretreatment of different dietary antioxidant. From the expression analysis of the oxidative stress genes, it is evident that the expression of these genes is modulated by the presence of antioxidants. The highest expression was found in the cells treated with Insulin in conjugation with an antioxidant. Resveratrol is the most potent modulator followed by Mangiferin, Estragole, and Capsaicin. This comparative analysis ascertains the potency of Resveratrol along with Insulin in scavenging the reactive oxygen species (ROS) generated under induced stress conditions through antioxidant defense mechanism against excessive ROS production, contributing to the prevention of oxidative damage in L6 myotubes. [ABSTRACT FROM AUTHOR]

There are several three-dimensional (3D) skeletal muscle (SkM) tissue engineered models reported in the literature. 3D SkM tissue engineering (TE) aims to recapitulate the structure and function of native (in vivo) tissue, within an in vitro environment. This requires the differentiation of myoblasts into aligned multinucleated myotubes surrounded by a biologically representative extracellular matrix (ECM). In the present work, a new commercially available 3D SkM TE culture chamber manufactured from polyether ether ketone (PEEK) that facilitates suitable development of these myotubes is presented. To assess the outcomes of the myotubes within these constructs, morphological, gene expression, and ECM remodeling parameters were compared against a previously published custom-built model. No significant differences were observed in the morphological and gene expression measures between the newly introduced and the established construct configuration, suggesting biological reproducibility irrespective of manufacturing process. However, TE SkM fabricated using the commercially available PEEK chambers displayed reduced variability in both construct attachment and matrix deformation, likely due to increased reproducibility within the manufacturing process. The mechanical differences between systems may also have contributed to such differences, however, investigation of these variables was beyond the scope of the investigation. Though more expensive than the custombuilt models, these PEEK chambers are also suitable for multiple use after autoclaving. As such this would support its use over the previously published handmade culture chamber system, particularly when seeking to develop higher-throughput systems or when experimental cost is not a factor. [ABSTRACT FROM AUTHOR]

We hypothesised that skeletal muscles of healthy young people have a large variation in oxidative capacity and fibre-type composition, and aimed therefore to investigate glucose metabolism in biopsies and myotubes isolated from musculus vastus lateralis from healthy males with varying degrees of maximal oxygen uptake. Trained and intermediary trained subjects showed higher carbohydrate oxidation in vivo. Fibre-type distribution in biopsies and myotubes did not differ between groups. There was no correlation between fibre-type I expression in biopsies and myotubes. Myotubes from trained had higher deoxyglucose accumulation and fractional glucose oxidation (glucose oxidation relative to glucose uptake), and were also more sensitive to the suppressive action of acutely added oleic acid to the cells. Despite lack of correlation of fibre types between skeletal muscle biopsies and cultured cells, myotubes from trained subjects retained some of their phenotypes in vitro with respect to enhanced glucose metabolism and metabolic flexibility. [ABSTRACT FROM AUTHOR]

Background Elevated levels of inflammatory molecules are key players in muscle wasting/atrophy leading to human morbidity. TNFα is a well-known pro-inflammatory cytokine implicated in the pathogenesis of muscle wasting under diverse clinical settings. S -allyl cysteine (SAC), an active component of garlic ( Allium sativum ), has established anti-oxidant and anti-inflammatory effects in various cell types. However, the impact of SAC on skeletal muscle pathology remains unexplored. Owing to the known anti-inflammatory properties of SAC, we investigated whether pre-treatment with SAC has a protective role in TNFα-induced atrophy in cultured myotubes. Methods and results C2C12 myotubes were treated with TNFα (100 ng/ml) in the presence or absence of SAC (0.01 mM). TNFα treatment induced atrophy in myotubes by up-regulating various proteolytic systems i . e . cathepsin L, calpain, ubiquitin-proteasome E3-ligases (MuRF1/atrogin1), caspase 3 and autophagy (Beclin1/LC3B). TNFα also induced the activation of NFκB by stimulating the degradation of IκBα (inhibitor of NFκB), in myotubes. The alterations in proteolytic systems likely contribute to the degradation of muscle-specific proteins and reduce the myotube length, diameter and fusion index. The SAC supplementation significantly impedes TNFα-induced protein loss and protects myotube morphology by suppressing protein catabolic systems and endogenous level of inflammatory molecules namely TNFα, IL-6, IL-1β, TNF-like weak inducer of apoptosis (TWEAK), fibroblast growth factor-inducible 14 (Fn14) and Nox. Conclusion and general significance Our findings reveal anti-atrophic role for SAC, as it prevents alterations in protein metabolism and protects myotubes by regulating the level of inflammatory molecules and multiple proteolytic systems responsible for muscle atrophy. [ABSTRACT FROM AUTHOR]

Background: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. Methods: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. Results: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. Conclusions: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function. [ABSTRACT FROM AUTHOR]

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High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 μM) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 μM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts. [ABSTRACT FROM AUTHOR]

Muscle atrophy occurs in various catabolic conditions, including hormone imbalance, severe injury, sepsis, cancer, and aging. Dexamethasone (DEX) is a synthetic glucocorticoid and is used an anti-inflammatory agent. However, when chronically used, it is accompanied by side effects, such as, muscle atrophy, diabetes mellitus, and obesity. In this study, we investigated the effect of sulforaphane (SFN) on DEX-induced muscle atrophy and the underlying mechanisms involved. DEX induced muscle atrophy was accompanied by increased muscle specific ubiquitin E3 ligase markers, such as, Atrogin-1 and myostatin, and decreased MyoD in C2C12 myotubes. To investigate the role played by SFN in DEX-induced muscle atrophy, we quantified mRNA levels of muscle atrophy markers, protein synthesis using a puromycin incorporation assay, protein degradation by ubiquitination, and myotube diameters by PAS staining in C2C12 myotubes co-treated with DEX and SFN. Interestingly, SFN effectively prevented myostatin and Atrogin-1 mRNA upregulations by DEX, increased the mRNA level of MyoD, and consequently, reduced protein degradation. Furthermore, SFN enhanced protein synthesis through a Foxo-dependent pathway by activating Akt, and thus, increased myotube diameters. These results show SFN inhibits DEX-induced muscle atrophy in C2C12 myotubes via Akt/Foxo signaling. [ABSTRACT FROM AUTHOR]

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho-functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders. [ABSTRACT FROM AUTHOR]

Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4μ8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12 myotubes. [ABSTRACT FROM AUTHOR]

To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat – a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments. • Clinostats rotate cell cultures to average-out the vector of gravitational forces. • Rotational movements induce complex fluid motions in cell culture vessels. • Suppression of myotube formation during clino-rotation is due to fluid motion. • Cell biological results from clino-rotation may not be attributed to microgravity. • Additional control experiments to determine fluid motion effects are mandatory. [ABSTRACT FROM AUTHOR]

Cachexia is strongly associated with a poor prognosis in cancer patients but the biological trigger is unknown and therefore no therapeutics exist. The loss of skeletal muscle is the most deleterious aspect of cachexia and it appears to depend on secretions from tumor cells. Models for studying wasting in cell culture consist of experiments where skeletal muscle cells are incubated with medium conditioned by tumor cells. This has led to candidates for cachectic factors but some of the features of cachexia in vivo are not yet well-modeled in cell culture experiments. Mouse myotube atrophy measured by myotube diameter in response to medium conditioned by mouse colon carcinoma cells (C26) is consistently less than what is seen in muscles of mice bearing C26 tumors with moderate to severe cachexia. One possible reason for this discrepancy is that in vivo the C26 tumor and skeletal muscle share a circulatory system exposing the muscle to tumor factors in a constant and increasing way. We have applied Transwell®-adapted cell culture conditions to more closely simulate conditions found in vivo where muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. C26 cells were incubated on a microporous membrane (a Transwell® insert) that constitutes the upper compartment of wells containing plated myotubes. In this model, myotubes are exposed to a constant supply of cancer cell secretions in the medium but without direct contact with the cancer cells, analogous to a shared circulation of muscle and cancer cells in tumor-bearing animals. The results for myotube diameter support the idea that the use of Transwell® inserts serves as a more physiological model of the muscle wasting associated with cancer cachexia than the bolus addition of cancer cell conditioned medium. The Transwell® model supports the notion that the dose and kinetics of cachectic factor delivery to muscle play a significant role in the extent of pathology.

Background/Aims: Deleterious effects of saturated fatty acids in skeletal muscle cells are well known but their impact on mitochondrial respiration has not been well studied. Mitochondrial oxidative damage has been implicated to play a role in their effect. The purpose of this study was to evaluate viability, mtDNA integrity and mitochondrial respiration in C2C12 myoblasts and myotubes exposed to palmitate and to test the effect of mitochondria-targeted antioxidants MitoQ and MitoTEMPOL in preventing palmitate-induced damage. Methods: Cells were treated with tested compounds, mtDNA damage was detected by quantitative PCR and mitochondrial respiration was measured using an extracellular flux analyzer XF24. Results: Palmitate caused mtDNA damage, which was associated with reduced mitochondrial respiration and cell death in myoblasts but not in myotubes. MitoTEMPOL was able to prevent palmitate-induced mtDNA damage in myoblasts but failed to prevent cell death. MitoQ did not show any protective effect and both compounds markedly inhibited mitochondrial respiration. Conclusion: Our results indicate that skeletal muscle progenitor cells could be the first target of the deleterious action of palmitate, as myoblasts appeared to be more sensitive to its effects than myotubes possibly in part due to a lower spare respiratory capacity in the former. Only MitoTEMPOL prevented palmitate-induced mtDNA damage but neither antioxidant was able to prevent cell death and both antioxidants had a marked negative effect on respiration.

Previous studies have shown that chronic hyperglycemia impairs glucose and fatty acid oxidation in cultured human myotubes. To further study the hyperglycemia-induced suppression of oxidation, lactate oxidation, mitochondrial function and glycolytic rate were evaluated. Further, we examined the intracellular content of reactive oxygen species (ROS), production of lactate and conducted pathway-ANOVA analysis on microarray data. In addition, the roles of the pentose phosphate pathway (PPP) and the hexosamine pathway were evaluated. Lactic acid oxidation was suppressed in hyperglycemic versus normoglycaemic myotubes. No changes in mitochondrial function or ROS concentration were observed. Pathway-ANOVA analysis indicated several upregulated pathways in hyperglycemic cells, including glycolysis and PPP. Functional studies showed that glycolysis and lactate production were higher in hyperglycemic than normoglycaemic cells. However, there were no indications of involvement of PPP or the hexosamine pathway. In conclusion, hyperglycemia reduced substrate oxidation while increasing glycolysis and lactate production in cultured human myotubes.

Background: Exercise induces many physiological adaptations. Recently, it has been proposed that some of these adaptations are induced by exercise-mediated lactate production. In this study, we aimed to investigate in vitro the effect of lactate in cultured myotubes and whether antioxidants could inhibit the effect. Methods: Differentiated myotubes were cultured at different concentrations of L-lactate (0, 10, 30, 50 mM) in the absence or presence of an antioxidant, N-acetyl-L-cysteine (Nac). The temporal effect of lactate exposure in myotubes was also explored. Results: Two hours of exposure to 50 mM L-lactate and six hours of exposure to 30 or 50 mM L-lactate caused a significant increase in PGC1-alpha (peroxisome proliferator-activated receptor γ coactivator-1α) expression in the myotubes. This up-regulation was suppressed by 2 mM Nac. Intermittent and continuous lactate exposure caused similar PGC1-alpha up-regulation. These results suggest that the increase in PGC1-alpha expression is mediated by reactive oxygen species (ROS) production from lactate metabolism and that both continuous and intermittent exposure to L-lactate can cause the up-regulation.

Data from our studies, and those of others, support the proposal that there is a role for skeletal muscle in the maintenance of vitamin D status. We demonstrated that skeletal muscle is able to internalise extracellular vitamin D binding protein, which then binds to actin in the cytoplasm, to provide high affinity binding sites which accumulate 25-hydroxyvitamin D 3 (25(OH)D 3 ) [1] . This study investigated the concentration- and time-dependent effects of parathyroid hormone (PTH) on the capacity of muscle cells to take up and release 3 H-25(OH)D 3 . Uptake and retention studies for 3 H-25(OH)D 3 were carried out with C2C12 cells differentiated into myotubes and with primary mouse muscle fibers as described [1] . The presence of PTH receptors on mouse muscle fibers was demonstrated by immunohistochemistry and PTH receptors were detected in differentiated myotubes, but not myoblasts, and on muscle fibers by Western blot. Addition of low concentrations of vitamin D binding protein to the incubation media did not alter uptake of 25(OH)D 3 . Pre-incubation of C2 myotubes or primary mouse muscle fibers with PTH (0.1 to 100 pM) for 3 h resulted in a concentration-dependent decrease in 25(OH)D 3 uptake after 4 or 16 h. These effects were significant at 0.1 or 1 pM PTH (p < 0.001) and plateaued at 10 pM, with 25(OH)D 3 uptake reduced by over 60% (p < 0.001) in both cell types. In C2 myotubes, retention of 25(OH)D 3 was decreased after addition of PTH (0.1 to 100 pM) in a concentration-dependent manner by up to 80% (p < 0.001) compared to non-PTH treated-C2 myotubes. These data show that muscle uptake and retention of 25(OH)D 3 are modulated by PTH, a physiological regulator of mineral homeostasis, but the cell culture model may not be a comprehensive reflection of vitamin D homeostatic mechanisms in whole animals. [ABSTRACT FROM AUTHOR]

Cachexia is strongly associated with a poor prognosis in cancer patients but the biological trigger is unknown and therefore no therapeutics exist. The loss of skeletal muscle is themost deleterious aspect of cachexia and it appears to depend on secretions from tumor cells. Models for studying wasting in cell culture consist of experiments where skeletal muscle cells are incubated with medium conditioned by tumor cells. This has led to candidates for cachectic factors but some of the features of cachexia in vivo are not yet well-modeled in cell culture experiments. Mouse myotube atrophy measured by myotube diameter in response to medium conditioned by mouse colon carcinoma cells (C26) is consistently less than what is seen in muscles of mice bearing C26 tumors with moderate to severe cachexia. One possible reason for this discrepancy is that in vivo the C26 tumor and skeletal muscle share a circulatory system exposing the muscle to tumor factors in a constant and increasing way. We have applied Transwell®-adapted cell culture conditions to more closely simulate conditions found in vivo where muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. C26 cells were incubated on a microporous membrane (a Transwell® insert) that constitutes the upper compartment of wells containing plated myotubes. In this model, myotubes are exposed to a constant supply of cancer cell secretions in the medium but without direct contact with the cancer cells, analogous to a shared circulation of muscle and cancer cells in tumor-bearing animals. The results for myotube diameter support the idea that the use of Transwell® inserts serves as a more physiological model of the muscle wasting associated with cancer cachexia than the bolus addition of cancer cell conditioned medium. The Transwell® model supports the notion that the dose and kinetics of cachectic factor delivery to muscle play a significant role in the extent of pathology. [ABSTRACT FROM AUTHOR]

Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H 2 O 2 ) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H 2 O 2 -induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H 2 O 2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H 2 O 2 -stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H 2 O 2 -activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance. [ABSTRACT FROM AUTHOR]

Muscle atrophy occurs when there is a net loss of muscle mass, leading to a change in the balance between protein synthesis and protein degradation. Igf1 is important for protein synthesis in muscle cells and can induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy via the PI3K/Akt pathway in mice, consequently restoring and improving muscle mass and strength. In this study, we show that miR-18a expression is down-regulated during C2C12 myoblast differentiation and mouse tibialis anterior muscle postnatal development. Functional studies show that forced expression of miR-18a induces myotubes atrophy and increases the expression of MuRF1, Atrogin-1 and CTSL. miR-18a also decreases the phosphorylation of both Akt and FoxO3, and an inhibitor of the PI3K/Akt pathway blocks the function of miR-18a. An analysis of miR-18a targets reveals that Igf1 is regulated by miR-18a. miR-18a suppresses the expression of Igf1 in a 3′UTR-dependent manner. These findings strongly support the idea that miR-18a has a functional role in muscle physiology and suggest that miR-18a is a potential novel therapeutic target for skeletal muscle atrophy. [ABSTRACT FROM AUTHOR]

We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3–5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo . Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application. [ABSTRACT FROM AUTHOR]

Much attention has been given to insulin-like growth factor (Igf) pathways that regulate the balance of skeletal muscle protein synthesis and breakdown in response to a range of extrinsic and intrinsic signals. However, we have a less complete understanding of how the same signals modulate muscle mass upstream of such signalling, through a family of functionally-diverse Igf-binding proteins (Igfbps) that modify the availability of Igfs to the cell receptor Igf1r. We exposed cultured myotubes from Atlantic salmon ( Salmo salar L.) to treatments recapturing three catabolic signals: inflammation (interleukin-1β), stress (dexamethasone) and fasting (amino acid deprivation), plus one anabolic signal: recovery of muscle mass post-fasting (supplementation of fasted myotubes with Igf-I and amino acids). The intended phenotype of treatments was confirmed by significant changes in myotube diameter and immunofluorescent staining of structural proteins. We quantified the mRNA-level regulation of the full expressed Igf and Igfbp gene complement across a post-treatment time course, along with marker genes for muscle structural protein synthesis, as well as muscle breakdown, via the ubiquitin-proteasome and autophagy systems. Our results highlight complex, non-overlapping responses of Igfbp family members to the different treatments, suggesting that the profile of expressed Igfbps is differentially regulated by distinct signals promoting similar muscle remodelling phenotypes. We also demonstrate divergent regulation of salmonid-specific gene duplicates of igfbp5b1 and igfbp5b2 under distinct catabolic and anabolic conditions. Overall, this study increases our understanding of the regulation of Igfbp genes in response to signals that promote remodelling of skeletal muscle. [ABSTRACT FROM AUTHOR]

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) increase insulin signaling in skeletal muscle. In the current study, we investigated the effect of eicosapentaenoic acid (EPA) on insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes. We showed that EPA did not affect basal and insulin-induced mTOR phosphorylation in myotubes. However, EPA abolished lipopolysaccharide (LPS) -induced deficiency in insulin signaling ( P < 0.05). Pre-incubation of nuclear factor κB (NF-κΒ) and c-Jun N-terminal kinases (JNK) inhibitors prevented the decreased insulin-induced mTOR phosphorylation elicited by LPS ( P < 0.05). In addition, in protein tyrosine phosphatase-1B (PTP1B) knockdown myotubes, LPS failed to decrease insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes ( P > 0.05). In myotubes, LPS stimulated PTP1B expression via NF-κB and activation protein-1 (AP1). Pre-incubation of 50 μM EPA prevented the LPS-induced activation of AP1 and NF-κΒ as well as PTP1B expression ( P < 0.05). Interestingly, incubation of peroxisome proliferator-activated receptor γ (PPARγ) antagonist (GW9662) prior to EPA treatment, the effect of EPA on insulin-induced mTOR phosphorylation was blocked. Accordingly, EPA did not inhibit the LPS-induced activation of AP1 or NF-κΒ as well as PTP1B expression when incubation of GW9662 prior to EPA treatment. The in vivo study showed that EPA prevented LPS-induced PTPT1B expression and a decrease in insulin-induced mTOR phosphorylation in muscle of mice. In summary, EPA abolished LPS inhibition of insulin-induced mTOR phosphorylation in myotubes, and one of the key mechanisms was to inhibit AP1 and NF-κB activation and PTP1B transcription. [ABSTRACT FROM AUTHOR]

[Display omitted] • A photo-affinity probe pMY was designed to identify the targets of myricanol (MY). • Nampt was identified as a direct target of MY via activity-based protein profiling. • MY sensitizes insulin action in C2C12 myotubes by activating Nampt. • pMY is the first probe to label Nampt. Herein, we synthesized an affinity-based probe of myricanol (pMY) with a photo-affinity cross-linker to initiate a bioconjugation reaction, which was applied for target identification in live C2C12 myotubes. Pull-down of biotinylated pMY coupled with mass spectroscopy and Western blotting revealed that pMY can bind with nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the nicotinamide adenine dinucleotide salvage pathway. Cellular thermal shift assay, drug affinity responsive target stability assay and recombinant protein labeling further validated the direct interaction between myricanol and Nampt. Myricanol did not affect the protein expression of Nampt, but enhanced its activity. Knock-down of Nampt totally abolished the promoting effect of myricanol on insulin-stimulated glucose uptake in C2C12 myotubes. Taken together, myricanol sensitizes insulin action in myotubes through binding with and activating Nampt. [ABSTRACT FROM AUTHOR]

The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 µM) and DPA (10 µM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte’s CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes.