Skeletal Muscle Cell Model Research Articles

AMPK and glucose deprivation exert an isoform-specific effect on the expression of Na+,K+-ATPase subunits in cultured myotubes

In skeletal muscle, Na+,K+-ATPase (NKA), a heterodimeric (α/β) P-type ATPase, has an essential role in maintenance of Na+ and K+ homeostasis, excitability, and contractility. AMP-activated protein kinase (AMPK), an energy sensor, increases the membrane abundance and activity of NKA in L6 myotubes, but its potential role in regulation of NKA content in skeletal muscle, which determines maximum capacity for Na+ and K+ transport, has not been clearly delineated. We examined whether energy stress and/or AMPK affect expression of NKA subunits in rat L6 and primary human myotubes. Energy stress, induced by glucose deprivation, increased protein content of NKAα1 and NKAα2 in L6 myotubes, while decreasing the content of NKAα1 in human myotubes. Pharmacological AMPK activators (AICAR, A-769662, and diflunisal) modulated expression of NKA subunits, but their effects only partially mimicked those that occurred in response to glucose deprivation, indicating that AMPK does not mediate all effects of energy stress on NKA expression. Gene silencing of AMPKα1/α2 increased protein levels of NKAα1 in L6 myotubes and NKAα1 mRNA levels in human myotubes, while decreasing NKAα2 protein levels in L6 myotubes. Collectively, our results suggest a role for energy stress and AMPK in modulation of NKA expression in skeletal muscle. However, their modulatory effects were not conserved between L6 myotubes and primary human myotubes, which suggests that coupling between energy stress, AMPK, and regulation of NKA expression in vitro depends on skeletal muscle cell model.

Mechanism of static loading injury in human skeletal muscle cells.

To establish a cellular-level mechanical injury model for human skeletal muscle cells and investigate changes in the mechanical effect mechanism after such injuries. The FX-5000™ Compression System was used to apply constant static mechanical pressure to human skeletal muscle cells. A factorial design analysis was conducted to discover the optimal injury model by evaluating the correlation between the amount of pressure, the duration of mechanical stimulation, and the number of days of observation. Skeletal muscle cell injury was evaluated by measuring cell metabolism, morphology, and calcium homeostasis. Mechanical injury was modeled as continuous pressure of 1 MPa for 2 hours with observation for 3 days. The results show that mechanical injury increased creatine kinase, intracellular Ca2+ concentration, and malondialdehyde content, decreased superoxide dismutase, and caused cell swelling and severe cytoplasmic vacuolization (all P < 0.05). This model of mechanically-injured human skeletal muscle cells provides an experimental model for the clinically common skeletal muscle injury caused by static loading pressure. It may be used to study the mechanism of action of treatment methods for mechanically injured skeletal muscle.

Perch essence prevents cell death to improve skeletal muscle mass and strength: Evidence from in vitro and human model

Background:Fish protein supplementation may maintain muscle strength and prevent sarcopenia as it contains a complex array of macro- and micronutrients essential for building the skeletal muscle. Objective:The aim of this study was to evaluate the influence of perch essence (PE) supplementation on muscle mass and muscle function of through human and cell model. Methods:The open label clinical trial was conducted to assess the therapeutic effect of PE on muscle mass improvement. The mouse skeletal muscle cell (C2C12) model of muscle atrophy was analyzed for cell viability. Results:Our results showed PE contained abundant branched chain amino acid, taurine, hydroxyproline and collagen. After one month of supplementation with PE in our human model, there was a significant increase in muscle mass in the whole body and all parts of the body, with an increase of 1.55 % in the whole body, 1.79% in the trunk, 1.64% in the arms and 1.38% in the legs. The percentage of subcutaneous fat in the trunk, arms and legs also decreased significantly by 2.49%, 3.21% and 3.40% respectively. PE supplementation also improves muscle grip strength, especially with the dominant hand. The cell model results demonstrated that PE could effectively prevent skeletal muscle cell from death induced by dexamethasone. Conclusion:This study suggests that the branched chain amino acids, taurine, hydroxyproline and collagen in PE have the potential to serve as a good source of dietary supplements for the improvement of skeletal muscle mass and strength through cell protection.Keywords:branched chain amino acid, collagen, perch, skeletal muscle, sarcopenia

Pathological modeling of glycogen storage disease type III with CRISPR/Cas9 edited human pluripotent stem cells.

Introduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches.

Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies.

Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

Regulation of voltage-induced Ca&lt;sup&gt;2+&lt;/sup&gt; release machinery by cell-cell fusion in skeletal myogenesis

One of the most dynamic processes during skeletal myogenesis is cell-cell fusion, which promotes skeletal myoblast to form a large multi-nucleated myofiber equipped with EC-coupling machinery. Recently discovered transmembrane protein Myomixer (Mymx) executes cell-cell fusion, although the physiological significance of Mymx-mediated fusion on myogenic cell differentiation remains largely unknown. The current study focuses on the intracellular Ca2+ signaling mechanism to understand the physiological importance of Mymx mediated cell fusion in regulating voltage-induced Ca2+release in the developing skeletal muscle cells. C2C12 cells were used as an in vitro myogenesis model of skeletal muscle cells. We investigated intracellular Ca2+ release upon electric field stimulation applied to differentiated Mymx-KO cells and Mymx-rescued cells. We found that the efficiency of Ca2+ response was dependent on the Mymx gene expression. Importantly, expression levels of MyoD and myogenin were almost unaltered by the gene rescue of Mymx, suggesting that the Mymx-dependent Ca2+ response is regulated independently of these transcription factors. In conclusion, we found a novel regulatory linkage between Mymx expression and the voltage-induced Ca2+ release essential for EC coupling.

Development of a primary skeletal muscle cell culture model from human muscle biopsies

Purpose: This work aimed to develop and validate a culture model of primary human skeletal muscle cells. Methods: Muscle biopsies were obtained post-mortem from the vastus lateralis. The biopsies were enzymatically digested with collagenase 1A (1mg/mL) and dispase II (4,8mg/mL) for 60 minutes at 37°C in order to isolate fibroblasts and satellite cells. Then, the satellite cells (CD56+) were separated from fibroblasts (CD56-) by two methods: pre-plating or immunomagnetic cell sorting using an anti-CD56 antibody, a transmembrane glycoprotein expressed on the surface of satellite cells. Indeed, some human muscle biopsies contain a high proportion of fibroblasts that can contaminate the myoblasts cell culture if the separation method is not efficient. The performance of these cell separation methods was evaluated by FACS. We also compared the effects of two culture media on myoblasts proliferation (10%FBS vs. 10%FBS + 2%Ultroser G (UG)) using cell doubling time as parameter. Two differentiation media (2%FBS vs. 0.5%UG) were tested on the myogenic index, which corresponds to the proportion of cells that have fused to form myotubes. This index was determined through the detection of the myosin heavy chain (MYH) by immunofluorescence. Finally, we studied the expression of several muscle cell markers during myogenesis by qRT-PCR and immunofluorescence. Results: The cells obtained from the enzymatic digestion of the biopsy contained a varying proportion of CD56+ satellite cells (86,15 ± 12,09%; 95%CI:73,45 to 98,84). The cell population obtained after immunomagnetic cell sorting method contained more than 92% of CD56+ cells (96,11 ± 3,27%; 95%CI:92,68 to 99,54). In comparison, the ratio of CD56+ obtained by the preplating method was lower (84,19 ± 9,94%; 95%CI:73,76 to 94,62), but no significant difference was observed between both methods. The cell doubling time was significantly shorter with culture media containing 10%FBS and 2%UG than with the media containing only 10%FBS (25.70 ± 3,96h vs. 70.64 ± 21,68h, p = 0.0313). The myogenic index was slightly higher with the 0.5%UG than 2%FBS differentiating medium (62,75% ± 8,57% vs. 56,39 ± 16,45%). Finally, the increase of MyoD1, myogenin and MYH, three markers of differentiation, indicated the phenotypic transition of myoblasts to a myogenic phenotype. Conclusions: Immunomagnetic cell sorting allows better satellite cells and fibroblasts separation than the conventionally used preplating technique. Ultoser G serum substitute accelerates cell proliferation and improves the differentiation of myoblasts into myotubes. The markers MyoD1, myogenin and MYH are good markers of the myogenic differentiation of myoblasts. This in vitro model makes it possible to purify efficiently muscle satellite cells from human muscle biopsies. These cells can be used in in vitro studies to study the pathophysiological mechanisms of muscle disease and the mechanisms of action of treatments.

Comparative study of callus culture and leaves of Thunbergia laurifolia for their bioactive constituents and the activation of AMPK and GLUT‐dependent glucose uptake on rat skeletal muscle (L6) cells

Thunbergia laurifolia (TL) is plant widely distributed in South East Asia. TL leaves have been used as herbal infusion tea for treating fever and chemical intoxication. In this study, we examined the content of bioactive phenolic acids and flavonoids from leaves of TL in different solvent extraction. Besides, in vitro callus culture of TL was established. The rat skeletal muscle (L6) cell model was performed to investigate the glucose uptake stimulation. TL leaves extract with absolute methanol produced higher glucose uptake stimulation than those of the other solvent extracts. Interestingly, callus of TL was successful in producing high rosmarinic acid content over than intact leaves (increase up to 96-fold). The enriched rosmarinic acid callus extract significantly enhanced glucose uptake activity and elevated AMPK-α2 and GLUT4 mRNA expression. The established TL callus culture can be used as a promising source for production of biologically active phenolic acid with in vitro antidiabetic effect. Practical applications The boiled water extraction via hot infusion as herbal drinking tea could extract bioactive constituents, caffeic acid, rutin, and apigenin from Thunbergia laurifolia (TL) leaves, which enhanced glucose uptake activity. Moreover, the enriched rosmarinic acid in TL callus culture can be applied for promoting as a new source of rosmarinic acid, which exhibited antidiabetic activity.

Exposure of low-concentration arsenic induces myotube atrophy by inhibiting an Akt signaling pathway

Arsenic, a widely distributed toxic metalloid, has been found to be associated with the low-birth-weight infants and the impairment of muscle regenerative capacity in areas with high levels of arsenic in drinking water. The distal muscular atrophy is one of side effects of arsenic trioxide (As2O3) for acute promyelocytic leukemia therapy. We hypothesized that arsenic may be a potential risk factor for skeletal muscle atrophy. Here, we investigated the action and molecular mechanism of low-dose arsenic on the induction of skeletal muscle atrophy in a skeletal muscle cell model. The differentiated C2C12 myotubes were treated with As2O3 (0.25–1 μM) for 48 h without apparent effects on cell viability. The signaling molecules for myotube atrophy were assessed. Submicromolar-concentration As2O3 dose-dependently triggered C2C12 myotube atrophy and increased the protein expressions of atrogenes Atrogin1 and MuRF1 and inhibited the upstream phosphorylated proteins Akt and FoxO1, while As2O3 dose-dependently increased AMPK phosphorylation in myotubes. Akt activator SC79 could significantly reverse the As2O3-induced myotube atrophy. These results suggest that arsenic is capable of inducing myotube atrophy by inhibiting an Akt signaling pathway.

Naringenin improves insulin sensitivity in gestational diabetes mellitus mice through AMPK

BackgroundGestational diabetes mellitus (GDM) is a temporary form of diabetes during pregnancy, which influences the health of maternal-child in clinical practice. It is still urgent to develop new effective treatment for GDM. Naringenin is a bioactive ingredient with multiple activities including anti-diabetic. In current study, the effects of naringenin on GDM symptoms, insulin tolerance, inflammation, and productive outcomes were evaluated and the underlying mechanisms were explored.MethodsWe administrated naringenin to GDM mice and monitored the GDM symptoms, glucose and insulin tolerance, inflammation and productive outcomes. We established tumor necrosis factor alpha (TNF-α)-induced insulin resistance skeletal muscle cell model and evaluated the effects of naringenin on reactive oxygen species (ROS) production, glucose uptake and glucose transporter type 4 (GLUT4) membrane translocation.ResultsWe found that naringenin ameliorated GDM symptoms, improved glucose and insulin tolerance, inhibited inflammation, and improved productive outcomes. It was further found that naringenin inhibited TNF-α-induced ROS production, enhanced GLUT4 membrane translocation, and glucose uptake, which were abolished by inhibition of AMP-activated protein kinase (AMPK).ConclusionNaringenin improves insulin sensitivity in gestational diabetes mellitus mice in an AMPK-dependent manner.

Bisphenol A exposure under metabolic stress induces accelerated cellular senescence in vivo in a p53 independent manner

Senescence is an irreversible process that is a characteristic of age-associated disease like Type 2 diabetes (T2D). Bisphenol-A (BPA), one of the most common endocrine disruptor chemicals, received special attention in the development of insulin resistance and T2D. To understand the role played by BPA in cellular senescence under metabolic stress, zebrafish embryos were exposed to BPA in the absence and presence of hyperglycaemia. Transcriptional levels of the senescence markers p15, p53, Rb1 and β-galactosidase were increased when BPA was combined with metabolic stress. In addition, zebrafish embryos that were exposed to combination of hyperglycaemia and BPA exhibited increased levels of apoptosis. However, cellular senescence remained induced by a combination of hyperglycaemia and BPA exposure even in the absence of a translated p53 protein suggesting that senescence is primarily independent of it but dependent on the p15-Rb1 pathway under our experimental conditions. To confirm that our results hold true in adult mammalian tissues, we validated our embryonic experiments in an adult mammalian metabolic model of skeletal muscle cells. Our work reveals a novel and unique converging role of senescence and apoptosis axis contributing to glucose dyshomeostasis. Thus, we conclude that BPA exposure can exacerbate existing metabolic stress to increase cellular senescence that leads to aggravation of disease phenotype in age-associated diseases like type 2 diabetes.

Multitargeted kinase inhibitors imatinib, sorafenib and sunitinib perturb energy metabolism and cause cytotoxicity to cultured C2C12 skeletal muscle derived myotubes

Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment and prognosis but have also resulted in adverse effects such as fatigue, diarrhea, hypothyroidism, and other toxicities. We investigated TKI effects on skeletal muscle as a possible explanation of TKI induced fatigue. Changes in mitochondrial function due to inhibition of oxidative phosphorylation complexes, generation of superoxides, and inhibition of key transporters involved in uptake of glucose and/or nucleosides may result in alteration of energy metabolism and/or mitochondrial function. We investigated effects of imatinib, sorafenib and sunitinib on these processes in cultured C2C12 murine skeletal muscle cells. Imatinib, sorafenib and sunitinib were cytotoxic to C2C12 cells with IC50 values of 20, 8 and 8 µM, respectively. Imatinib stimulated glucose uptake and inhibited complex V activity by 35% at 50 µM. Sorafenib inhibited complex II/III and V with IC50 values of 32 and 28 µM, respectively. Sorafenib caused activation of caspase 3/7 and depolarization of mitochondrial membranes occurred very rapidly with complete loss at 5–10 µM. Sunitinib inhibited Complex I with an IC50 value of 38 µM and caused ATP depletion, caspase 3/7 activation, an increase in reactive oxygen species (ROS), and decreased nucleoside and glucose uptake. In conclusion, imatinib, sunitinib and sorafenib caused changes in mitochondrial complex activities, glucose and nucleoside uptake leading to decreased energy production and mitochondrial function in a skeletal muscle cell model, suggesting that these changes may play a role in fatigue, one of the most common adverse effects of TKIs.

High throughput screening of mitochondrial bioenergetics in human differentiated myotubes identifies novel enhancers of muscle performance in aged mice

Mitochondrial dysfunction is increasingly recognized as a contributor to age-related muscle loss and functional impairment. Therefore, we developed a high throughput screening strategy that enabled the identification of compounds boosting mitochondrial energy production in a human skeletal muscle cell model. Screening of 7949 pure natural products revealed 22 molecules that significantly increased oxygen consumption and ATP levels in myotubes. One of the most potent compounds was the flavanone hesperetin. Hesperetin (10 µM) increased intracellular ATP by 33% and mitochondrial spare capacity by 25%. Furthermore, the compound reduced oxidative stress in primary myotubes as well as muscle tissue in vivo. In aged mice administration of hesperetin (50 mg/kg/d) completely reverted the age-related decrease of muscle fiber size and improved running performance of treated animals. These results provide a novel screening platform for the discovery of drugs that can improve skeletal muscle function in patients suffering from sarcopenia or other disorders associated with mitochondrial dysfunction.

PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells

To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12 skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The results showed that the survival rates of C2C12 cells decreased gradually with an increasing fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling pathway.

Mifepristone enhances insulin-stimulated Akt phosphorylation and glucose uptake in skeletal muscle cells

Mifepristone is the only FDA-approved drug for glycaemia control in patients with Cushing's syndrome and type 2 diabetes. Mifepristone also has beneficial effects in animal models of diabetes and patients with antipsychotic treatment-induced obesity. However, the mechanisms through which Mifepristone produces its beneficial effects are not completely elucidated. PurposeTo determine the effects of mifepristone on insulin-stimulated glucose uptake on a model of L6 rat-derived skeletal muscle cells. ResultsMifepristone enhanced insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane and Akt Ser473 phosphorylation in L6 myotubes. In addition, mifepristone reduced oxygen consumption and ATP levels and increased AMPK Thr172 phosphorylation. The knockdown of AMPK prevented the effects of mifepristone on insulin response. ConclusionsMifepristone enhanced insulin-stimulated glucose uptake through a mechanism that involves a decrease in mitochondrial function and AMPK activation in skeletal muscle cells.

The morphological analysis of autophagy in primary skeletal muscle cells infected with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.

Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line.

Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS) levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

Suppression of phosphatase and tensin homolog protects insulin-resistant cells from apoptosis.

In the present study, a glucosamine-induced model of insulin-resistant skeletal muscle cells was established in order to investigate the effect of inhibition of phosphatase and tensin homolog (PTEN)/5'-adenosine monophosphate-activated protein kinase (AMPK) on these cells. The glucosamine-induced insulin-resistant skeletal muscle cells were produced and the rate of glucose uptake was measured using the glucose oxidase-peroxidase method. The expression levels of PTEN and phosphorylated PTEN (p-PTEN) were assessed using western blotting. Glucose transporter 4 (GLUT4) translocation was detected by immunofluorescence. Cell apoptosis was evaluated using flow cytometry. Following insulin stimulation, the rate of glucose uptake was significantly reduced in the cells with glucosamine-induced insulin-resistance in comparison with those in the control group. The expression and translocation of GLUT4 were reduced in the insulin-resistant muscle cells. By contrast, the expression of PTEN and p-PTEN as well as apoptosis were significantly increased. Following treatment with bisperoxopicolinatooxovanadate (BPV) or metformin in the insulin-resistant skeletal muscle cells, there was an increase in the rate of glucose uptake, an increase in GLUT4 expression and its translocation, a reduction in the expression of PTEN and p-PTEN, and a decrease in cell apoptosis compared with untreated insulin-resistant cells. Glucosamine may be used to produce an effective model of insulin-resistant skeletal muscle cells. Cells with glucosamine-induced insulin-resistance exhibited a reduced expression of GLUT4 and dysfunction in GLUT4 translocation, as well as increased activation of PTEN and increased cell apoptosis. Inhibition of PTEN or its upstream regulator, AMPK, protects glucosamine-induced insulin-resistant skeletal muscle cells from apoptosis.

Nerve growth factor-induced myoprotection in C2C12 muscle cells is mediated by α9β1 integrin via release of PGE2.

Nerve growth factor (NGF) mediates a wide range of activities in the central nervous system including neuronal differentiation, synaptic plasticity, and neuroprotection. In addition, NGF places an important role in skeletal muscle physiology by some unknown mechanisms. We recently demonstrated that NGF conferred myoprotection toward ischemia in C2C12 skeletal muscle cell model, establishing an important trophic role for NGF in skeletal muscle. In this report, using ELISA and oxygen-glucose deprivation (OGD) assays, we investigated the potential contribution of prostaglandin E2 (PGE2) to NGF myoprotective effects toward C2C12 cultures exposed to OGD insults. Vipera lebetina obtusa disintegrin 5 (VLO5), a selective antagonist of α9β1 integrin, was used as an experimental tool to clarify α9β1 integrin role in NGF action. NGF-induced mitogen-activated protein kinase type 1 or 2 (ERK1/2) phosphorylation in C2C12 cells and in a dose-response fashion stimulated PGE2 release, both effects antagonized by VLO5 and PD98059. NGF-induced myoprotection of the cells exposed for 7 h to OGD, followed by 18 h of reoxygenation, was reversed by VLO5 treatment. These results suggest that NGF activation of α9β1 integrin induced myoprotection by stimulation of ERK phosphorylation and release of cytoprotective PGE2 mediator. This effect may be also relevant for NGF-induced pain and hyperalgesia in the skeletal muscle.

S143 Premature Ageing And Skeletal Muscle Dysfunction In Copd Patients: Development Of A Cell Culture Model

Introduction COPD is a disease of accelerated ageing, as increased cellular-ageing (senescence) occurs in the lungs of these patients. We aim at developing a primary skeletal muscle cell culture (Human skeletal muscle satellite cells [HSKMC]) model of muscle ageing to study the cascade of events that occur in muscle senescence in vitro and to explore the effect of inflammation (TNF alpha) and oxidative stress (H 2 O 2 ), two of the putative mechanisms related to muscle dysfunction and/wasting, on muscle differentiation and on protein loss in differentiated cells. Methods and results HSKMC were cultured to senescence when the cells stopped replicating. DNA was isolated from cells in serial passages of culture. Telomere length, a marker of biological ageing, was measured by qPCR and expressed as the ratio of telomere repeat copy number to single gene copy number in the experimental sample relative to a control sample (relative T/S ratio) (n = 3). Preliminary results show a progressive shortening of telomere length with cellular ageing when comparing early (passage 2, 1.433 ± 0.05 relative T/S ratio) with a later passage (passage 15, 0.340 ± 0.2 relative T/S ratio) (Fig 1). Conclusion We have developed a novel in vitro model of ageing skeletal muscle cells, which will help us to assess the role of accelerated ageing in muscle dysfunction and/wasting in COPD patients. Dr Lakhdar was funded by an LTERS fellowship grant.