Involvement of the FoxO1/MuRF1/Atrogin-1 Signaling Pathway in the Oxidative Stress-Induced Atrophy of Cultured Chronic Obstructive Pulmonary Disease Myotubes.

Oxidative stress is thought to be one of the most important mechanisms implicated in the muscle wasting of chronic obstructive pulmonary disease (COPD) patients, but its role has never been demonstrated. We therefore assessed the effects of both pro-oxidant and antioxidant treatments on the oxidative stress levels and atrophic signaling pathway of cultured COPD myotubes. Treatment of cultured COPD myotubes with the pro-oxidant molecule H2O2 resulted in increased ROS production (P = 0.002) and protein carbonylation (P = 0.050), in association with a more pronounced atrophy of the myotubes, as reflected by a reduced diameter (P = 0.003), and the activated expression of atrophic markers MuRF1 and FoxO1 (P = 0.022 and P = 0.030, respectively). Conversely, the antioxidant molecule ascorbic acid induced a reduction in ROS production (P<0.001) and protein carbonylation (P = 0.019), and an increase in the myotube diameter (P<0.001) to a level similar to the diameter of healthy subject myotubes, in association with decreased expression levels of MuRF1, atrogin-1 and FoxO1 (P<0.001, P = 0.002 and P = 0.042, respectively). A significant negative correlation was observed between the variations in myotube diameter and the variations in the expression of MuRF1 after antioxidant treatment (P = 0.047). Moreover, ascorbic acid was able to prevent the H2O2-induced atrophy of COPD myotubes. Last, the proteasome inhibitor MG132 restored the basal atrophy level of the COPD myotubes and also suppressed the H2O2-induced myotube atrophy. These findings demonstrate for the first time the involvement of oxidative stress in the atrophy of COPD peripheral muscle cells in vitro, via the FoxO1/MuRF1/atrogin-1 signaling pathway of the ubiquitin/proteasome system

Impaired training-induced adaptation of blood pressure in COPD patients: implication of the muscle capillary bed

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Effects of a human microenvironment on the differentiation of human myoblasts

International audienceMyogenic differentiation mechanisms are generally assessed using a murine cell line placed in low concentrations of an animal-derived serum. To more closely approximate in vivo pathophysiological conditions, recent studies have combined the use of human muscle cells with human serum. Nevertheless, the in vitro studies of the effects of a human microenvironment on the differentiation process of human myoblasts require the identification of the culture conditions that would provide an optimal and reproducible differentiation process of human muscle cells. We assessed the differentiation variability resulting from the use of human myoblasts and serums from healthy subjects by measuring the myotube diameter, fusion index and surface covered by myotubes. We showed the preserved cell-dependent variability of the differentiation response of myoblasts cultured in human serums compared to FBS. We found that using a pool of serums reduced the serum-dependent variability of the myogenic response compared to individual serums. We validated our methodology by showing the atrophying effect of pooled serums from COPD patients on healthy human myotubes. By replacing animal-derived tissues with human myoblasts and serums, and by validating the sensitivity of cultured human muscle cells to a pathological microenvironment, this human cell culture model offers a valuable tool for studying the role of the microenvironment in chronic disease

A pericyte‐glia scarring develops at the leaky capillaries in the hippocampus during seizure activity

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Oxidative stress regulates autophagy in cultured muscle cells of patients with chronic obstructive pulmonary disease

International audienceThe proteolytic autophagy pathway is enhanced in the lower limb muscles of patients with chronic obstructive pulmonary disease (COPD). Reactive oxygen species (ROS) have been shown to regulate autophagy in the skeletal muscles, but the role of oxidative stress in the muscle autophagy of patients with COPD is unknown. We used cultured myoblasts and myotubes from the quadriceps of eight healthy subjects and twelve patients with COPD (FEV1% predicted: 102.0% and 32.0%, respectively; p < 0.0001). We compared the autophagosome formation, the expression of autophagy markers, and the autophagic flux in healthy subjects and the patients with COPD, and we evaluated the effects of the 3-methyladenine (3-MA) autophagy inhibitor on the atrophy of COPD myotubes. Autophagy was also assessed in COPD myotubes treated with an antioxidant molecule, ascorbic acid. Autophagosome formation was increased in COPD myoblasts and myotubes (p = 0.011; p < 0.001), and the LC3 2/LC3 1 ratio (p = 0.002), SQSTM1 mRNA and protein expression (p = 0.023; p = 0.007), BNIP3 expression (p = 0.031), and autophagic flux (p = 0.002) were higher in COPD myoblasts. Inhibition of autophagy with 3-MA increased the COPD myotube diameter (p < 0.001) to a level similar to the diameter of healthy subject myotubes. Treatment of COPD myotubes with ascorbic acid decreased ROS concentration (p < 0.001), ROS-induced protein carbonylation (p = 0.019), the LC3 2/LC3 1 ratio (p = 0.037), the expression of SQSTM1 (p < 0.001) and BNIP3 (p < 0.001), and increased the COPD myotube diameter (p < 0.001). Thus, autophagy signaling is enhanced in cultured COPD muscle cells. Furthermore, the oxidative stress level contributes to the regulation of autophagy, which is involved in the atrophy of COPD myotubes in vitro

Oxidative stress status after H₂O₂ treatment.

<p>(A) The variation of ROS production in COPD myotubes is represented after H₂O₂ treatment. (B) Representative Western blots showing levels of protein carbonylation with or without H₂O₂ treatment, for the myotubes cultures derived from 12 COPD patients (1–12). Coomassie blue-stained gels are also presented for loading control. (C) Variation of protein carbonylation relative to Coomassie blue-stained bands after H₂O₂ treatment. (D) Representative Western blots showing levels of lipid peroxidation with or without H₂O₂ treatment in COPD cultured myotubes derived from 12 COPD patients (1–12). Tubulin detection is used as a loading control. (E) Variation of lipid peroxidation relative to tubulin expression levels following H₂O₂ treatment. (n.s.) indicates statistically non-significant. (*) and (**) indicate statistical significance at P≤0.05 and P<0.01, respectively. The medians are indicated.</p

Neurovascular multiparametric MRI defines epileptogenic and seizure propagation regions in experimental mesiotemporal lobe epilepsy

International audienceObjective: Improving the identification of the epileptogenic zone and associated seizure-spreading regions represents a significant challenge. Innovative brain-imaging modalities tracking neurovascular dynamics during seizures may provide new disease biomarkers.Methods: With use of a multi-parametric magnetic resonance imaging (MRI) analysis at 9.4 Tesla, we examined, elaborated, and combined multiple cellular and cerebrovascular MRI read-outs as imaging biomarkers of the epileptogenic and seizure-propagating regions. Analyses were performed in an experimental model of mesial temporal lobe epilepsy (MTLE) generated by unilateral intra-hippocampal injection of kainic acid (KA).Results: In the ipsilateral epileptogenic hippocampi, tissue T1 and blood-brain barrier (BBB) permeability to gadolinium were increased 48-72 hours post-KA, as compared to sham and contralateral hippocampi. BBB permeability endured during spontaneous focal seizures (4-6 weeks), along with a significant increase of apparent diffusion coefficient (ADC) and blood volume fraction (BVf). Simultaneously, ADC and BVf were augmented in the contralateral hippocampus, a region characterized by electroencephalographic seizure spreading, discrete histological neurovascular cell modifications, and no tissue sclerosis. We next asked whether combining all the acquired MRI parameters could deliver criteria to classify the epileptogenic from the seizure-spreading and sham hippocampi in these experimental conditions and over time. To differentiate sham from epileptogenic areas, the automatic multi-parametric classification provided a maximum accuracy of 97.5% (32 regions) 48-72 hours post-KA and of 100% (60 regions) at spontaneous seizures stage. To differentiate sham, epileptogenic, and seizure-spreading areas, the accuracies of the automatic classification were 93.1% (42 regions) 48-72 hours post-KA and 95% (80 regions) at spontaneous seizure stage.Significance: Combining multi-parametric MRI acquisition and machine-learning analyses delivers specific imaging identifiers to segregate the epileptogenic from the contralateral seizure-spreading hippocampi in experimental MTLE. The potential clinical value of our findings is critically discussed

Expression levels of protein synthesis markers after H₂O₂ treatment.

<p>(A) Variation of IGF-1 mRNA expression in COPD myotubes after H₂O₂ treatment. (B) Representative Western blots showing expression levels of phosphorylated-AKT (P-AKT) and AKT in cultured COPD myotubes derived from 12 COPD patients (1–12), with or without H₂O₂ treatment. Tubulin expression is used as a loading control. (C) Variation of the P-AKT/AKT ratio relative to tubulin expression levels following H₂O₂ treatment. (n.s.) indicates statistically non-significant. The medians are indicated.</p

Expression levels of protein synthesis markers after ascorbic acid treatment.

<p>(A) Variation of IGF-1 mRNA expression in COPD myotubes after ascorbic acid treatment. (B) Representative Western blots showing expression levels of phosphorylated-AKT (P-AKT) and AKT in cultured COPD myotubes derived from 12 COPD patients (1–12), with or without ascorbic acid treatment. Tubulin expression is used as a loading control. (C) Variation of the P-AKT/AKT ratio relative to tubulin expression levels following ascorbic acid treatment. (n.s.) indicates statistically non-significant. The medians are indicated.</p

Characteristics of the study groups.

<p>Characteristics of the study groups.</p