Table 1.
Primers used.
Table 2.
Characteristics of the study groups.
Fig 1.
Oxidative stress status after H2O2 treatment.
(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.
Fig 2.
Diameter of COPD and healthy subject myotubes after H2O2 treatment.
Representative images of COPD myotubes (A) or healthy subject myotubes (C) with or without H2O2 treatment, showing fluorescence double-labelling using an anti-troponin T antibody (green) and Hoechst (blue). Bar = 200 μm. Analysis of the variation of the myotube diameter after H2O2 treatment of the COPD myotubes (B) or the healthy subject myotubes (D). (**) indicates statistical significance at P<0.01. (n.s.) indicates statistically non-significant. The medians are indicated.
Fig 3.
Expression levels of protein synthesis markers after H2O2 treatment.
(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.
Fig 4.
Expression levels of protein breakdown markers after H2O2 treatment.
Variation of MuRF1 (A), atrogin-1 (B), FoxO1 (C), FoxO3 (E) and myostatin (F) RNA expression in COPD myotubes after H2O2 treatment. (D) Variation of FoxO1 protein expression relative to tubulin expression in COPD myotubes after H2O2 treatment, and representative Western blots showing expression levels of FoxO1 and tubulin in cultured COPD myotubes derived from 12 COPD patients (1–12), with or without H2O2 treatment. (n.s.) indicates statistically non-significant, and (*) indicates statistical significance at P≤0.05. The medians are indicated.
Fig 5.
Oxidative stress status after ascorbic acid treatment.
(A) The variation of ROS production in COPD myotubes is shown after ascorbic acid treatment. (B) Representative Western blots showing levels of protein carbonylation with or without ascorbic acid treatment for the myotubes cultures derived from 12 COPD patients (1–12). Coomassie blue-stained gels are used for loading control. (C) Variation of protein carbonylation relative to Coomassie blue-stained bands after ascorbic acid treatment. (D) Representative Western blots showing levels of lipid peroxidation with or without ascorbic acid 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 ascorbic acid treatment. (n.s.) indicates statistically non-significant. (*) and (***) indicate statistical significance at P≤0.05 and P<0.001, respectively. The medians are indicated.
Fig 6.
Diameter of COPD and healthy subject myotubes after ascorbic acid treatment.
Representative images of COPD myotubes (A) or healthy subject myotubes (C) with or without ascorbic acid treatment, showing fluorescence double-labeling using an anti-troponin T antibody (green) and Hoechst (blue). Bar = 200 μm. Analysis of the variation of the COPD myotube diameter (B) or the healthy subject myotube diameter (D) after ascorbic acid treatment. (E) Diameter of myotubes from healthy subjects and COPD patients, cultured with or without ascorbic acid. (*) and (***) indicate statistical significance at P≤0.05 and P<0.001, respectively. (n.s.) indicates statistically non-significant. The medians are indicated.
Fig 7.
Expression levels of protein synthesis markers after ascorbic acid treatment.
(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.
Fig 8.
Expression levels of protein breakdown markers after ascorbic acid treatment.
Variation of MuRF1 (A), atrogin-1 (C), FoxO1 (E), FoxO3 (G) and myostatin (H) RNA expression levels in COPD myotubes after ascorbic acid treatment. Variations of MuRF1 (B), atrogin-1 (D) and FoxO1 (F) protein expression relative to tubulin expression in COPD myotubes after ascorbic acid treatment, and the representative Western blots showing their expression levels as well as tubulin expression in cultured COPD myotubes derived from 12 COPD patients (1–12). (n.s.) indicates statistically non-significant. (*), (**) and (***) indicate statistical significance at P≤0.05, P<0.01 and P<0.001, respectively. The medians are indicated. Statistical analysis showing correlations between the variations in the RNA expression of MuRF1 and: (I) the variations in myotube diameter, and (J) the variations in the RNA expression of FoxO1, after ascorbic acid treatment.
Fig 9.
ROS production and COPD myotube diameter after both H2O2 and ascorbic acid treatment.
(A) The variation of ROS production in myotubes derived from 4 COPD patients is shown after H2O2 treatment, and both H2O2 and ascorbic acid treatment. (B) Representative images of COPD myotubes from one patient, in absence of treatment, after H2O2 treatment or after both H2O2 and ascorbic acid treatment, showing fluorescence double-labeling using an anti-troponin T antibody (green) and Hoechst (blue). Bar = 200 μm. (C) Analysis of the variation of the myotube diameter to a reference value of 100after H2O2, and both H2O2 and ascorbic acid treatments. (n.s.) indicates statistically non-significant. (*) indicates statistical significance at P≤0.05. The medians are indicated.
Fig 10.
COPD myotube diameter after MG132 treatment.
Analysis of the variation of the diameter of COPD myotubes derived from one patient after treatment with increasing concentrations of the proteasome inhibitor MG132, in absence (white bars) or in presence (black bars) of the pro-oxidant molecule H2O2. Results are from one of two separate experiments that yielded similar results. The mean diameter of COPD myotubes cultured in absence of MG132 and H2O2 is considered as the reference value (100%).