Oxidised proteins and superoxide anion production in the diaphragm of severe COPD patients

Abstract

In the diaphragms of chronic obstructive pulmonary disease (COPD) patients, the nature of oxidatively modified proteins and superoxide anion production were explored.Diaphragm specimens were obtained through thoracotomy because of localised lung lesions in COPD patients (16 severe and eight moderate) and 10 control subjects. Lung and respiratory muscle functions were evaluated. Oxidised proteins were identified using immunoblotting and mass spectrometry. Protein and activity levels of the identified proteins were determined using immunoblotting and activity assays. Lucigenin-derived chemiluminescence signals in a luminometer were used to determine superoxide anion levels in muscle compartments (mitochondria, membrane and cytosol) using selective inhibitors.In severe COPD patients compared with controls, respiratory muscle function was impaired; creatine kinase, carbonic anhydrase III, actin and myosin were oxidised; myosin carbonylation levels were increased five-fold; creatine kinase content and activity and myosin protein were reduced; superoxide anion levels were increased in both mitochondria and membrane compartments; and the percentage of superoxide anion inhibition achieved by rotenone was significantly greater.In severe COPD patients, oxidation of diaphragm proteins involved in energy production and contractile performance is likely to partially contribute to the documented respiratory muscle dysfunction. Furthermore, generation of the superoxide anion was increased in the diaphragms of these patients.

Redox Imbalance in Lung Cancer of Patients with Underlying Chronic Respiratory Conditions

“…Plasma levels of the ratio of oxidized (GSSG) to reduced glutathione (GSH) were determined using the OxiSelect total glutathione GSSG/GSH ELISA kit (Cell Biolabs Inc.) following the precise manufacturer's instructions and previous methodologies (34).…”

“…Two-D electrophoresis and silver staining. Carbonylated lung proteins were separated and identified in the samples as published elsewhere (23,34).…”

“…Identification of carbonylated proteins was conducted in the Proteomics Laboratory at Universitat de Barcelona following the quality criteria established by ProteoRed standards (Instituto Nacional de Proteómica) and procedures previously published (23,34).…”

Does oxidative stress modulate limb muscle atrophy in severe COPD patients?

“…Detection of superoxide anion radicals in muscle compartments Superoxide anion levels were detected in cytosolic, membrane and mitochondrial compartments following previous published methodologies [23].…”

“…On this basis, it was hypothesised that oxidative stress may differentially regulate protein loss within the muscles of patients with severe COPD exhibiting a wide range of body composition. Therefore, in the present study, our objectives were to assess the following potentially interrelated molecular events within the limb muscles of a population of severe COPD patients exhibiting the following different degrees of body composition: 1) redox balance (protein oxidation, superoxide anion content in myonuclei and muscle fibre compartments, antioxidant enzymes and reduced glutathione (GSH)); 2) levels of redox-sensitive signalling pathways, total protein ubiquitination and markers of the ubiquitin-proteasome system; 3) inflammation; 4) the content of key contractile and functional proteins, previously shown to be consistently oxidised and prone to degradation in COPD muscles [12,15,23], such as myosin heavy chain (MyHC), actin, creatine kinase and carbonic anhydrase-3; and 5) muscle structural abnormalities. A group of healthy sedentary control subjects was also recruited in the investigation.…”