Expression and Carbonylation of Creatine Kinase in the Quadriceps Femoris Muscles of Patients with Chronic Obstructive Pulmonary Disease

Abstract

Oxidative protein modification involving carbonylation has recently been identified as an important factor in skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD). However, the exact identity of modified proteins inside limb muscles of patients with COPD remains unknown. We used 2D electrophoresis, immunoblotting, and mass spectrometry to identify carbonylated proteins in the vastus lateralis muscle of 12 patients with COPD and 6 control subjects. Both creatine kinase (CK) and carbonic anhydrase III (CAIII) were identified as being strongly carbonylated in this muscle in both groups of subjects. Total CK activity, CK protein expression, and the intensity of CK carbonylation were significantly greater in the muscles of patients with COPD as compared with control subjects, whereas CAIII protein expression and intensity of carbonylation were similar in the two groups. In patients with COPD, CK activity and protein expression correlated positively with FEV(1) and V O(2)max, whereas the intensity of CK carbonylation correlated negatively with the same parameters. These results indicate that oxygen radicals selectively target CK and CAIII inside limb muscles of humans. The observation that the intensity of CK carbonylation correlates negatively with CK activity in limb muscles of patients with COPD suggests that carbonylation may have a deleterious effect on CK activity, and may contribute to impaired CK function in the limb muscles of these patients.

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

“…Immunoblotting of one-dimensional electrophoresis The levels of oxidative and nitrosative stress and those of muscle creatine kinase, carbonic anhydrase III, a-actin and MyHC were assessed, as described elsewhere [13,14,18].…”

“…Identification of carbonylated proteins: one-dimensional and twodimensional electrophoresis and mass spectrometry Oxidised proteins (protein carbonylation) were separated and identified in the diaphragms of COPD patients and control subjects, as previously described [11,13,18].…”

“…Creatine kinase activity assay Total muscle creatine kinase activity was measured with a commercial kit (Diagnostic Chemicals Ltd, Charlotte Town, PE, Canada), as described elsewhere [13,18].…”

“…Over the past decade, a growing body of evidence has shown that oxidative stress is one of the mechanisms clearly involved in the skeletal muscle dysfunction of COPD patients [12][13][14]. Carbonyl formation is an important detectable marker of protein oxidation in tissues.…”

“…However, there is growing evidence that in vitro fibre contractile function is also impaired in the diaphragms of patients with COPD [9], even at early stages of their disease, as shown by reduced myosin heavy chain (MyHC) content and increased protein degradation via the ubiquitin-proteasome pathway [10,11]. Oxidative stress, among other factors, was also suggested to be a contributor to this process of muscle dysfunction and wasting [12][13][14], although its specific role is yet to be fully elucidated. The oxidative post-translational modifications of proteins may cause loss of protein function as a result of oxidation of residues in the protein active site.…”