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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0114OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 636-642, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0114OC

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

Esther Barreiro, Joaquim Gea, Ghassan Matar and Sabah N.A. Hussain

Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; Muscle and Respiratory System Research Unit and Experimental Health Sciences Department, Institut Municipal d'Investigació Mèdica–Universitat Pompeu Fabra, Respiratory Medicine Department, Hospital del Mar, Barcelona, Catalonia, Spain

Correspondence and requests for reprints should be addressed to Dr. S. Hussain, Room L3.05, 687 Pine Ave. West, Montreal, PQ, H3A 1A1 Canada. E-mail: sabah.hussain{at}muhc.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
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 FEV1 and V·O2max, 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.

Key Words: carbonic anhydrase • COPD • creatine kinase • protein oxidation • skeletal muscle.


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Limb muscle dysfunction is a major factor in poor exercise performance in patients with chronic obstructive pulmonary disease (COPD) (1). Although many factors are likely to be involved in causing muscle dysfunction in these patients, recent studies implicate oxidative stress, which develops as a result of increased production of reactive oxygen species (ROS) and reduced antioxidant capacity (2–6). In addition, there is also evidence of intrinsic defects in energy metabolism of limb muscles in patients with COPD. For example, measurements of the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi), which is closely related to the ATP/ADP ratio, suggest that limb muscles of patients with COPD have lower PCr/Pi ratios during exercise, and experience slower recovery of this ratio as compared with control subjects (7–9). A major regulator of the PCr/Pi ratio in skeletal muscle is creatine kinase (CK). This enzyme catalyses the reversible transfer of a phosphoryl group from ATP to creatine (Cr) to produce ADP and PCr: MgATP + Cr {iff} MgADP + PCr.

CK is localized in both the mitochondria and the cytosol. The main function of mitochondrial CK is this conversion of ATP synthesized by oxidative phosphorylation to PCr, which is then transported from the mitochondria to the cytosol and reconverted to ATP by cytosolic CK. Cytosolic CK is loosely bound to actin filaments, and its function is coupled to myosin ATPase in such a way that the latter uses ATP that has been rephosphorylated by CK (10). CK function is also linked to sarcolemmal Na-K ATPase and sarcoplasmic reticulum Ca2+-ATPase (11).

Despite the importance of CK in regulating skeletal muscle energy metabolism, there is little information about CK protein levels and activity in limb muscles of patients with COPD. In addition to protein abundance, CK activity is also determined by post-translational modifications, including oxidation. Several reports have shown that in vitro exposure of CK to ROS or derivatives of nitric oxide, such as peroxynitrite, leads to inhibition of CK activity as a result of modifications of critical residues within the enzyme (12–14). In addition, carbonylation (a form of protein oxidation) of CK has recently been shown to increase inside the diaphragm during the course of severe sepsis in septic rats (15). These results, along with the fact that the intensity of CK carbonylation is inversely related to CK activity inside the diaphragm, suggest that oxidation of CK protein exerts an inhibitory effect on muscle CK activity (15). Whether oxidative modifications of CK occur in limb muscles of patients with COPD remains unknown. The recent detection by our group of several strongly carbonylated protein bands in the molecular weight range of CK inside limb and ventilatory muscles of patients with COPD suggests that CK may indeed be the target for oxidative modifications in the muscles of these patients (4, 16).

The first objective of this study was to compare CK protein expression and activity in limb muscles of patients with COPD and control subjects. Our second objective was to determine whether limb muscle CK expression and activity correlate with the degree of airflow limitation (FEV1) or exercise performance (V·O2max) in patients with COPD, as these are prime indices of the severity of the condition. Our third objective was to determine whether CK is a target of ROS action in limb muscles of patients with COPD, given the relationship between contractile dysfunction and ROS production, and whether the intensity of CK carbonylation in COPD correlates with FEV1 and V·O2max. Finally, we investigated whether CK activity is determined in part by the intensity of carbonylation of the enzyme.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Patient Characteristics
In this study, we evaluated the same 12 patients with COPD and the 6 control individuals with normal pulmonary functions who were included in our previous study (4). The COPD diagnosis was established based on a clinical history compatible with chronic bronchitis and/or emphysema, a long history of cigarette smoking, and pulmonary function testing revealing fixed airflow obstruction (FEV1/FVC ratio < 70% and FEV1 < 75% predicted). As described in our previous study (4), all subjects underwent thoracotomy for a localized lung neoplasm. The following exclusion criteria were instituted: chronic respiratory failure, positive bronchodilator test, bronchial asthma, coronary disease, undernourishment (body mass index[BMI] < 20 kg/m2), chronic metabolic diseases, orthopedic diseases, suspected paraneoplastic or myopathic syndromes, previous abdominal or thoracic surgery, and/or treatment with drugs known to alter muscle structure and/or function.

Study Design
All experiments were approved by the Ethics Committee on Human Investigation at Hospital del Mar-Institut Municipal d'Investigació Mèdica (IMIM) and were performed according to the World Medical Association guidelines for research in humans. Informed written consent was obtained from all individuals after full explanation of the purposes and characteristics of the study. Pulmonary and muscle functions, exercise capacity, and nutritional status were assessed at study entry. Seven to fifteen days later, samples from the quadriceps muscle were obtained before thoracotomy. BMI was calculated in all subjects as the ratio of weight per height. Serum cholesterol, triglycerides, total protein, albumin, globulins, albumin/globulins index, and prothrombin consumption time were determined in all subjects.

Pulmonary Function Tests
Forced spirometry was performed using a pneumotachograph and standard procedures. Static lung volumes and airway resistance were determined using standard body plethysmography. Carbon monoxide transfer was used to assess diffusion capacity.

Peripheral Muscle Function
All subjects performed the classical nondominant handgrip maneuver using a Collins dynamometer (Herrera, Barcelona, Spain). At least three maneuvers were performed in each case, and the highest value was chosen. In addition, a standardized incremental exercise test to measure V·O2max was performed on each subject using a cycloergometer (Crescent 864; Monark Inc., Varberg, Sweden). Subjects performed a 3-min warm-up period at 25 watts, followed by 25-watt increments at 2-min intervals while pedaling at a constant frequency of 60 rpm until exhaustion. Predicted VO2max values were derived according to Jones and coworkers (17).

Biological Muscle Studies
Muscle samples were obtained from the quadriceps (vastus lateralis) by open muscle biopsy, and were immediately frozen in liquid nitrogen and stored at –80°C for further analysis, as previously described (4). Frozen muscle samples were homogenized in a buffer containing tris-maleate 10 mM, EGTA 3 mM, sucrose 275 mM, DTT 0.1 mM, leupeptin 2 µg/ml, PMSF 100 µg/ml, aprotinin 2 µg/ml, and pepstatin A 1mg/100 ml (pH 7.2). Samples were then centrifuged at 1,000 x g for 10 min. The pellet was discarded and the supernatant was designated as a crude homogenate. Total muscle protein level in each sample was determined with the Bradford technique (BioRad Inc., Hercules, CA).

Detection of Protein Carbonyls Using 2D Electrophoresis
This technique was used to separate and identify carbonylated proteins in crude muscle homogenates of two patients with COPD, as previously described (15). Briefly, 4 volumes of 10 mM 2,4-dinitrophenyl hydrazine (DNPH) (in 2 M HCl) were added to 200 µg of protein per sample and incubated for 30 min at room temperature. Ice-cold 100% trichloroacetic acid (TCA) was added to yield a final TCA concentration of 15%, and the sample was then incubated for 10 min on ice. The sample was centrifuged for 10 min at 14,000 x g, and the pellet was washed three times with ethanol ethyl acetate, centrifuged at 14,000 x g, and then resuspended in 2D rehydration buffer. Each derivatized muscle fraction was separated into two portions (100 µg total in each), and both portions underwent 2D electrophoresis. First-dimensional protein separation was performed with a protean isoelectric focusing cell (Bio-Rad Inc., Hercules, CA) using immobilized pH gradient strips (17 cm, nonlinear, pH 3–10). Isoelectric focusing was performed at 10,000 V/h for up to a total of 60–100 kVh. The IPG strips were then equilibrated with equilibration buffer (6 M urea, 2% SDS, 0.05 mM Tris-HCl, 20% glycerol) to which 2% DTT was added before use. The strips were then embedded in 0.7% agarose on top of 10% acrylamide slab gels, and the second-dimension SDS-PAGE was performed for 5 hr. One of the resulting 2D gels for each muscle fraction was stained with silver stain, as described previously (15). The second gel derived from a given sample underwent electrophoretic transfer to polyvinylidene difluoride (PVDF) membrane and immunoblotting with an anti-DNP antibody (see below). Gels and PVDF membranes were imaged with a digital camera and aligned (ImagePro Plus; Media Cybernetics, Silver Spring, MD) to identify carbonylated protein spots on the gels.

Mass Spectrometry
Carbonylated protein spots were cut out of the gels and taken for in-gel digestion using 6 ng/µl trypsin on a robotic MassPrep Workstation (MicroMass; Waters Corp., Milford, MA) (15). Peptides were then extracted with 1% formic acid/2% acetonitrile. Identification of the digested proteins was completed using a liquid chromatography–quadrupole–time of flight (LC-Q-Tof) mass spectrometer (MicroMass). Digests were loaded into 10 cm capillary PicoFrit columns filled with C18 stationary phase and eluted by linear gradient of 5–70% acetonitrile in 0.1% formic acid at a flow rate of 200 µl/min. The eluted peptides were electrosprayed into Q-Tof, and the precursor ions were selected and subjected to fragmentation by collision with argon (MS/MS). The MS/MS data were submitted to Mascot (Matrix Science, London, UK) for a search against the National Center for Biotechnology Information nonredundant database.

Total and Carbonylated CK and Carbonic Anyhdrase III Intensities
Protein carbonylation in crude muscle homogenates was first measured with 1D electrophoresis and a commercial kit (Oxyblot; Intergen Inc., Purchase, NY). Carbonyl groups in the protein side chains were derivatized to DNPH by reaction with DNPH. In brief, 15 µg of protein was used per derivatization reaction. Proteins were then denatured by addition of 12% SDS. The samples were subsequently derivatized by adding 10 µl of 1x DNPH solution and incubated for 15 min. Finally, 7.5 µl of neutralization solution and 2-mercaptoethanol were added to the sample mixture. To evaluate the selectivity of carbonyl measurements, muscle protein samples also underwent a protein carbonyl detection procedure without the derivatization step (negative controls). DNP-derivatized proteins were loaded onto 12% tris-glycine SDS polyacrylamide gels and separated by electrophoresis. Proteins were transferred electrophoretically to methanol-presoaked PVDF membranes, and then blocked with 5% nonfat dry milk for 1 hr at room temperature. PVDF membranes were subsequently incubated with a polyclonal anti-DNP moiety antibody, followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibody. Specific proteins were detected with a chemiluminescence kit (Roche Inc., Basel, Switzerland). The blots were scanned with an imaging densitometer. The primary anti-DNP moiety and the secondary antibodies were stripped by incubation with 0.2 N NaOH solution for 5–60 min, and the membranes were then probed with goat polyclonal anti-CK and rabbit polyclonal anti-CAIII antibodies. Specific proteins were then detected with HRP-conjugated secondary antibodies and an enhanced chemiluminescence kit (Amersham Biosciences, Bucks, UK). Specific protein blots were then scanned with an imaging densitometer. The optical densities of carbonylated (measured from the Oxyblot) and total CK and CAIII proteins were quantified using ImagePro Plus.

CK Activity Assay
Total muscle CK activity was measured with a coupled assay system that is based on the conversion of creatine phosphate and ADP by CK to creatine and ATP. The ATP and glucose are then converted to ADP and glucose-6-phosphate by hexokinase. Glucose-6-phosphate dehydrogenase then oxidizes glucose-6-phosphate and reduces the NAD to NADH. The rate of NADH formation, measured at 340 nm, is, therefore, directly proportional to creatine kinase activity. We added 3 µg of muscle protein to 500 µl of reagent buffer containing D-glucose, Mg2+, AMP, creatine phosphate, ADP, oxidized NADH, glucose- 6-phosphate dehydrogenase, hexokinase, and EDTA. The reaction was maintained for 2 min at 37°C. Absorbance was then measured at 340 nm and converted to creatine kinase activity units using the equation:CK activity (IU/L)= ({Delta}abs x TV x 1,000)/(d x x SV), where TV = total reaction volume, d = light path length in cm, = millimolar absorptivity of NADH, and SV = sample volume in ml.

Statistical Analysis
Data are presented as mean ± SE. Analysis of variance was used to compare optical densities and enzyme activities between patients with COPD and control subjects. Relationships between various parameters were studied by calculating the Spearman's correlation coefficient. A P value <= 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
The characteristics of patients with COPD and control subjects were previously described (4). No differences were observed in age, weight, BMI, nutritional status, and arterial blood gases between the two groups. In patients with COPD, as compared with control subjects, FEV1 (54 ± 14 versus 86 ± 9% predicted, P < 0.05), FVC (62 ± 12 versus 85 ± 3% predicted, P < 0.05), and FEV1/FVC (61 ± 7 versus 74 ± 3% predicted, P < 0.05) were significantly lower. Furthermore, both peripheral muscle strength (74 ± 16 versus 92 ± 15% predicted, P < 0.01) and exercise capacity (V·O2max: 73 ± 11 versus 87 ± 11% predicted, P < 0.05; and WRmax: 71 ± 14 versus 88 ± 11% predicted, P < 0.05) were mildly reduced in patients with COPD.

Figure 1 shows a representative example of a 2D protein map of crude homogenate of vastus lateralis muscle obtained from a single patient with COPD in which carbonylated proteins were detected using anti-DNP antibody. Seven strongly positively carbonylated proteins were detected in this muscle biopsy (Figure 1). Four strongly carbonylated protein spots with a molecular mass of ~ 43 kD (spots 1, 2, 3, and 4) were identified to be muscle creatine kinase (CK, accession no. NP036662), whereas three strongly carbonylated protein spots with a molecular mass of ~ 29 kD (spots 5, 6, and 7) were identified to be carbonic anhydrase III (CAIII, accession no. 1FLJA). Reprobing the 2D blot with selective antibodies to CK and CAIII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) confirmed the identity of the two strongly carbonylated proteins in this muscle biopsy (Figure 1).



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  		Figure 1. Detection of protein carbonylation in crude homogenate of vastus lateralis muscle of a patient with chronic obstructive pulmonary disease (COPD). Representative 2D anti-DNP blot (top left) shows seven strongly carbonylated protein spots. Reprobing the 2D blot with anti–creatine kinase (top right) and anti–carbonic anhydrase III (CAIII) (bottom) antibodies has confirmed the identity of these proteins as strongly carbonylated proteins.

 
Figure 2A shows representative examples of the changes in total and carbonylated CK protein levels in the vastus lateralis muscles. Total and carbonylated CK levels were significantly greater in muscles of patients with COPD when compared with control subjects (Figures 2B and 2C). Total muscle CK activity was significantly greater in patients with COPD as compared with that of control subjects (Figure 2D). Within the patients with COPD, CK activity correlated positively and significantly with the degree of airflow limitation (FEV1) but not with V·O2max (Figure 3). Muscle CK protein expression correlated significantly and positively with both V·O2max and FEV1 (Figure 3). In comparison, the intensity of carbonylated CK (normalized per total CK) in the vastus lateralis muscles correlated negatively and significantly with both V·O2max and FEV1 values (Figure 3). In addition, there was a significant (P < 0.05) negative correlation between muscle CK activity and the intensity of carbonylated CK (normalized per total CK) in patients with COPD (Figure 4).



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  		Figure 2. (A) Representative examples of changes in CK protein expression and intensity of carbonylation (detected with the Oxyblot kit) in the vastus lateralis muscle of control subjects and patients with COPD. (B, C, and D) Mean and SEM values of optical densities (OD) of total CK proteins, carbonylated CK, and CK activity in the vastus lateralis muscle of control subjects and patients with COPD. *P < 0.05 as compared with control subjects.

 


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  		Figure 3. Correlation analysis between CK activity, total CK protein expression and intensity of CK carbonylation (normalized per total CK level) in the vastus lateralis muscle and FEV1 (% predicted) and maximum oxygen consumption (VO2max) (% predicted) of patients with COPD.

 


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  		Figure 4. Correlation analysis between CK activity and the intensity of CK carbonylation (normalized per total CK level) in the vastus lateralis muscle of patients with COPD.

 
In a post hoc analysis, in which the patients with severe COPD (FEV1 <= 50% predicted, n = 5) were compared with patients with moderate disease (n = 7) (18), we found that muscle CK expression and activity were significantly lower, whereas the degree of CK carbonylation was significantly greater, in the former group compared with those detected in the latter (Figure 5).



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  		Figure 5. Mean and SEM values of CK activity, total CK OD and intensity of CK carbonylation (normalized per total CK level) in the vastus lateralis muscle of patients with moderate COPD (FEV1 >= 50% predicted) and severe COPD (FEV1 < 50% predicted). *P < 0.05 as compared with patients with severe COPD.

 
Figure 6 shows representative examples of the changes in total and carbonylated CAIII protein levels in the vastus lateralis muscles of patients with COPD and control subjects. No significant differences were observed in the degree of muscle CAIII expression or carbonylation of CAIII between the two groups. Moreover, within patients with COPD, no significant correlations could be detected between FEV1 and the intensity of muscle CAIII expression and CAIII carbonylation (data not shown).



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  		Figure 6. (A) Representative examples of changes in CAIII protein expression and intensity of carbonylation (detected with the Oxyblot kit) in the vastus lateralis muscle of control subjects and patients with COPD. (B) Mean and SEM values of OD of total CAIII proteins and carbonylated CAIII in the vastus lateralis muscle of control subjects and patients with COPD.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The main findings of this study are that in the vastus lateralis muscle: (1) CK activity and expression are significantly greater in patients with COPD as compared with control subjects; (2) both CK and CAIII proteins are carbonylated inside these muscles in both groups of subjects; (3) in patients with COPD, muscle CK expression and activity correlate positively with FEV1, whereas carbonylation intensity of CK correlates negatively with FEV1; (4) there is a negative correlation between the intensity of carbonylation and activity of CK in patients with COPD.

Critique of the Study
Two major limitations could be identified in our study. First, the sample sizes of both patients with COPD and control subjects are relatively small (12 patients with COPD and 6 control subjects). Moreover, we studied patients with COPD with a limited range of disease severity (7 out of 12 patients had FEV1 > 50% predicted). This is because we employed very strict criteria for inclusion and exclusion of subjects. Yet, despite the relatively small number of patients with COPD studied, we found consistent results in terms of carbonylation and expression of CK and CAIII. We were also able to identify statistically significant correlations between muscle CK expression, activity, and the degree of carbonylation and resting FEV1 and V·O2max values in patients with COPD. In addition, we obtained muscle samples through an open biopsy procedure, which is technically more difficult than the commonly used needle biopsy technique. However, open biopsies provide significantly larger and better quality muscle samples relative to those obtained with needle biopsies.

Second, we only assessed one form of oxidative modification of muscle protein in this study—namely, carbonylation, which is the result of modification of lysine residues by reducing sugars or their oxidation production (glycation/glycoxidation reactions). It is also formed by the reaction of lysine, cysteines, or histidine with {alpha}- and {beta}-unsaturated aldehydes formed during fatty acid oxidation (19). Carbonyl formation is a widely used index of protein oxidative modification, and is usually detected with either a spectrophotometric carbonyl assay or by using the Oxyblot kit that employs an antibody against 2,4-dinitrophenol groups. The Oxyblot technique that was used in the current study provides several advantages over the carbonyl assay, among which are the use of a relatively small protein sample (as little as 15 µg) and the ability to identify the true identity of carbonylated proteins using immunoblotting techniques (20).

The CK System
Recent studies suggest that skeletal muscle energy metabolism of patients with COPD is impaired, as indicated by the significant reduction in PCr/Pi ratio during and after exercise and more than doubling of the time for resynthesis of PCr as compared with control subjects (7–9). Furthermore, gastrocnemius muscles of patients with severe COPD showed significantly more rapid PCr depletion during exercise as compared with control subjects (21). These alterations are indicative of impaired mitochondrial oxidative phosphorylation and/or depressed CK activity. Despite these suggestions, there is little information regarding the status of the CK system in limb muscles of patients with COPD. Puente-Maestu and colleagues (22) have reported that bicycle training reduces limb muscle CK activity in patients with severe COPD. In the deltoid and intercostals muscles, CK activities were similar between patients with severe to mild COPD and control subjects (23, 24). Our current study shows that this is not the case in the vastus lateralis muscles, where CK protein and activity levels were significantly greater in patients with COPD compared with control subjects. We should emphasize, however, that this elevation in CK activity and protein levels is observed mainly in patients with moderate COPD (Figure 5). The mechanisms behind this finding are unclear. We propose that enhanced CK expression and activity in these patients represent an adaptive response designed to cope with increased muscle ATP utilization and production. One likely cause of this rise in ATP requirement is a switch in myosin ATPase from the slow to the fast isoform in limb muscles of patients with COPD. Additionally, an increase in sarcoplasmic reticulum Ca2+-ATPase activity as a result of greater abundance of type II fibers would also likely trigger a rise in ATP requirements, and hence increased CK expression in limb muscles of patients with moderate COPD.

It is also notable that CK expression and activity in the vastus lateralis muscles of patients with severe COPD were significantly lower than those of patients with moderate COPD (Figure 5). One possible explanation for this finding is that, as the severity of COPD disease progresses, the pattern and intensity of limb muscle recruitment changes in such a way that reduces the reliance of muscle fiber on PCr to buffer ATP. The use of PCr in skeletal muscle energetics allows for high-power output; however, muscle fibers rely on other metabolic pathways to buffer ATP during prolonged contractions that require the generation of low-power outputs. One possible substitute for the PCr/CK system is the adenylate kinase family proteins that catalyze the "myokinase reaction" (2ADP->ATP+AMP), which buffers ATP levels in skeletal muscles (25). The expression of adenylate kinases in skeletal muscle fibers increases when CK expression is relatively low (26). At this time, there is no information concerning the preferential use of CK versus adenylate kinase for ATP buffering in limb muscles of patients with COPD. Another possible cause of decreased CK activity in limb muscles of severe patients with COPD is oxidative modifications of CK protein. Many reports have described that in vitro CK activity is inhibited by oxidative modifications elicited by exposure to oxygen radicals (13, 27, 28). In vivo experiments have confirmed that CK is carbonylated and oxidized in heart muscles, in the brain of patients with Alzheimer's disease, and, more recently, in the diaphragm of septic rats (15, 29, 30). Our assertion that oxidative modifications may have been behind the reduction in CK activity in limb muscles of patients with severe COPD is supported by the observations that the ratio of carbonylated CK to total CK is elevated in muscles of patients with severe COPD, and by the negative correlation between CK activity and the intensity of carbonylation of CK protein. It is clear that further studies are needed to elucidate the exact mechanisms responsible for alterations of CK activity and expression in patients with COPD.

The functional implications of reduced CK activity and expression in limb muscles of patients with COPD remain to be determined. Reduction in CK activity is likely to have a deleterious effect on the rate of ATP utilization of critical enzymes, such as sarcolemmal Na-K ATPase, SR Ca2+-ATPase, and myosin ATPase, resulting in impairment of conduction, excitation- contraction coupling, and contractile machinery, respectively. In addition, inhibition of mitochondrial-CK activity as a result of carbonylation of this enzyme is likely to result in major defects in recycling of mitochondrial ATP to PCr and diminished supply of PCr to myofibrillar CK.

Carbonylation of CAIII
In addition to CK, CAIII was also carbonylated in the vastus lateralis muscles of both patients with COPD and control subjects. This observation is in agreement with our recent study in rat diaphragms in which we reported that CAIII is a target for ROS actions (15). CAIII is a member of a class of zinc metallo-enzymes that catalyze the reversible hydration of CO2 (31). CAIII has other activities as well, including carboxyl esterase and tyrosine phosphatase activities, and is involved in carbohydrate utilization (32, 33). Recent studies have reported that glutathione interacts with CAIII by forming a disulfide link with two of the five cysteine residues of CAIII in a process termed S-glutathiolation (34). This observation suggests that CAIII may play an important role in the cellular response to oxidative stress, and may also participate in antioxidant defenses against ROS formation inside skeletal muscle fibers (35). An interesting observation in our study is that, unlike CK, the intensity of CAIII carbonylation was not different in vastus lateralis muscles of patients with COPD and control subjects. These results suggest that excessive ROS formation in COPD muscles selectively target CK as compared with CAIII. The mechanisms behind this selective targeting are unclear, but they may be related to the proximity of CK to molecular sources of ROS generation, such as mitochondrial oxidative phosphorylation enzymes and sarcolemmal-associated enzymes, such as NADPH oxidase.

In summary, we report here that CK activity and expression are significantly greater in the vastus lateralis muscles of patients with COPD as compared with control subjects, and that both CK activity and expression correlate positively with FEV1 and V·O2max. This suggests that, as disease severity progresses, this protective mechanism is lost in the limb muscles of the patients with severe COPD. Our other important observation is that both CK and CAIII are carbonylated in the vastus lateralis muscles of patients with COPD and control subjects. The intensity of CK carbonylation in the muscles of patients with COPD correlates negatively with FEV1 and CK activity, whereas the degree of CAIII carbonylation does not differ between patients with COPD and control subjects.


   Footnotes
 
This work was supported by the Canadian Institute of Health Research and Plan Nacional I+D (SAF 2001–0426), Spanish Respiratory Network for Research among different centers (RESPIRA) (RTIC C03/11) (Spain), and European Network for Investigating the Global Mechanisms of Muscle Abnormalities (ENIGMA) in COPD (QLRT-2002–02285) (European Union).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0114OC on September 15, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 22, 2005

Accepted in final form August 5, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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