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Protein Carbonyl Formation in the Diaphragm

Critical Care and Respiratory Divisions, Royal Victoria Hospital, McGill University Health Centre, and Meakins-Christie Laboratories, McGill University; McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada; Muscle and Respiratory System Research Unit, IMIM, CEXS, Universitat Pompeu Fabra; and 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

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Although protein carbonyl formation is an index of oxidative stress in skeletal muscles, the exact proteins, which undergo oxidation in these muscles, remain unknown. We used 2D electrophoresis, immunoblotting, and mass spectrometry to identify carbonylated proteins in the diaphragm in septic animals. Rats were injected with saline (control) or Escherichia coli lipopolysaccharides (LPS) and killed after various intervals. Diaphragm protein carbonylation increased significantly and peaked 12 h after LPS injection, and it was localized both inside muscle fibers and in blood vessels supplying muscle fibers. Aldolase A, glyceraldehyde 3-phosphate dehydrogenase, enolase 3ß, mitochondrial and cytosolic creatine kinases, -actin, carbonic anyhdrase III, and ubiquinol-cytochrome c reductase were all carbonylated in septic rat diaphragms. In addition, we found significant negative correlations between the intensity of carbonylation and creatine kinase and aldolase activities. We conclude that glycolysis, ATP production, CO₂ hydration, and contractile proteins are targeted by oxygen radicals inside the diaphragm during sepsis.

Key Words: carbonyl formation • muscle contraction • nitric oxide • protein oxidation • sepsis

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
It has been well established in both humans and animals that sepsis and septic shock elicit a significant deterioration in the ability of ventilatory and limb muscles to generate force and sustain work loads (1). Although many factors have been implicated, there is increasing evidence that increased levels of reactive oxygen species (ROS) and nitric oxide (NO) are in part responsible for sepsis-induced muscle dysfunction, as indicated by the improvement of skeletal muscle contractility in septic animals treated with antioxidants and inhibitors of nitric oxide synthases (NOSs) (2, 3).

In normal skeletal muscle fibers ROS are produced at relatively low levels, mainly in the mitochondria, and play a positive role in maintaining muscle contractility (4). However, increased production of these species to levels significantly greater than those that can be neutralized by intracellular antioxidant defenses leads to the development of a state of oxidative stress, which has profound effects on many processes inside skeletal muscles, including action potential conduction, excitation–contraction coupling, contractile proteins, and mitochondrial respiration (4). Proteins constitute one of the major targets of ROS, and oxidation of proteins can lead to a loss of protein function as well as conversion of proteins to forms that are more susceptible to degradation by proteinases (5). ROS elicit a variety of modifications in amino acid residues, including cysteines, methionine, tryptophan, arginine, lysine, proline, and histidine (5). Among amino acid modifications by ROS is the formation of carbonyls as a result of oxidation of arginine, lysine, threonine, or proline amino acids. Protein carbonylation is the result of secondary reactions of amino groups of lysine residues with reducing sugars or their oxidation production (glycation/glycoxidation reactions) and also by reactions of lysine, cysteines, or histidine amino acids with - and ß-unsaturated aldehydes formed during the peroxidation of polyunsaturated fatty acids (6).

Carbonyl formation, usually detected by a reaction with 2,4-dinitrophenylhydrazine (DNPH) and the conversion to hydrazones, has been widely used as an important marker of protein oxidation in skeletal muscles (7–10). In the ventilatory muscles, increased protein carbonyls have been detected in response to chronic resistive loading, after several hours of mechanical ventilation and in response to sepsis induced by cecal ligation (11–13). Despite this wide use of protein carbonyl detection as an index of oxidative modification of proteins, little information is yet available about the exact identity of proteins targeted by ROS. With the more recent availability of techniques such as two-dimensional protein fingerprinting, investigators have described carbonylation of several cytosolic proteins including ß-tubulin, ß-actin, and creatine kinase in brain samples of patients with Alzheimer's disease (14). No information is yet available as to the exact protein targets for carbonylation inside limb or ventilatory muscles during the course of severe sepsis. Goto and coworkers (15) suggested, on the basis of abundance of carbonyl signals, that -actin and myosin heavy chain might be carbonylated in skeletal muscles.

The main objectives of this study were: (1) to evaluate the time course of protein carbonylation and the localization and identity of carbonylated proteins inside the diaphragm of septic rats; and (2) to correlate the time courses of diaphragm contractile dysfunction and muscle protein carbonylation in septic rats.

	MATERIALS AND METHODS

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Animal Preparation
The Animal Research Committee of McGill University approved all procedures. Pathogen-free male Sprague-Dawley rats (250–275 g) were housed in the animal facility of the hospital, were fed food and water ad libitum and were studied 1 wk after arrival. All animals were killed with an overdose of sodium pentobarbital and the diaphragm was quickly excised and was either flash-frozen in liquid nitrogen and stored at –80°C for immunoblotting analysis or embedded in paraffin and used for immunohistochemistry. In a few animals, diaphragm contractility was assessed in vitro using isolated muscle strips.

Experimental Protocol
Six groups of rats (n = 5 in each group) were studied. Group 1 was injected intraperitoneally with normal saline (control group) and killed 24 h later. Groups 2, 3, 4, 5, and 6 were injected intraperitoneally with Escherichia coli LPS (serotype 055:B5, 20 mg/kg; Sigma Inc., St. Louis, MO) and killed 1, 3, 6, 12, and 24 h later. The diaphragm was excised from all animals and preserved as described above.

Muscle Preparation
Frozen diaphragm samples (100 mg) were homogenized in 1 mg of buffer A (HEPES 20 mM, pH 7.4, 0.2 mM PMSF, 1 µM leupeptin, 1 µM pepstatin A, 0.4 mM EDTA, 0.2 mM sodium orthovanadate, 30 mM sodium fluoride) and then centrifuged at 700 x g for 20 min. The supernatant was collected and designated as crude muscle homogenate. For fractionation of muscle samples into cytosolic and myofibrillar-mitochondrial fractions, the crude homogenates were centrifuged at 14,000 x g for 20 min and the supernatant was collected and designated as the cytosolic fraction. The pellet was reconstituted in buffer B (1% trifluoroacetic acid, 1mM tris[2-carboxyethyl phosphine hydrochloride]) and centrifuged at 14,000 x g for 20 min. The supernatant was collected and designated as the myofibrillar-mitochondrial fraction.

Detection of Protein Carbonyls using 1D Electrophoresis
Changes in protein carbonylation in crude diaphragm homogenates were detected using a commercial kit (Oxyblot Protein Oxidation Detection; Intrgen Inc., Purchase, NJ). Carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophenylhydrazine (DNPH) according to the manufacturer's instructions. In brief, 15 µg of protein were used per derivatization reaction. Proteins were then denatured by addition of 12% sodium dodecylsulfate (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 polyvinylidene difluoride (PVDF) membranes, and then blocked with 5% nonfat dry milk for 1hr at room temperature. The PVDF membranes were subsequently incubated overnight at 4°C with a polyclonal anti-DNP moiety antibody. The PVDF membranes were then washed several times with washing buffer and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies for 1 h. Specific proteins were detected with a chemiluminescence (ECL) kit (Roche Inc., Laval, Quebec, Canada). The blots were scanned with an imaging densitometer and optical densities (OD) of protein bands were quantified using ImagePro Plus software (Media Cybernetics, Carlsbad, CA). Total protein carbonylation OD in a given sample was calculated by adding OD of individual carbonylated protein bands.

Detection of Protein Carbonyls using 2D Electrophoresis
This technique was used to separate and identify carbonylated proteins in the cytosolic and myofibrillar-mitochondrial diaphragm muscle fractions obtained after 12 h of LPS injection. Briefly, 4 vols of 10 mM DNPH (in 2 M HCl) were added to 200 µg protein per sample and incubated for 30 min at room temperature. Ice-cold 100% trichloroacetic acid (TCA) was then added to yield a final TCA concentration of 15% and the sample was then incubated for 10 min on ice. The sample was then centrifuged for 10 min at 14,000 x g and the pellet was then washed three times with ethanol ethyl acetate and centrifuged at 14,000 x g for 15 min. The pellet was resuspended in 2D re-hydration buffer (8 M urea, 4% CHAPS, 0.2% ampholytes [pH 3–10], and 0.5 M DTT). Each derivatized muscle fraction was then separated into two portions (100 µg total each) and both portions underwent 2D electrophoresis. First-dimensional protein separation was performed with Protean IEF Cell (Bio-Rad Inc., Hercules, CA). Samples were applied to immobilized pH gradient strips (17-cm nonlinear pH 3–10; Bio-Rad Inc.) for 1 h at room temperature. The strips were then covered with mineral oil overnight and isoelectric focusing was performed at 10,000 V/1 h for up to a total of 60 to 100 kVh. For the second dimension, the IPG strips were equilibrated in room temperature for 10 min in equilibration buffer (6 M urea, 2% SDS, 0.05 mM tris-HCl, 20% glycerol) to which 2% DTT was added before use. An additional 10-min equilibration period was then used with equilibration buffer to which 2.5% iodoacetamide was added. The strips then were embedded in 0.7% agarose on the top of 10% acrylamide slab gels (23.5 x 18 x 0.15 cm) containing a 4% stacking gel. The second dimension SDS/PAGE was performed for 5 h, 30 mA per gel at 300 V. One of the resulting 2D gels for each muscle fraction was then stained with silver stain. Gels were fixed overnight in a fixation solution (10% acetic acid, 45% methanol), then rinsed twice in water, sensitized for 1 min in 0.02% sodium thiosulfate, followed by rinsing in water and immersion for 1 h in a silver nitrate solution (0.5 mM silver nitrate, 0.026% formaldehyde). Gels were then rinsed twice in water and developed in a developer solution (0.1 M sodium carbonate, 0.01% formaldehyde, 0.00125% sodium thiosulfate). A stop solution (0.1 M tris, 2% acetic acid) was then added for 30 min followed by rinsing with water for 5 min. Gels were then stored in 2% acetic acid. The second gel derived from a given sample underwent electrophoretical transfer to PVDF membrane and immunoblotting with an anti-DNP antibody as described above. Gels and PVDF membranes were imaged with a digital camera and aligned (ImagePro Plus) so as to identify positive carbonylated protein spots on the gels.

Mass Spectrometry
Carbonylated protein spots were cut out of the gels and taken for in-gel digestion on a robotic MassPrep Workstation (Micromass; Waters Corp. Milford, MA). In brief, the gel pieces were destained, reduced with 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide, and incubated with 6 ng/µl trypsin for 5 h at 37°C. 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). The digests were loaded into a 10-cm capillary PicoFrit column filled with C18 stationary phase and eluted by linear gradient of 5–70% acetonitrile in 0.1% formic acid at the flow rate 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 NCBI nonredundant database.

Localization of Protein Carbonyls
Muscle samples were immersed in subsequent baths of different degrees of alcohol, formol, and xylol, before being embedded in paraffin. Slides were then fixed in amino propyl-triethoxilane and acetone, and dried by heat (60°C). Muscle paraffin-embedded sections (3 µm) were obtained using a microtome. All sections were deparaffinized and incubated with a citric acid solution in a pressure cooker (antigen retrieval protocol). Carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with DNPH followed by the addition of 7.5 µl of neutralization solution. Sections were then incubated overnight with a polyclonal anti-DNP moiety antibody. After several washes in phosphate-buffered saline (PBS), slides were incubated for 1 h with biotinylated secondary antibodies followed by HRP-conjugated streptavidin and diaminobenzidine as a substrate. Slides were counterstained with hematoxylene, dehydrated, and mounted for conventional microscopy. Aside from those slides that were exposed only to secondary antibodies, slides not subjected to the derivatization process were also used as negative controls in this case.

Carbonyl Assay
In addition to the use of Oxyblot, we measured protein carbonylation by using the modified technique of Fagan and coworkers (16). In brief, aliquots of crude muscle homogenates (100 µg per sample) were precipitated with 10 vols of HCl–acetone (3:100) (vol/vol), then washed with 5 ml HCl–acetone to remove chromophores. The protein pellet was then washed twice with 5 ml 10% TCA. The protein pellets were disintegrated by vortexing during each wash and the supernatant was decanted after each centrifugation (800 x g, 20 min). Protein pellets were resuspended in 500 µl of homogenization buffer to which 500 µl of 10 mM DNPH (in 2 M HCl) was added and vortexed every 5 min for 15 min at room temperature. Protein blanks were prepared by adding 500 µl of 2 M HCl instead of DNPH to the assay tubes containing protein sample. After mixing, 500 µl of 30% TCA was added to each tube, and the samples were vortexed and then placed on ice for 10 min. After centrifugation for 20 min at 800 x g, the supernatant was discarded and the pellets were washed with 5 ml 20% TCA followed by three 5-ml ethanol–ethylacetate (1:1) (vol/vol) washes to remove any unreacted DNPH. The pellets were solubilized in 1 ml of 6 M guanidine hydrochloride and 20 mM potassium dihydrogen phosphate (pH 2.3) in 37°C water bath for 30–60 min. The final solution was centrifuged to remove any insoluble material. Carbonyl content was calculated from the absorbance measurement at 380 nm and absorbance coefficient of 22,000 M^–1 cm^–1.

Time Course of Specific Protein Carbonylation
We evaluated the influence of LPS injection on carbonylation of specific proteins by first detecting protein carbonylation in the cytosolic fraction of the diaphragms of control and septic animals using 1D electrophoresis and the Oxyblot kit as described above. Proteins (15 µg per sample) were derivatized with DNPH solution, neutralized, separated on 12%, 10%, and 8% tris-glycine polyacrylamide gels, transferred to PVDF membranes, and were probed with a polyclonal anti-DNP moiety antibody followed by incubation with HRP-conjugated anti-rabbit secondary antibody and the ECL kit. The primary and secondary antibodies were then stripped from the PVDF membranes by incubation with 0.2N NaOH solution for 5–60 min. After several washes, the membranes were then probed with goat polyclonal anti-creatine kinase-M, aldolase A, and enolase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) as well as rabbit polyclonal anti-carbonic anyhdrase III antibody (17). Specific proteins were then detected with HRP-conjugated secondary antibodies and ECL kit. Protein carbonylation and specific protein blots were then scanned with an imaging densitometer. The ODs of carbonylated and specific proteins were quantified using ImagePro Plus.

Creatine Kinase Activity Assay
Total muscle creatine kinase activity was measured with a commercial kit (Teco Diagnostics, Anaheim, CA) using a coupled assay system. This assay is based on the conversion of creatine phosphate and ADP by creatine kinase to creatine and ATP. The ATP and glucose are then converted to ADP and glucose-6-phosphate by hexokinase (HK). Glucose-6-phosphate dehydrogenase (G6PD) then oxidizes glucose-6-phosphate and reduces the nicotinamide adenine dinucleotide (NAD) to NADH. The rate of NADH formation, measured at 340 nm, is therefore, directly proportional to creatine kinase activity. The assay reaction involves the addition of 12 µl of muscle lysate (containing 3 µg protein) to 500 µl of reagents buffer containing D-glucose (20 mM), Mg²⁺ (10 mM), AMP (50 mM), creatine phosphate (30 mM), ADP (2 mM), oxidized NADH (2 mM), G6PD (300 U/l), HK (3,000 U/l), EDTA (2 mM). 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:

where TV is total reaction volume; d is light path length in cm; represents millimolar absorptivity of NADH; and SV indicates sample volume in ml.

Aldolase Activity Assay
Aldolase activity in crude muscle lysates was measured by following NADH oxidation at 340 nm at 22°C using a coupled assay system (18). We added 2 µl of coupling enzymes triose-phosphate isomerase (1 µg/ml)/ glycerol-3-phosphate dehydrogenase (10 µg/ml) and 10 µl of crude diaphragm fraction were added in a quartz cuvet, 988 µl assay buffer containing Tris (50 mM), EDTA (10 mM), NADH (0.3 mM), and fructose-1,6 bisphosphate (1 mM). Absorbance at 340 nm was measured every 10 s for 4 min. The slope of absorbance versus time was then converted to units of aldolase activity using the equation:

Diaphragmatic Muscle Contractility
Diaphragmatic strips (3–4 mm wide) were excised from control and septic (6, 12 and 24 h after LPS) rats. Strips were placed in an equilibrated (95% O₂–5% CO₂; pH 7.38) Krebs solution chilled at 4°C that had the following composition (mM): 118.0 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1 KH₂PO₄, 25 NaHCO₃, and 11.0 glucose. The strips were mounted in a muscle chamber and the muscle chamber was mounted vertically into a double jacket gut bath. A 4.0 silk thread was used to secure the central tendon to the isometric force transducer. Muscle strips were electrically stimulated at constant currents via platinum electrodes mounted in the muscle chamber that were connected to a square wave pulse stimulator (Model S48; Grass Instruments, Quincy, MA). After an equilibration period of 30 min (temperature of 22–25°C), the organ bath temperature was increased to 35°C and the maximum current necessary to elicit maximum force was then identified. Muscle length was then gradually adjusted to the optimal value (Lo). Force–frequency relationships of diaphragmatic strips were then constructed at frequencies of 10, 50, and 120 Hz while holding supramaximal current and stimulation duration (600 ms) constant. Tetanic contractions were digitized and analyzed with a personal computer. At the end of the experiment, the strips were blotted dry and weighed. Muscle length (cm) and weight (g) were measured and used to calculate the cross-sectional area. Isometric forces were normalized for muscle cross-sectional area estimated by using the value of 1.056 g/cm³ for muscle density.

Statistical Analysis
Values are presented as means ± SEM. Differences in OD of individual carbonylated proteins, total muscle carbonyl OD, and content were compared with one-way ANOVA followed by the Tukey test for multiple comparisons. P values < 5% were considered significant. Statistical analyses were performed with SigmaStat software (Jandel Scientific, Chicago, IL).

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Figure 1 illustrates the time course of carbonyl formation in crude diaphragm homogenates of septic rats. Weakly carbonylated proteins ranging in molecular masses between 50 and 29 kD were detected in control diaphragms (Figure 1A). The intensity of carbonyl groups rose significantly after 1 h of LPS administration with total carbonyl OD exceeding 200% of that detected in control diaphragm (Figure 1B). There was a further increase in diaphragm protein carbonylation measured 12 h after LPS administration reaching 300% of that detected in control diaphragms (Figures 1A and 1B). The rise in total diaphragm protein carbonylation of septic rats was due to an increase in both the intensity of carbonylated proteins already detected in the control diaphragm, particularly proteins of 29, 36, 40, and 48 kD molecular masses, and the appearance of new carbonylated protein bands with molecular masses > 50 kD and < 29 kD (Figure 1A). The increase in diaphragm protein carbonyl ODs after LPS injection was also verified by measuring carbonyl contents using the modified DNPH assay (16), which showed a similar time course to that detected with the Oxyblot kit (Figure 1C).

View larger version (21K): [in this window] [in a new window]   Figure 1. (A) A representative immunoblot showing the time course of protein carbonylation in crude diaphragm homogenates detected with the Oxyblot kit during the course of sepsis in rats. C refers to control rats (injected with saline and killed 24 h later). (B) Mean values (n = 5 in each group) of total carbonyl OD (expressed as percentage of control rats) in crude diaphragm homogenates during the course of sepsis in rats. *P < 0.05 as compared with control values. (C) Diaphragm carbonyl contents detected in 100 µg of crude homogenates using the modified DNPH assay.

View larger version (164K): [in this window] [in a new window]   Figure 2. Localization of protein carbonylation in the diaphragm of control (A) and septic (B) rat diaphragm detected with a selective antibody. Positive staining is shown in brown color. (C) Positive protein carbonylation in endothelial cells of vessels traversing rat diaphragms. Replacement of the primary anti-DNP antibody with a nonspecific antibody or omission of the derivatization processes completely eliminated positive carbonyl group staining (D).

View larger version (69K): [in this window] [in a new window]   Figure 3. Detection of protein carbonylation in the cytosolic fraction of septic rat diaphragm using 2D electrophoresis. Representative silver-stained 2D gel (top panel) and 2D anti-DNP blot (bottom panel) performed on cytosolic fraction of septic rat diaphragm. Fifteen positively carbonylated proteins were detected in the 2D blot and corresponding protein spots are indicated by circles in the 2D gel. Identity of each carbonylated protein spot is listed in Table 1.

View larger version (70K): [in this window] [in a new window]   Figure 4. Detection of protein carbonylation in the myofibrillar-mitochondrial fraction of septic rat diaphragm using 2D electrophoresis. Representative silver-stained 2D gel (top panel) and 2D anti-DNP blot (bottom panel) performed as described in MATERIALS AND METHODS. Four strongly carbonylated protein spots were identified in the myofibrillar-mitochondrial fraction (bottom panel). Circles in the 2D gel indicate corresponding protein spots. Identity of each carbonylated protein spot is listed in Table 1.

View larger version (31K): [in this window] [in a new window]   Figure 5. Representative examples of the changes in protein carbonylation of enolase, aldolase, creatine kinase and carbonic anyhdrase III in the cytosolic fraction of rat diaphragms during the course of sepsis. Protein carbonyl levels were measured with the Oxyblot kit, whereas specific protein levels were measured with selective primary antibodies.

View larger version (17K): [in this window] [in a new window]   Figure 6. Correlation analysis between creatine kinase and aldolase activities and the intensities of protein carbonylation of these enzymes (measured with Oxyblot) in the diaphragms of control and septic rats.

View larger version (15K): [in this window] [in a new window]   Figure 7. Changes in diaphragmatic isometric force in response to LPS injection in rats. In vitro isometric force of diaphragmatic strips was measured in response to 10, 50, and 120 Hz stimulation frequencies at 0 (control, filled circles), 6 (open circles), 12 (closed squares), and 24 (open squares) h of LPS treatment. * P < 0.05 compared with control animals.

	DISCUSSION

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Time Course of Protein Carbonyl Formation
Cellular oxidative damage develops when the balance between ROS-generating systems and ROS-scavenging systems tilts in favor of the former. It has long been proposed that oxidative stress contributes significantly to ventilatory and limb muscle dysfunction in sepsis (2, 19). In the current study, we measured protein carbonyl formation as an index of oxidative damage in the diaphragm on the basis of the idea that formation of reactive carbonyl groups (ketones and aldehydes) represents a major manifestation of oxidative modifications of proteins and reflects cellular damage induced by multiple forms of ROS. In limb muscles of humans and animals, protein carbonyl contents increase significantly in response to acute bouts of exercise, chronic exercise training, and in muscles of patients with Duchenne and Becker muscular dystrophies (7, 8, 10). To our knowledge, there are only two published reports describing protein carbonyl formation in limb and ventilatory muscles in septic animals. Fagan and colleagues (13) used biochemical measurement of carbonyl contents and reported 60–70% increases in protein carbonylation in the extensor digitorium longus and soleus muscles after 8–16 h of cecal ligation in rats. Our group has recently documented a significant rise in protein carbonyls in the diaphragm 24 h after LPS injection in rats (20). We described in this study for the first time the time course of protein carbonylation in the diaphragm in response to LPS injection in rats. Our results are in general agreement with previous studies confirming increased ROS formation in the diaphragm of septic rats. Many sources are likely to be involved in enhanced ROS production inside the diaphragm during sepsis, including mitochondrial respiratory chain complexes, xanthine dehydrogenase/xanthine oxidase system, enzymes involved in arachidonic acid oxidation, cytochrome P450 related enzymes, and the recently discovered nonphagocytic NADPH oxidase (21). Our group (21) has recently confirmed the contribution of the latter to ROS generation in the diaphragm of septic rats.

Nature of Carbonylated Proteins
Relatively little work has been performed regarding the interactions between ROS and proteins in skeletal muscles. Haycock and coworkers (22) confirmed in human limb muscle sections that cytochrome c oxidase, succinate dehydrogenase, dystrophin, ß-spectrin, and myosin heavy chain are sensitive to oxidation by exogenously generated O₂^–• and HO^–•. Our study uncovers, for the first time, that protein carbonylation in septic rat diaphragm involves key proteins involved in glycolysis (aldolase, enolase, and GAPDH), regulation of ATP metabolism (creatine kinases and ubiquinol-cytochrome c reductase), hydration of CO₂ (carbonic anhydrase III) and a major myofilament protein (-actin).

Creatine kinases (CKs), enzymes that catalyze the reversible transfer of a phosphoryl group from ATP to creatine to produce ADP and phosphocreatine, are localized both in the cytosol (cytosolic CKs) and the mitochondria (mitochondrial CK). CKs play a critical role in energy metabolism of various cells including skeletal and cardiac muscles. Recent studies have revealed that in vitro exposure to ROS inactivates CKs (23). In in vivo experiments, a decrease in heart myofibrillar CK activity has been attributed to modification of this enzyme by ROS (24). The deleterious effect of ROS on CK activity has been attributed to oxidation of a specific cystein residue (Cys²⁸² of MM-CK), which is essential for normal enzyme activity (23). We measured in this study both creatine kinase activity and the degree of carbonylation of this enzyme inside the diaphragm during the course of sepsis. Our results clearly illustrate that creatine kinase is weakly carbonylated in control rat diaphragms and that the intensity of creatine kinase carbonylation rises significantly 6 h after LPS injection (Figure 5). We also found that muscle creatine kinase activity correlated negatively with the intensity of carbonylation (Figure 6) suggesting that protein oxidation is associated with depressed enzyme activity as previously documented in cardiac myocytes (24). We should emphasize, however, that our experiments do not exclude the involvement of other yet to be determined factors in the inhibition of creatine kinase activity in the diaphragm of septic rats.

Our results also reveal that three key glycolysis enzymes (enolase, aldolase, and GAPDH) are targeted for carbonylation in septic diaphragms. Among these three enzymes, GAPDH has been the most widely studied in terms of oxidative modifications (6). GAPDH derived from rabbit skeletal muscle is irreversibly inactivated by peroxynitrite as a result of oxidation of several amino acids, including a critically important cysteine residue (Cys¹⁴⁹) (25). Little data is available with respect to the susceptibility of aldolase and enolase for oxidation. Aulack and colleagues (26) identified aldolase among tyrosine-nitrated proteins in the liver and lungs of septic rats, and suggested that this protein is a target of reactive nitrogen species. Similarly, enolase was recently identified to be tyrosine-nitrated proteins in the brains of patients with Alzheimer's disease (27). Our current results indicate that both enolase and aldolase are targeted by ROS inside the diaphragm and both show increased protein carbonylation in septic rats. However, the time courses of protein carbonylation of these two enzymes during the course of sepsis are different. While enolase carbonylation increased significantly within 1 h of LPS injection, aldolase carbonylation was clearly enhanced 6 h after LPS injection (Figure 5). Our study indicates, as with creatine kinase, that enhanced protein carbonylation correlates negatively with aldolase activity, suggesting that increased ROS may elicit significant inhibition of aldolase activity.

It has long been proposed that cytoskeletal elements represent one of the major cellular targets of ROS. Oxidation of actin has been documented in the brain of patients with Alzheimer's disease (14), in ischemic myocardium (28), and in macrophages exposed to hyperoxia (29). Our results show that -actin is strongly oxidized in the septic rat diaphragm. The nature of oxidative modifications of actins in response to ROS exposure is quite complex and involves oxidation of at least seven methionine residues (30). Oxidation is associated with profound disruption of actin filaments and inhibition of polymerization, and impaired interaction with the myosin protein (31). More recently, O'Reilly and coworkers (29) have reported that exposure of macrophages to hyperoxia resulted in increased actin oxidation and polymerization and inhibition of macrophage antibacterial function.

Another important target for protein carbonylation in the diaphragm is carbonic anhydrase (CA III), which is a member of zinc metallo-enzymes that catalyze the reversible hydration of carbon dioxide. CA III has many functions such as CO₂ hydratase, carboxyl esterase, and tyrosine phosphatase activities, as well as a role in skeletal muscle carbohydrate use (32). Glutathione interacts with CA III by forming a disulfide link with two of the five cysteine residues of CAIII in a process termed S-glutathionylation (33). This observation suggests that CA III may participate in antioxidant defenses against ROS formation inside skeletal muscle fibers.

Only one mitochondria-derived carbonylated protein was detected in the myofibrillar-mitochondrial fraction of septic rat diaphragm, namely, ubiquinol-cytochrome c reductase (complex III), which donates electrons from ubiquinol to cytochrome c, and thereby reduces cytochrome c. It is well known that the activities of complex I and IV of the mitochondrial respiratory chain are sensitive to both ROS and RNS (34). More recent studies have reported that complex III is also sensitive to the detrimental effects of ROS and RNS. Indeed, complex III activity was inhibited by 40% in response to exogenous HO^–• (generated by ascorbate/iron) (35). Similarly, Bolanos and coworkers (36) reported that complex III activity is strongly inhibited by relatively high levels of NO. However, no direct link between carbonyl formation and complex III inhibition has yet been made (35). Our results of significant complex III carbonylation in the diaphragm of septic rats is in general agreement with the notion that this complex is a target for ROS and RNS.

Our study indicates that enhanced protein carbonylation inside the diaphragm of septic rats precedes the contractile dysfunction of this muscle. Although this finding may give the impression that protein carbonylation is not directly responsible for depressed muscle function, we propose that protein carbonylation is an early form of ROS-derived post-translational modifications of muscle proteins, and that other modifications including hydroxynonenal formation and tyrosine nitration may follow a slower time course and may also target important intracellular processes inside muscle fibers resulting eventually in depressed muscle contractility. The importance of protein oxidation in the regulation of muscle contractility in sepsis needs to be confirmed in future experiments in which specific protein oxidation is significantly attenuated by antioxidant or selective inhibition of ROS-producing enzymes.

In summary, our study indicates that protein carbonyl formation increases significantly in the diaphragm during the course of severe sepsis in rats and that several important enzymes involved in glycolysis, ATP production, CO₂ hydration, and a myofilament protein are the targets for protein carbonylation inside diaphragmatic muscle fibers in septic animals. Our results also revealed that increased carbonylation of creatine kinase and aldolase in the septic rat diaphragm correlated negatively with the activities of these two enzymes.

	Acknowledgments

 
The authors are grateful to Mr. Luigi Franchi and Ms. Anna Llorens for their technical assistance, and to Ms. Anne Gatensby for editing the manuscript. Dr. Sabah Hussain is a scholar of the FRSQ. FUCAP, SOCAP and the Fondo de Investigación Sanitaria (RTIC C03/11 [Spain]) and BIOMED (BMTH4-CT98-3406 [E.U.]) supported Dr. Esther Barreiro.

	Footnotes

 
This study was funded by a grant from the Canadian Institute of Health Research.

Conflict of Interest Statement: E.B. has no declared conflicts of interest; J.G. has no declared conflicts of interest; M.D.F. has no declared conflicts of interest; L.K. has no declared conflicts of interest; S.J. has no declared conflicts of interest; and S.N.A.H. has no declared conflicts of interest.

Received in original form January 22, 2004

Received in final form September 27, 2004

	References

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 

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