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Am J Physiol Lung Cell Mol Physiol 290: L996-L1003, 2006; doi:10.1152/ajplung.00337.2005
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Modifications of proteins by 4-hydroxy-2-nonenal in the ventilatory muscles of rats

Sabah N. A. Hussain,1 Ghassan Matar,1 Esther Barreiro,2 Maria Florian,1 Maziar Divangahi,1 and Theodoros Vassilakopoulos3

1Critical Care and Respiratory Divisions, Royal Victoria Hospital, McGill University Health Centre, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; 2Muscle and Respiratory System Research Unit, Institut Municipal d'Investigació Mèdica, Departament de Ciències Experimental de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain; and 3Department of Critical Care and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece

Submitted 2 August 2005 ; accepted in final form 13 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although 4-hydroxy-2-nonenal (HNE, a product of lipid peroxidation) is a major cause of oxidative damage inside skeletal muscles, the exact proteins modified by HNE are unknown. We used two-dimensional electrophoresis, immunoblotting, and mass spectrometry to identify selective proteins targeted by HNE inside the diaphragm of rats under two conditions: severe sepsis [induced by E. coli lipopolysaccharides (LPS)] and during strenuous muscle contractions elicited by severe inspiratory resistive loading (IRL). Diaphragm HNE-protein adduct formation (detected with a polyclonal antibody) increased significantly after 1 and 3 h of LPS injection with a return to baseline values thereafter. Similarly, HNE-protein adduct formation inside the diaphragm rose significantly after 6 but not 3 h of IRL. Mass spectrometry analysis of HNE-modified proteins revealed enolase 3b, aldolase and triosephosphate isomerase 1, creatine kinase, carbonic anyhdrase III, aconitase 2, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein-beta. Measurements of in vitro enolase activity in the presence of pure HNE revealed that HNE significantly attenuated enolase activity in a dose-dependent fashion, suggesting that HNE-derived modifications have inhibitory effects on enzyme activity. We conclude that lipid peroxidation products may inhibit muscle contractile performance through selective targeting of enzymes involved in glycolysis, energy production as well as CO2 hydration.

oxygen radicals; protein oxidation; carbonyl formation; sepsis; skeletal muscle; enolase

IN RESTING SKELETAL MUSCLES, reactive oxygen species (ROS) are generated at relatively low levels and have been shown to promote muscle contractile force (30). However, under certain conditions such as during intense physical exercise and during systemic inflammatory pathologies such as severe sepsis, increased production of ROS causes oxidative stress inhibiting muscle contractility by disrupting action potential conduction, excitation-contraction coupling, contractile proteins, and mitochondrial respiration (30, 34, 41).

Proteins constitute one of the major targets of ROS and oxidation of proteins can lead to alterations in protein function including loss of enzymatic activity and increased susceptibility to protein degradation (10). One of the major amino acid modifications by ROS is the formation of carbonyl groups which are generated as a result of oxidation of arginine, lysine, threonine, or proline residues by ROS (10). Our group has recently confirmed the rise in protein carbonylation in the diaphragm of septic rats and identified several important enzymes such as enolase, aldolase, and creatine kinase to be targets for carbonylation inside diaphragm muscle fibers (4). In addition to proteins, polyunsaturated fatty acids of membrane lipids are highly susceptible to ROS-mediated damage (15). Moreover, elevated rates of lipid peroxidation are usually observed along with increased ROS production (15). Peroxidation of membrane lipids results in the fragmentation of polyunsaturated fatty acids resulting in the production of various cytotoxic and highly reactive aldehydes, alkenals, and hydroxyalkenals such as malonaldehyde and 4-hydroxy-2-nonenal (HNE) (15).

HNE is an {alpha},beta-unsaturated aldehyde and is considered to be the most reactive lipid peroxidation product and the most damaging to tissues (11). Increased HNE formation has been detected in the brain of patients with Alzheimer disease (25), in the spinal cord following traumatic injury (33), in the plasma of children with autoimmune diseases (14), and in the lungs following ozone exposure (42). HNE exerts broad biological toxicity effects including inhibition of DNA and protein synthesis, modification of low-density lipoprotein, and modulation of gene expression (11). Moreover, HNE can form adducts with specific proteins and chemically modifies several amino acid residues of these proteins including the sulphydryl group of cysteine, the imidazole moiety of histidine and the e-amino group of lysine. These modifications usually result in the inactivation of enzyme activity (18, 19, 28). In neurons, HNE has also been shown to form adducts with glutamate transporter Glt-1 (21), the cytoskeleton protein Tau (26), and apoliprotein E (27). Adduct formation with these proteins was associated with cross-linking and depressed function. In skeletal muscles, formation of HNE-protein adduct has been used along with protein carbonylation as an important marker of ROS formation and lipid peroxidation. In the ventilatory muscles, increased HNE protein adduct formation has been detected in the diaphragm of septic rats (40), in the diaphragm of humans with chronic obstructive lung diseases, (3) and in rat diaphragms exposed to peroxynitrite, a highly reactive ROS derived from the reaction of nitric oxide and O2– anions (38). Despite the increasing use of HNE detection as an index of lipid peroxidation-derived protein modifications, little information is yet available about the exact identity of proteins targeted by HNE in skeletal muscles in general and in the ventilatory muscles in particular.

This study had two main objectives: first, to assess whether HNE production increases significantly inside diaphragm muscle fibers under conditions where oxidative stress is known to develop, namely, severe sepsis and during strenuous muscle contractions; second, to identify the exact nature of proteins targeted by HNE inside the diaphragm.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Preparations

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 pentobarbital sodium, and the diaphragm was quickly excised, flash-frozen in liquid nitrogen, and stored at –80°C for immunoblotting analysis.

Experimental Protocol

Sepsis experiments. Four groups of rats (n = 5 in each group) were studied. Group 1 was injected intraperitoneally with normal saline [control group (C)] and killed 12 h later. Groups 2, 3, and 4 were injected intraperitoneally with Escherichia coli lipopolysaccharide (LPS, serotype 055:B5, 20 mg/kg; Sigma) and killed 1, 3, and 12 h later. The diaphragm and gastrocnemius muscles were excised from all animals and preserved as described above.

Inspiratory resistive loading experiments. These experiments were designed to elicit strenuous diaphragm contraction and to evaluate whether oxidative stress, which develops in response to these contractions, is associated with increased HNE-protein adduct formation. Three groups of pathogen-free, adult male Sprague-Dawley rats (300–325 g) were studied. Animals were lightly anesthetized with pentobarbital sodium (30 mg/kg ip) and were given supplemental doses (10 mg/kg) as needed. The animals were tracheostomized with polyethylene tubing (inner diameter 2.2 mm) that was sutured firmly in place with a silk tie. The cannula was connected to a two-way nonrebreathing valve (model 2300; Hans Rudolph, Kansas City, MO). Tracheal pressure was measured with a differential pressure transducer connected to the tracheal cannula via a side port. The inspiratory line was connected to a 5-liter bag, which was continuously filled with 100% O2 so as to prevent the development of hypoxemia. An arterial catheter (22 gauge) was placed into the internal carotid artery for sampling of arterial blood. Another catheter, placed into the jugular vein, was used to access the venous circulation. At the end of the surgical procedure, a 30-min stabilization period was allowed. The animals (n = 6 in each group) were then randomly assigned to a period of 3 or 6 h of inspiratory resistive loading (IRL) or left unloaded (quiet breathing group, see below). In each animal, maximum tracheal pressure was measured before loading by occluding the inspiratory port of the Hans Rudolph valve for a period of 30 s. We initiated IRL by inserting a needle into the inspiratory line so as to elicit tracheal pressure value of ~50% of maximum tracheal pressure. The average pressure time index was calculated in each animal as an index of the load achieved in each animal. At the end of the period of loading, the inspiratory resistance was removed, maximum tracheal pressure was remeasured, and the animals were killed with an overdose of pentobarbital sodium. The chest and the abdomen were opened, and the diaphragm was quickly excised and frozen in liquid nitrogen. The third group of animals was left to breathe spontaneously without any inspiratory resistance (quiet breathing group) and was killed 6 h later.

Muscle Preparation

Frozen muscle samples were homogenized in a buffer containing 10 mM Tris-maleate, 3 mM EGTA, 275 mM sucrose, 0.1 mM dithiothreitol (DTT), 2 µg/ml leupeptin, 100 µg/ml PMSF, 2 µg/ml aprotinin, and 1 mg/100 ml pepstatin A (pH 7.2). Samples were then centrifuged at 1,000 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 (Bio-Rad, Hercules, CA).

Detection of HNE-Protein Adduct Formation Using One-dimensional Electrophoresis

Crude muscle homogenates (30 µg per sample) were loaded onto 12% Tris-glycine sodium dodecyl sulfate (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 1 h at room temperature. Membranes were then incubated overnight with a selective polyclonal anti-HNE antibody (Calbiochem, San Diego, CA) (42). Specific proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit (Roche). Negative control experiments were also performed in which primary antibodies were omitted and membranes were probed with secondary antibodies only. Blots were scanned with an imaging densitometer, and optical densities (OD) of specific proteins were quantified with ImagePro Plus (Media Cybernetics, Silver Spring, MD). We calculated total HNE-protein adduct formation for each sample by adding OD of individual positive protein bands. To validate equal protein loading among various lanes, PVDF membranes were stripped and reprobed with a monoclonal antisarcomeric {alpha}-actinin antibody (Sigma).

Detection of HNE-Protein Modification By Two-dimensional Electrophoresis

Crude homogenates of diaphragms obtained after LPS injection in rats (400 µg of protein per sample) were added to ice-cold trichloroacetic acid (15% final concentration). The samples were then incubated for 10 min on ice and centrifuged for 10 min at 14,000 g, and the pellets were then washed three times with ethanol ethyl acetate and centrifuged at 14,000 g for 15 min. The pellets were resuspended in two-dimensional rehydration buffer [8 M urea, 4% CHAPS, 0.2% ampholytes (pH 3–10), and 0.5 M DTT]. Each muscle sample was then separated into two portions (100 µg total each), and both portions underwent two-dimensional electrophoresis. First-dimensional protein separation was performed with Protean IEF Cell (Bio-Rad, Hercules, CA). Samples were applied to immobilized pH gradient strips (17-cm nonlinear pH 3–10, Bio-Rad) 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 60–100 kVh. For the second dimension, the immobilized pH gradient 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 two-dimensional gels for each muscle sample 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-HNE 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

HNE-modified protein spots were cut out of the gels and taken for in-gel digestion on a robotic MassPrep Workstation (Micromass, Waters Milford, MA). In brief, the gel pieces were destained, reduced with 10 mM DTT, 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 with a Liquid Chromatography-Quadrupole-Time of Flight (LC-Q-Tof) mass spectrometer (MicroMass). The digests were loaded into 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 National Center for Biotechnology Information nonredundant database.

To confirm the time course of changes in HNE-derived modifications of specific proteins, we first detected HNE-protein adduct formation in crude diaphragm lysates by one-dimensional electrophoresis as described above. The primary and secondary antibodies were then stripped from the PVDF membranes by incubation with 0.2 N 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) as well as rabbit polyclonal anticarbonic anyhdrase III antibody (12). 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 with ImagePro Plus.

Influence of HNE on Enolase Activity

To evaluate the effect of HNE on enolase enzyme activity, we performed in vitro pure enolase activity assay in the presence and absence of increasing HNE concentrations. Pure enolase [in units (U)] purified from rabbit muscle was purchased from Sigma. A single unit of enolase is defined as the amount of enzyme that produces 1 µmol of phosphoenol pyruvate from phospho-D-glycerate/min in standard assay (13). Enolase activity assay was measured at 37°C by incubating pure enolase (3–9 U) in a buffer containing 50 mM imidazole-HCl (pH 6.8), 2.0 mM MgSO4 and 400 mM KCl (13) in the absence or presence of 10–60 µM of pure HNE (Sigma). The reaction was initiated by adding 1 µmol of 2-phospho-D-glycerate, and the OD was measured after 10 min of reaction time with a spectrophometer at 240 nm.

Statistical Analysis

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


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Sepsis Experiments

Figure 1 illustrates the time course of HNE-protein adduct formation in crude diaphragm homogenates of septic rats. Anti-HNE antibody detected several HNE-modified proteins; however, five of these bands (apparent molecular masses of 90.8, 83.3, 49.3, 46.9, and 30 kDa) have relatively higher intensities than the remaining bands (Fig. 1A). The intensities of several HNE-modified protein bands rose significantly after 1 and 3 h of LPS administration with a subsequent decline 12 h later to levels similar to those observed in control diaphragms (Fig. 1, A and B). Several HNE-modified protein bands were also detected in the gastrocnemius muscles; however, the intensities of these bands remained unchanged during the course of sepsis (Fig. 1, B and C).


Figure 1
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  		Fig. 1. A: representative immunoblot showing the time course of 4-hydroxy-2-nonenal (HNE)-protein adduct formation in crude diaphragm homogenates detected with anti-HNE antibody. C, control rats. B: mean ± SE values of total HNE-protein adduct optical densities (expressed in arbitrary units) in crude diaphragm homogenates during the course of sepsis in rats. *P < 0.05 compared with control values. OD, optical density. C and D: representative immunoblot and mean ± SE values of total HNE-protein adduct formation in the gastrocnemius muscle during the course of sepsis.

 
Figure 2 illustrates a representative example of a two-dimensional protein map of crude diaphragm homogenate obtained after 3 h of LPS injection (stained with silver stain in Fig. 2A) and probed with anti-HNE antibody (Fig. 2B). Excision of HNE-modified proteins from silver-stained gel followed by mass spectrometry analysis identified 12 HNE-modified protein with varying intensities (Table 1, Fig. 2B). The glycolysis enzyme enolase 3b was identified to be strongly HNE-modified (spots 3, 4, 5, and 6 in Fig. 2B). Two other glycolysis enzymes, namely aldolase (spot 9) and triosephosphate isomerase 1 (spot 12) are also modified by HNE (Fig. 2B). In addition, creatine kinase (spots 7 and 8) and carbonic anyhdrase III (spot 10) are also identified to be modified by HNE (Fig. 2B). Finally, our analysis also revealed that three mitochondrial enzymes involved in energy production (aconitase, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein) were also targeted by HNE inside the diaphragm of septic rats (spots 1, 2, and 11, Fig. 2; and Table 1). To verify the identity of enolase and creatine kinase as HNE-modified proteins in the diaphragm of septic rats, the two-dimensional HNE immunoblots of rat diaphragms were stripped and reprobed with selective antibodies to enolase and creatine kinase. Figure 3 shows that the strongest HNE-modified protein in septic rat diaphragms is indeed the glycolysis enzyme enolase. This figure also indicates that creatine kinase was only weakly HNE-modified in these samples.


Figure 2
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  		Fig. 2. Detection of HNE-derived protein modification in crude diaphragm homogenates of septic rats using 2-dimensional (2D) electrophoresis. Representative silver-stained 2D gel (top) and 2D anti-HNE blot (bottom) are shown. We detected 12 positive HNE-modified proteins in the 2D blot; circles in the 2D gel indicate corresponding protein spots. Each these HNE-modified proteins is identified in Table 1.

 

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  		Table 1. HNE-modified proteins in rat diaphragms obtained after 12 h of LPS injection

 

Figure 3
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  		Fig. 3. Confirmation of the identity of enolase and creatine kinase (CK) as HNE-modified proteins in the crude diaphragm lysate of septic rat diaphragm. HNE-modified proteins were first detected by probing the 2D blot with anti-HNE antibody followed by horseradish peroxidase-conjugated secondary anti-rabbit antibody. These antibodies were then stripped and the blot was then probed with antienolase and anti-CK antibodies.

 
Figure 4 illustrates representative examples of changes in HNE-protein adduct formation and total protein concentration of enolase, aldolase, creatine kinase, and carbonic anyhdrase III in crude diaphragmatic homogenates of septic rats. Total levels of enolase, aldolase, creatine kinase, and carbonic anyhdrase III during sepsis remained similar to those detected in control diaphragms (Fig. 4). In control diaphragms, HNE-product adduct formation was detected mainly in enolase, creatine kinase, and carbonic anyhdrase III. After 3 h of LPS injection, the intensity of HNE-derived modifications increased in the four proteins mentioned above. However, HNE-derived modification of aldolase and carbonic anyhdrase III declined whereas that of enolase and creatine kinase remained strong 12 h after LPS injection (Fig. 4).


Figure 4
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  		Fig. 4. Representative examples (A) and mean ± SE OD (B) of HNE-derived modification of enolase, aldolase, CK, and carbonic anyhdrase III (CAIII) in crude homogenates of rat diaphragms during the course of sepsis. HNE-derived modification was detected with anti-HNE antibody, whereas specific protein levels were measured with selective primary antibodies.

 
IRL

Maximum peak tracheal pressure measured (at baseline) before IRL averaged 75.2 ± 11.7 cmH2O. Peak inspiratory tracheal airway pressure developed by the animals during 3 and 6 h of loading averaged 35.5 ± 1.96 cmH2O (46 ± 8% of maximum peak tracheal pressure), whereas pressure time index averaged ~0.18. In the quietly breathing animals, arterial pH, PaO2, and PaCO2 during quiet breathing averaged 7.40 ± 0.02, 357 ± 34 mmHg, and 39 ± 5 mmHg, respectively. By comparison, these values averaged 7.04 ± 0.12, 305 ± 29 mmHg, and 95 ± 11 mmHg at the end of 3 h of IRL, respectively. Similarly, arterial pH, PaO2, and PaCO2 averaged 6.93 ± 0.11, 245 ± 31 mmHg, and 109 ± 25 mmHg, respectively at the end of 6 h of IRL. Figure 5 shows the influence of IRL on HNE-protein adduct formation in the diaphragm. Only after 6 h of IRL did the intensity of HNE-protein adduct formation increase significantly inside the diaphragm (P < 0.05 compared with quiet breathing, Fig. 5). This significant rise in total HNE-protein adduct formation in response to IRL appears to have been due to an increase in HNE-derived modification of enolase, carbonic anyhdrase III, and aldolase and to a lesser extent of creatine kinase (Fig. 5C). We should emphasize that our results do not exclude the possibility that other proteins other than enolase, aldolase, carbonic anyhdrase III, and creatine kinase might have contributed to the rise in total HNE-derived modifications in the diaphragm during IRL.


Figure 5
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  		Fig. 5. A: representative immunoblot showing the time course of HNE-protein adduct formation in crude diaphragm homogenates obtained from quiet breathing (QB) and rats exposed to 3 and 6 hrs of severe inspiratory resistive loading (IRL). B: mean ± SE values of total HNE-protein adduct ODs (expressed in arbitrary units) in crude diaphragm homogenates of QB rats and rats exposed to 3 and 6 h of IRL. *P < 0.05 compared with QB values. C and D: mean ± SE ODs of HNE-derived modification of enolase, CK, aldolase, and CAIII during the course of IRL in rats.

 
Regulation of Enolase Activity by HNE

Figure 6 shows the influence of increasing HNE concentrations on pure enolase activity. Three units of pure enolase per tube were incubated with 10–60 µM of HNE for 10 min. Exposure to physiological concentrations of HNE resulted in a significant a dose-dependent decline in enolase activity [P < 0.05 compared with control (no HNE)].


Figure 6
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  		Fig. 6. Effect of increasing concentrations of HNE on enolase activity measured after 10 min of in vitro incubation at 37°C. Enolase activity was expressed as percentage of control (0 HNE concentration). *P < 0.05 compared with control values.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study are 1) the intensity of HNE-protein adduct formation in the diaphragm increases significantly within 1 and 3 h of LPS injection and after 6 h of severe IRL; 2) HNE-derived protein modification involves several key enzymes involved in glycolysis (enolase, aldolase, and triosephosphate isomerase), ATP production (creatine kinase, dihydrolipoamide dehydrogenase, electron transfer flavoprotein, and aconitase), and a regulator of CO2 hydration (carbonic anhydrase III); and 3) direct exposure to pure HNE results in a significant and a dose-dependent decline in enolase activity.

Cellular oxidative damage develops when the level of ROS generation exceeds the capacity of endogenous ROS-scavenging systems. Many investigators have proposed that oxidative stress contributes significantly to ventilatory and limb muscle dysfunction in sepsis (29, 34). In the current study, we measured HNE-protein adduct formation as an index of oxidative damage in the diaphragm on the basis that formation of HNE-adduct represents a major manifestation of lipid peroxidation-derived modifications of proteins and reflects an aspect of tissue damage induced by many sources of ROS. HNE can be produced at relatively large levels (up to 5 mM) and is more stable than other lipid peroxidation product (estimated half-life of 2.5 min) (5, 11). The presence of lipophilic and hydrophilic components enables HNE to cross through cellular compartments and target many molecules (11). Our observation that LPS administration elicited a significant elevation in diaphragm HNE-protein adduct formation is consistent with the notion that oxidative stress develops in the ventilatory muscles during sepsis (34, 41). Despite the importance of lipid peroxidation products as major regulators of many intracellular processes, only a few studies have addressed the alterations in lipid peroxidation inside the ventilatory muscles in septic animals. Shindoh et al. (32) and Supinski et al. (35) described a significant increase in diaphragm malondialdehyde and 8-isoprostane (indexes of lipid peroxidation) levels after 48 h of LPS injection in hamsters. By comparison, the same research group failed to detect any change in levels of conjugated diene (another index of lipid peroxidation) in the diaphragm of septic rats (34). Our results clearly illustrates that HNE-protein adduct formation is transiently elevated inside the diaphragm of septic rats. Although we did not identify the molecular sources of increased HNE formation inside the diaphragm during sepsis, many enzymes including NAPDH oxidase, xanthine oxidase, arachidonic acid metabolism enzymes, and mitochondrial respiratory chain complexes are likely to have contributed to increased lipid peroxidation in sepsis. As to the mechanisms responsible for the decline in HNE-protein OD after 12 h of LPS administration, we speculate that antioxidant pathways might have undergone a compensatory upregulation in response to the initial rise in ROS production. One likely pathway that might have attenuated lipid peroxidation during the course of sepsis is heme oxygenases (HO). Both HO-1 and HO-2 exert important antioxidant properties, and both are known to be upregulated inside the diaphragm within several hours of LPS administration in rats (2, 43). The importance of these enzymes in regulating protein oxidative modification in the diaphragm was evidenced by the significant rise in carbonyl formation and HNE-protein adduct formation when HO-1 and HO-2 enzymes were selectively inhibited in rats (2, 43). We should emphasize that the importance of other antioxidant pathways in reversing elevated HNE-protein adduct formation could not be excluded.

It has been well established that production of ROS and lipid peroxidation products is elevated in the ventilatory muscles during strenuous muscle contractions (36, 37, 39). Supinski et al. (36) investigated the association between increased lipid peroxidation and the development of muscle fatigue in dogs. These authors reported that antioxidant pretreatment significantly reduced diaphragm levels of thiobarbituric acid reactive substances (TBAR, an index of lipid peroxidation) and delayed the development of diaphragm fatigue elicited by electrical stimulation (36). Our results confirm that strenuous diaphragmatic contractions elicited by inspiratory resistive loading in rats leads to a significant increase in lipid peroxidation as indicated by HNE-protein adduct formation. However, this rise in HNE-protein adduct formation was detectable only after 6 h of IRL, whereas others have reported significant elevation of diaphragm malondialadehyde after 1 h of very severe loading in rats (37). This difference in time course of lipid peroxidation inside the diaphragm may be related to differences in the intensity of diaphragm contraction elicited by resistive loading.

There is little information available regarding ROS-mediated posttranslational modifications of proteins inside skeletal muscle fibers. Using gamma radiation as a source of ROS, Haycock et al. (17) reported that structural and contractile proteins such as dystrophin, dystroglycan, spectrin, myosin heavy chain, cytochrome c oxidase, and succinate dehydrogenase are more susceptible to oxidative damage by hydroxyl radicals than superoxide anions. More recently, our group used two-dimensional electrophoresis approach to identify carbonylated proteins inside the diaphragm of septic rats. These carbonylated proteins include glycolysis enzymes [aldolase, enolase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)], mitochondrial enzymes (creatine kinases and ubiquinol-cytochrome c reductase), enzymes involved in hydration of CO2 (carbonic anhydrase III), and a contractile protein ({alpha}-actin) (4). We report here that carbonylation of few of these enzymes might be mediated by lipid peroxidation in general and HNE in particular. Indeed, we found that enolase, aldolase, creatine kinase, and carbonic anyhdrase III are targets for HNE-derived modifications in the diaphragm both during severe sepsis and during inspiratory resistive loading. Our results also indicate that a third enzyme critical for normal glycolysis (triosephosphate isomerase-1) and three important mitochondrial enzymes involved in ATP production (aconitase, electron transfer flavoproteins, dihydrolipoamide dehydrogenase) are also modified by HNE in the diaphragm of septic rats. These results clearly indicate that lipid peroxidation products such as HNE selectively target several critical enzymes involved in energy production inside skeletal muscle fibers.

Our previous study on protein carbonylation and our current results suggest that glycolysis enzymes are major targets for the action of ROS inside skeletal muscle fibers both during the course of sepsis and in response to strenuous muscle activity. Previous studies have focused on GAPDH as the key glycolysis enzyme to be inhibited by both reactive oxygen and nitrogen species. For instance, Uchida and Stadtman (44) have reported that exposure of GAPDH to pure HNE resulted in inactivation of GAPDH as result of modifications of cysteines, histidine, and lysine residues. GAPDH and aldolase have recently been shown to be strongly carbonylated inside the diaphragm of septic rats. By comparison, we failed to detect HNE-derived modification in GAPDH protein, whereas strong HNE-derived modification of enolase and aldolase was detected in this study. In addition, we also report here that triosephosphate isomerase-1, an enzyme which converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a key reaction in glycolysis, is also a target for HNE-derived modification further confirming the influence of ROS on glycolysis inside the diaphragm. It should be emphasized that sepsis and strenuous muscle activity are not the only conditions that are associated with carbonylation of glycolysis enzymes. Oxidative modifications of enolase and aldolase by reactive oxygen and nitrogen species have been reported in the brains of patients with Alzheimer disease and in the liver and lungs of septic rats (1, 6). Previous studies have revealed that oxidative modifications of proteins are usually associated with inhibition of enzyme function as a result of conformational changes, oxidation of key cysteines residues, cross linking of proteins and due to increased degradation of oxidized proteins. Our current study reveals that physiological levels of HNE exert a direct negative influence on enolase activity (Fig. 6). This observation along with our previous report of a negative correlation between aldolase activity and the intensity of aldolase carbonylation in the diaphragm of septic rats (4) suggest that carbonylation and HNE-derived oxidative modifications may interfere with the activities of important glycolysis enzymes. However, whether these inhibitory effects on glycolysis enzyme are sufficient enough to alter glucose metabolism remains debatable. For instance, in erythrocytes, exposure to 0.1 mM of HNE had no effect on glycolysis (23). By comparison, Miwa et al. (24) have described significant inhibition of glucose-induced insulin secretion and glucose utilization by HNE in the pancreas. Clearly more studies are needed to address the selective influence of HNE on glycolysis and energy production inside skeletal muscle fibers.

We have previously reported that creatine kinase and carbonic anyhdrase III are strongly carbonylated inside the diaphragm of septic rats (4). Creatine kinases catalyses the reversible transfer of a phosphoryl group from ATP to creatine to produce ADP and phosphocreatine. Creatine kinase activity plays a critical role in energy metabolism of various cells including skeletal and cardiac muscles. By comparison, carbonic anhydrase III is a member of zinc metallo-enzymes and plays an important role in maintaining muscle carbohydrate metabolism as well as the reversible hydration of carbon dioxide (9). Recent studies indicate that, by interacting directly with glutathione, carbonic anyhdrase III participate in the cellular defense against ROS (8). Our current results indicate that both creatine kinase and carbonic anyhdrase III are modified by HNE in control diaphragms but more so in septic rat diaphragms (Figs. 2–4). These results confirm that carbonylation of these proteins inside the diaphragm is mediated in part through increased lipid peroxidation. As in the case of glycolysis enzymes, many in vitro and in vivo experiments on cardiac myocytes have shown that creatine kinase activity is inhibited by ROS (20, 22). Our group (4) has recently confirmed this inhibitory effect of ROS on creatine kinase activity in the diaphragm of septic rats.

There is increasing evidence that HNE exerts an important inhibitory influence on mitochondrial functions. Exposure of isolated cardiac muscle mitochondria to pure HNE elicited a rapid decline in respiratory activity and the ability to meet NADH demands during maximum rates of O2 consumption (19). Humphries and Szweda (18) have reported that depression of mitochondrial respiration by HNE is mediated through selective inhibition of {alpha}-ketogluatarate dehydrogenase (KGDH) and pyurvate dehydrogenase (PDC) enzyme complexes. KGDH is a TCA enzyme that catalyzes the conversion of {alpha}-ketogluarate to succinyl-CoA and plays a critical role for mitochondrial respiration and oxidative phosphorylation. Similarly, PDC provides a critical link between glycolysis and the TCA cycle by regulating the flux of pyurvate to the mitochondria. Inhibition of PDC and KGDH activities by HNE is thought to be the result of modifications of the reduced lipoyl moieties of the dihydrolipoamide acyltransferases (E2 component of the PDC and KGDH complexes) (18). Our results indicate that one of the HNE-modified proteins in septic rat diaphragm is dihydrolipoamide dehydrogenase, the E3 component of PDC and KGDH complexes. This protein is a flavoprotein disulphide oxidoreductase and is localized in the mitochondrial matrix. To our knowledge, this is the first evidence of HNE-derived modification of this component of the PDC and KGDH complexes and lends further credence to the notion that HNE and lipid peroxidation products might inhibit the activities of these complexes. Our study also reveals that the inhibitory effect of HNE on mitochondrial respiration may also be mediated through modifications of aconitase (Table 1). Aconitase catalyses the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, a reaction which is near equilibrium in vivo and is therefore of limited regulatory importance to the operation of the TCA cycle. However, in situations in which ROS production is increased as during exercise and sepsis, an inhibition of the enzyme could result in a low flux through the TCA cycle, low rates of ATP generation, and decreased performance. In vitro experiments on isolated organisms and on purified aconitase revealed that hydrogen peroxide, superoxide anions, and peroxynitrite strongly carbonylate and inactivate the activity of this enzyme (7, 16, 45). Finally, we found that a third mitochondrial protein, namely, electron transfer flavoprotein-beta is also HNE modified in septic rat diaphragm (Table 1). Electron transfer flavoproteins (also known as electron transfer flavoprotein:ubiquinone oxidoreductase, ETF-QO) are two mitochondrial enzymes that are required for the transfer of electrons between at least nine mitochonchdrial matrix dehydrogenases and the main electron transport chain (31). Electron transfer flavoprotein is a heterodimer consisting of {alpha}- and beta-subunits, both nuclear encoded and synthesized in the cytosol. Despite its central role in the oxidation of fatty acids and some amino acids, relatively little is known about the structure and the influence of ROS on ETF-QO function. In humans, deficiency of these proteins leads to metabolic disorders including glutaric aciduria. Our study suggests that the beta-subunit of ETF-QO inside rat diaphragms is susceptible to posttranslational modifications by HNE during the course of sepsis. Whether these modifications influence the ability of ETF-QO to transfer electrons to the respiratory chain remains to be determined.

In summary, our study indicates that HNE-protein adduct formation increases significantly in the diaphragm during the course of severe sepsis in rats and that several enzymes critical for glycolysis, ATP production, and mitochondrial respiration are targeted by HNE in the diaphragm of septic rats. Our results also revealed that enhanced HNE-protein adduct formation could also be detected inside the diaphragm after several hours of strenuous contractions of this muscle elicited by severe inspiratory resistive loading.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was funded by grants from the Canadian Institute of Health Research and the Canadian Cystic Fibrosis Foundation.


   ACKNOWLEDGMENTS
 
Dr. Sabah Hussain is a scholar of the Fonds de recherche en santé du Québec. RESPIRA (RTIC C03/11), and Fondo de Investigaciones Sanitarias (FIS 04/1165) (Spain) funded Dr. E. Barriero.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Hussain, Rm. L3.05, 687 Pine Ave. W, Montreal, Quebec H3A 1A1, Canada (e-mail: sabah.hussain{at}muhc.mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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