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Originally published In Press as doi:10.1074/jbc.M600837200 on March 27, 2006

J. Biol. Chem., Vol. 281, Issue 22, 15110-15120, June 2, 2006
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Redox Activation of Aldose Reductase in the Ischemic Heart*

Karin Kaiserova{ddagger}, Sanjay Srivastava{ddagger}, Joseph D. Hoetker{ddagger}, Sunday O. Awe§, Xian-Liang Tang{ddagger}, Jian Cai, and Aruni Bhatnagar{ddagger}1

From the {ddagger}Institute of Molecular Cardiology, Departments of §Physiology and Biophysics and Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202

Received for publication, January 27, 2006 , and in revised form, March 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2–4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldose reductase (AR)2 is an efficient catalyst for the reduction of glucose and a wide range of aldehydes derived from lipid peroxidation (1, 2). The enzyme also catalyzes the reduction of glutathione conjugates of unsaturated aldehydes such as acrolein, 4-hydroxy-trans-2-nonenal, and trans-2-hexenal with higher efficiency than the parent aldehyde (3, 4). Our recent studies suggest that AR participates also in the metabolism of 1-palmitoyl-2-oxovaleroyl phosphatidylcholine and related phospholipid ("core") aldehydes (5) generated upon oxidation of unsaturated fatty acids esterified to the sn-2 position of phospholipids. The efficacy of AR in reducing multiple aldehydes suggests that the enzyme may be involved in cellular defense against aldehydes produced by lipid peroxidation. The notion that AR is involved in antioxidant defense is supported by evidence showing that: the AR gene is induced by oxidants such as aldehydes (6, 7) and hydrogen peroxide (7, 8) and under conditions associated with oxidative stress such as myocardial ischemia (9), heart failure (10), vascular inflammation (11), and alcoholic liver disease (12); and that inhibition of AR increases aldehyde toxicity in rat vascular smooth muscle cell lines (7) and Chinese hamster fibroblast cell lines (8). An antioxidant role of AR is also in accord with our observation that inhibition of the enzyme increases 4-hydroxy-trans-2-nonenal accumulation in the ischemic heart and abolishes cardioprotection associated with the late phase of ischemic preconditioning (9). In addition, because of its ability to catalyze the reduction of glucose, AR also regulates the flux of glucose in the heart and inhibition of the enzyme has been shown to prevent myocardial ischemia-reperfusion injury by increasing glycolysis and preserving NADH/NAD+ levels (13–15). Perhaps the role of the enzyme in the heart is context-dependent and determined in part by transcriptional and post-translational mechanisms regulating its activity.

Aldose reductase is a NADPH-dependent monomeric protein that belongs to the aldo-keto reductase superfamily (2, 16). Whereas the protein is expressed basally in many tissues, its expression is enhanced by growth factors such as fibroblast growth factor and platelet-derived growth factor (6), and cytokines such as tumor necrosis factor-{alpha} (17, 18). The AR gene is markedly induced in high ionic strength media (19) via a NFAT-5-dependent mechanism (20), which stimulates the osmotic response element located at the promoter site of the AR gene. AR is also subject to post-translational modifications and protein kinase C-dependent phosphorylation of the enzyme has been described (21). However, it remains unclear whether such mechanisms regulate AR activity under conditions of oxidative stress or ischemia.

Several investigators have reported the presence of activated and non-activated forms of AR in normal and diabetic tissues (22–26) and in the ischemic heart (13). Native AR isolated from most tissues displays biphasic kinetics that could be attributed to the simultaneous presence of activated and non-activated forms of the enzyme (22–24, 26). In contrast, the kinetics of the activated form (high Vmax) of the enzyme is monophasic. A characteristic feature of an activated enzyme is its relative insensitivity to inhibition by drugs such as sorbinil. Whereas some kinetic features of the activated enzyme could be simulated by oxidizing its cysteine residues (27–31) or mutation of active site cysteine to alanine (32), the in vivo identity of activated AR has not been established. Therefore, to delineate the mechanism of AR activation, we examined changes in enzyme activity during ischemia. Our results show that ischemia activates AR by modifying its cysteine residues to sulfenic acids. Preliminary findings of this study have been published as an abstract (33).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Tiron, DL-glyceraldehyde, dithiothreitol (DTT), mammalian protease inhibitor mixture, {alpha}-cyanohydroxycinnamic acid, sorbitol, sorbitol dehydrogenase, dimedone, N-ethylmaleimide (NEM), CHAPS, N-(2-mercaptoproprionyl)glycine (MPG), SDS, lucigenin, urea, EDTA, EGTA, NADPH, and NAD+ were obtained from Sigma. Sorbinil was a gift from Pfizer. Polyvinylidene difluoride membranes, immobilized pH gradient strips, and Bio-Lyte 3/10 ampholytes were purchased from Bio-Rad. Primary polyclonal antibodies against rat AR were purchased from Santa Cruz Biotechnology. Sephadex G-25 columns, enhanced chemiluminescence (ECL) reagents, and horseradish peroxidase-linked secondary anti-goat antibodies were obtained from Amersham Biosciences. All other reagents were of analytical grade.


Figure 1
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  		SCHEME 1
 
Global Ischemia-Reperfusion ex Vivo—Hearts excised from male Sprague-Dawley rats (300–350 g, 3–4 months old) were cannulated and perfused in the Langendorff retrograde mode, as described (34, 35). Briefly, the hearts were perfused at a constant perfusion pressure of 80 mm Hg with Krebs-Henseleit buffer (KH buffer, pH 7.5) containing, in mM: NaCl 118, KCl 4.7, CaCl2 3, MgCl2 1.25, NaHCO3 25, KH2PO4 1.25, EDTA 0.5, glucose 10, and equilibrated with a mixture of 95% O2 and 5% of CO2 for 10 min. A latex fluid-filled balloon was placed in the left ventricle through an incision in the left atrial appendage and inflated to a pressure of 8–10 mm Hg and the perfusion flow rate was between 8 and 12 ml/min for all heart preparations.

Seven groups of rats (300–350 g; 4–8 rats per group) underwent the following experimental protocols (Scheme 1). In Protocols I–III isolated rat hearts were perfused for 40 min with Krebs-Henseleit (KH) buffer alone, (Protocol I), or KH buffer containing either 10 mM Tiron (Protocol II) or 5 mM MPG (Protocol III). In Protocols IV–VI the hearts were first perfused for 10 min either with KH buffer alone (Protocol IV), or KH buffer containing Tiron (Protocol V) or MPG (Protocol VI). In Protocol VII, the hearts were perfused with KH buffer for 10 min, subjected to global ischemia for 30 min, and reperfused for 30 min.

Regional Ischemia-Reperfusion in Situ—Male Sprague-Dawley rats (300–350 g) were anesthetized with an intraperitoneal injection containing ketamine (37 mg/kg) and xylazine (5 mg/kg) followed by continuous inhalation of 0.5–1.0% isofurane after endotracheal intubation. The animals were ventilated with oxygen-enriched room air using a rodent respirator (Harvard Rodent Ventilator model 683, 60–70 breaths/min). Tidal volume was set to 1.0 ml/100 g of body weight. The chest was opened via a left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. After pericardioctomy, a 6-0 proline (Ethicon, NJ) ligature was placed under the left main coronary artery, and the ends of the tie were threaded through a small plastic (PE50) tube to form a snare for reversible left coronary artery occlusion for 15 min (Protocol IX). For occlusion-reperfusion experiments (Protocol X), the ligature was removed and the hearts were subjected to 15 min of reperfusion. The border of the ischemic zone of the left ventricle was demarcated with a surgical marker during occlusion. Sham operated hearts served as controls (Protocol VIII). The hearts were excised and tissue samples were taken immediately from the ischemic (anterior wall) and non-ischemic (posterior wall) zones of the left ventricle. Samples were snap-frozen in liquid nitrogen and stored at –80 °C until use.

Measurement of Superoxide Anions—The hearts were pulverized and homogenized in 10 volumes of perchloric acid (10%, v/v) and centrifuged for 20 min at 13,000 x g. The protein-free supernatant (0.1 ml) was incubated with 0.25 mM lucigenin at room temperature for 5 min in the dark and chemiluminescence was measured in a microtiter plate at excitation and emission wavelengths of 430 and 452 nm, respectively. Lucigenin (0.25 mM) in 10% perchloric acid was used as a reagent blank.

Measurement of AR Activity—Left ventricular tissue was homogenized in 4 volumes of 0.01 M potassium phosphate (pH 7.0), containing 1 mM EDTA, 50 mM NEM, and protease inhibitor mixture (1:100, v/v). The homogenate was centrifuged at 105,000 x g for 1 h at 4°C and the supernatant was used for measuring AR activity. Wherever indicated the homogenate was reduced with 0.1 M DTTin0.1 M Tris-HCl (pH 8.0) for 1 h at 37 °C. Excess DTT was removed by gel filtration on a Sephadex G-25 (PD-10) column pre-equilibrated with N2-saturated 50 mM potassium phosphate (pH 6.0), containing 1 mM EDTA. Enzyme activity was measured at 25 °C in a 0.2-ml system containing 0.1 ml of tissue homogenate (1–1.5 mg of protein), 0.1 M potassium phosphate (pH 6.0), 0.15 mM NADPH, and 10 mM glyceraldehyde. The reaction was monitored by the rate of oxidation of NADPH at 340 nm for 3 min. The control cuvette (blank) contained all components of the reaction mixture except glyceraldehyde. To estimate steady-state kinetic parameters, initial velocity was measured at 7–10 different concentrations of glyceraldehyde.


Figure 2
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  		FIGURE 1.
Ischemia activates aldose reductase. A, hearts removed from adult Sprague-Dawley rats were perfused in the Langendorff mode ex vivo. After 10 min of equilibration with KH buffer, the hearts were subjected to either 10–30 min of ischemia (Protocol IV) or 30 min of ischemia (I) followed by 30 min of reperfusion (I/R; Protocol VII). Hearts perfused (P) for 40 min with KH buffer served as controls (Protocol I). AR activity and sorbitol levels in the left ventricle were measured as described under "Experimental Procedures." Inset shows changes in AR activity in hearts subjected to the indicated durations of ischemia, without reperfusion. B, hearts were subjected to regional ischemia in situ by ligating the left main coronary artery for 15 min (Protocol IX), or 15 min of occlusion (O) followed by 15 min of reperfusion (O/R; Protocol X). Tissue from the ischemic zone was excised and used for measuring AR activity and sorbitol levels. Sham operated rats (Protocol VIII) served as controls. Values are expressed as mean ± S.D. (n = 3); *, p < 0.05 versus the corresponding value of the parameter in sham operated hearts, or hearts perfused with KH buffer for 40 min.

 
Measurement of Myocardial Sorbitol Levels—Left ventricular tissue (0.1–0.2 g) was pulverized in liquid nitrogen and homogenized in 4 volumes of 6% perchloric acid. Homogenates were centrifuged at 2,400 x g for 10 min at 4 °C and the protein-free supernatant (1.0 ml) was mixed with 0.5 ml of glycine buffer (pH 9.4) containing 3.6 mM NAD+ and 6 units of sorbitol dehydrogenase. The sorbitol levels were measured spectrofluorometrically, as described previously (36).

Two-dimensional Electrophoresis, Western Blotting, and MALDI-TOF/MS Analyses—Left ventricular tissue (0.1–0.2 g) was homogenized in 4 volumes of the protein extraction buffer (20 mM Tris-Cl (pH 6.8) containing 2 mM EDTA, 50 mM NEM, and protease inhibitor mixture (1:100, v/v)). Homogenates (25%) were centrifuged at 105,000 x g for 1 h at 4 °C and the supernatants were aspirated and mixed with 10% trichloroacetic acid to precipitate the proteins. The pellet was washed three times with acetone and resuspended in 0.2 ml of resuspension buffer (20 mM Tris (pH 6.8) containing 8 M urea and 2 mM EDTA). Samples were diluted to 0.5 mg of protein/ml with base rehydration buffer (9.8 M urea, 4% CHAPS, 0.2% Bio-Lyte 3/10 ampholytes, and 0.002% bromphenol blue) and 100 µg of proteins were loaded onto the 11-cm immobilized pH gradient strips (pH 5–8). Proteins were focused at 35,000 volt-hours with a maximum current of 50 µA/strip. After focusing, the strips were incubated in base equilibration buffer (50 mM Tris (pH 8.8) containing 6 M urea, 2% SDS, 20% glycerol, and 50 mM NEM) for 15 min. The strips were then equilibrated in SDS-PAGE running buffer (25 mM Tris (pH 8.3) containing 0.2 M glycine and 1% SDS) and placed on the top of a 12% SDS-polyacrylamide gel, overlaid with agarose, and subjected to electrophoresis in the second dimension. The proteins were transferred onto polyvinylidene difluoride membranes and probed with polyclonal anti-AR antibodies (21). Parallel gels were silver-stained. Spots corresponding to a molecular mass of 36 kDa and pI ~ 6.28 were excised and used for MALDI-TOF/MS analysis. To extract peptides, the gel plugs were incubated for 15 min with 20 µlof 0.1 M NH4HCO3 followed by addition of 30 µl of acetonitrile. Solvents were removed and the gel plugs were dried under vacuum. Trypsin (200 ng) in 50 mM NH4HCO3 was added to the gel plugs and the mixture was incubated overnight at 37 °C. The resulting solution was co-crystallized with a matrix of a cyano-4-hydroxycinnamic acid in a spot on a stainless steel target plate. The TOF-Spec 2E (Micromass), which employs a nitrogen laser (337 nm), was used. Ionization was obtained by automated data acquisition using pulses at 20–505 power and raster array of 10 clusters of 10 pulses each. A standard mixture of cytochrome c, myoglobin, and trypsinogen was used for mass axis calibration. Accelerating potential was set to 20 kV with delayed extraction and data were collected at a rate of 2 GHz, by scanning from 500 to 3,000 atomic mass units in the positive ion reflector mode. Average data, represented as a centroid peak were compared with a battery of databases with 50–100 ppm resolution precision. Peptide m/z values were analyzed in silico for suspect modifications using MASCOT data base.

Electrospray Ionization MS (ESI+/MS)—For ESI+/MS analysis the protein was desalted on a Sephadex G-25 column equilibrated with N2-saturated ammonium acetate (10 mM). The desalted protein was diluted with the flow injection solvent (acetonitrile:H2O:formic acid; 50:50:1, v/v/v). The mixture was injected into a MicroMass ZMD spectrometer at a rate of 10 µl/min. The operating parameters were as follows: capillary voltage 3.1 kV; cone voltage 27 V; extractor voltage 4 V; source block temperature 100 °C, and desolvation temperature 200 °C. Spectra were acquired at the rate of 200 atomic mass units/s over the range of 20–2,000 atomic mass units. The instrument was calibrated with myoglobin (0.15 mg/ml) dissolved in 50% (v/v) acetonitrile containing 0.2% (v/v) formic acid.

Statistical Analysis—Individual saturation curves used to obtain steady-state kinetic parameters were fitted to a general Michaelis-Menten equation using EnzFitter (Biosoft, Cambridge, UK). In all cases, best fits to the data were chosen on the basis of the standard error of the fitted parameters. Data are expressed as mean ± S.D. and were analyzed by one-way analysis of variance for multiple comparisons or by Student's t test for unpaired data. Statistical significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ischemia Activates AR—Ischemia-induced changes in AR activity were determined in the tissue homogenates prepared from hearts subjected to ischemia or ischemia-reperfusion ex vivo or coronary occlusion or occlusion-reperfusion in situ (for specific protocols, see Scheme 1). As shown in Fig. 1A (inset), homogenates prepared from perfused hearts subjected to different durations of global ischemia displayed a time-dependent increase in AR activity. Maximal activation (3-fold) was observed after 30 min of ischemia (Protocol IV). The increase in AR activity in ischemic hearts was accompanied by a 5-fold increase in sorbitol content (Fig. 1A). No further change in AR activity was observed upon reperfusion (Protocol VII), although reperfusion did lead to a slight (25%) decrease in tissue sorbitol content.

Left ventricular tissue obtained from the ischemic zone of hearts subjected to 15 min of coronary occlusion (Protocol IX) displayed nearly a 3-fold increase in AR activity and more than a 2-fold increase in myocardial sorbitol content, as compared with sham operated hearts (Protocol VIII, Fig. 1B). The enzyme activity remained elevated in hearts reperfused for 15 min after 15 min of coronary occlusion (Protocol X), although the total sorbitol recovered from the ischemic zone was decreased, possibly due to further metabolism or efflux. Taken together, these results indicate that both, global ischemia ex vivo, or coronary occlusion in situ increase myocardial AR activity and sorbitol accumulation.


Figure 3
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  		FIGURE 2.
Ischemic activation of aldose reductase is due to sulfhydryl oxidation. A, activation of AR by sulfate. AR activity was measured in the perfused (Protocol I) and ischemic (Protocol IV) hearts in 0.1 M potassium phosphate (pH 6.0) in the absence or presence of 0.4 M ammonium sulfate (+SO2–4). To examine the contribution of sulfhydryl oxidation to AR activation, tissue homogenates were incubated with 0.1 M DTT in 0.1 M Tris (pH 8.0) for 1 h at 37°C. Excess DTT was removed by gel filtration and AR activity was measured in the reduced homogenates. B, inhibition of AR by sorbinil. Inhibition of AR activity by 1 µM sorbinil was measured in the homogenates prepared from perfused or ischemic hearts before and after reduction with DTT, as indicated. Values are expressed as mean ± S.D., n = 3; p < 0.05 for: * versus AR activity with the indicated additives without sulfate (A) or sorbinil (B); {dagger} versus AR activity without DTT; and # versus perfused hearts.

 
Activator/Inhibitor Sensitivity of AR from Ischemic Hearts—To examine how AR is affected by ischemia, we determined the kinetic properties and the sensitivity of the enzyme to inhibitors and activators. Previous study shows that AR is highly sensitive to divalent anions, such as sulfate (16). Consistent with this observation, we found that addition of 0.4 M (NH4)2SO4 into the assay buffer led to a 2–3-fold increase in AR activity in homogenates prepared from either perfused (Protocol I) and the ischemic (Protocol IV, Fig. 2A) hearts, suggesting that ischemic activation does not affect the sensitivity of the enzyme to sulfate. To test the sensitivity of AR to inhibition we used sorbinil, which inhibits AR with a Ki of ~1.5 µM (37). Although, sorbinil also inhibits aldehyde reductase with similar affinity (37), there is little, or no aldehyde reductase activity in the rat heart (35). In perfused heart homogenates, AR activity was inhibited by 70% in the presence of 1 µM sorbinil, but the same concentration of sorbinil inhibited AR in homogenates of ischemic hearts by only 30%, indicating that ischemia decreases the sensitivity of AR to inhibition by sorbinil.


Figure 4
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  		FIGURE 3.
Ischemic activation of aldose reductase is mediated by ROS. Perfused hearts were equilibrated with KH buffer for 10 min and then perfused with KH buffer alone (Protocol I) for 40 min (P), or with KH buffer containing 10 mM Tiron (A) or 5 mM MPG (B) for 40 min (Protocols II and III, respectively) or perfused with Tiron (Protocol V) or MPG (Protocol VI) for 10 min and then subjected to global ischemia for 30 min. AR activity and sorbitol levels were measured in the left ventricle. Inset to panel A shows changes in chemiluminescence in the hearts perfused with KH buffer alone (P; Protocol I), or Tiron in KH buffer (Protocol II), or hearts subjected to ischemia in the presence of KH buffer alone (I; Protocol IV) or KH buffer containing 10 mM Tiron (Protocol V). Values are expressed as mean ± S.D. (n = 3); *, p < 0.05 versus the corresponding parameter in the absence of Tiron or MPG; and # versus the corresponding parameter in the perfused hearts.

 
Our previous studies show that AR becomes relatively insensitive to sorbinil when its cysteine residues are modified (2, 16). Therefore we examined whether AR activation in the ischemic heart is due to oxidation of its cysteine residues. For this, we measured AR activity in homogenates prepared from perfused (Protocol I) and ischemic (Protocol IV) hearts that were reduced with 0.1 M DTT for 1 h at 37°C. Excess DTT was removed by gel filtration and AR activity was measured. As shown in Fig. 2, DTT abolished ischemia-induced activation of AR, and the reduced enzyme was inhibited to 42% by 1 µM sorbinil. The ability of DTT to reverse activation and partially restore sorbinil sensitivity of AR in ischemic heart homogenates indicates that ischemic activation of AR is due to the oxidative modification of its cysteine side chain(s).

Steady-state Kinetic Parameters of AR from the Ischemic Heart—To examine the catalytic properties of activated AR, steady-state kinetic parameters were determined in the tissue homogenates of perfused (Protocol I) and ischemic (Protocol IV) hearts. As listed in Table 1, no significant differences were observed between the Km glyceraldehyde values of AR from the ischemic (71 ± 16 µM) and perfused (84 ± 19 µM) hearts. However, the Vmax values in ischemic heart homogenates were 1.5-fold higher, as compared with those obtained from the perfused hearts, indicating that ischemic activation of AR is primarily due to an increase in Vmax. Reduction of the tissue homogenates with DTT had no effect on the Km glyceraldehyde of AR in perfused and ischemic hearts, but it decreased the Vmax values in both, perfused and ischemic hearts (Table 1), suggesting that a small fraction of AR in the perfused heart may be in the activated form, and that this form of the enzyme is significantly increased in the ischemic heart.


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  		TABLE 1
Kinetic parameters of aldose reductase from perfused and ischemic heart

The enzyme activity was measured in 0.1 M phosphate buffer (pH 6.0) containing 0.15 mM NADPH and different concentrations of DL-glyceraldehyde. Km glyceraldehyde values were calculated using the EnzFitter.

 
Ischemic Activation of AR Is Mediated by Reactive Oxygen Species—Next we examined whether activation of AR is mediated by the reactive oxygen species (ROS) generated in the ischemic heart. As shown in the inset to Fig. 3A, hearts subjected to 30 min of ischemia (Protocol IV) displayed a 5-fold increase in ROS generation (as measured by lucigenin chemiluminescence) over the levels observed in the perfused hearts. This increase in ROS generation was completely abolished in hearts pretreated with a superoxide radical scavenger, Tiron. Treatment of perfused hearts with Tiron (Protocol II) led to a slight decrease in AR activity, possibly due to inhibition of basal ROS generation. However, pre-treatment with Tiron abolished AR activation in the ischemic heart (Protocol V), and the values of AR activity and sorbitol recovered from Tiron-treated ischemic hearts were similar to those obtained from the hearts perfused with KH buffer alone. Similar results were obtained when the hearts were treated with another ROS scavenger, MPG. As shown in Fig. 3B, treatment with MPG (Protocol III) caused a slight decrease in AR activity and sorbitol accumulation in the perfused hearts; however, MPG treatment of the ischemic heart (Protocol VI) caused an even greater decrease in the AR activity and sorbitol accumulation. Collectively, these findings support the notion that AR is highly sensitive to oxidation and that the enzyme is activated in the ischemic heart by a ROS-dependent mechanism.

AR Is Activated by H2O2—Because hydrogen peroxide is the most abundant ROS in vivo (38), we determined whether H2O2 would activate recombinant AR in vitro. Incubation of human recombinant AR with 0.1 mM H2O2 for 1 h at 25°C resulted in a >2-fold increase in AR activity (Fig. 4). The activated enzyme was relatively insensitive to 1.0 µM sorbinil. Incubation of the activated protein with DTT completely abolished AR activation and restored the sensitivity of the enzyme to sorbinil, indicating that AR is activated upon incubation with H2O2 alone. To determine the chemical basis of AR activation, the reduced and the H2O2-modified protein was incubated with a sulfenic acid-specific reagent, dimedone (39). Treatment of the reduced enzyme with dimedone did not affect enzyme activity or sorbinil sensitivity, however, when the H2O2-modified enzyme was treated with dimedone a significant decrease in enzyme activity was observed and the remaining enzyme activity was relatively insensitive to inhibition by sorbinil (Fig. 4). These results suggest that H2O2 activates AR by oxidizing its cysteine residue(s) to sulfenic acid(s), which then react with dimedone.

To examine the chemical nature of AR modification, the H2O2-treated human AR was analyzed by ESI+/MS analysis. As shown in Fig. 5A, the ESI+/MS spectrum of the native, reduced AR displayed a major peak corresponding to a molecular mass of 37,880 Da, which is within an experimental error of the expected molecular mass of 37,883 Da (AR + His Tag). An additional small peak was observed at 37,916 Da. The chemical identity of this peak is unknown. Incubation of the reduced AR with H2O2 resulted in the appearance of a major peak with a molecular mass of 37,912 Da (Fig. 5B) and a minor peak with a molecular mass of 37,944 Da. The 32-Da increase in the major peak corresponds to the addition of 2 oxygen atoms to the protein, which could be due to the formation of either two sulfenic acids (AR-SOH) or one sulfinic acid (AR-SOOH) in the protein. To distinguish between them, H2O2-modified AR was treated with 0.5 mM dimedone and analyzed by ESI+/MS. As shown in Fig. 5C, H2O2-modified AR treated with dimedone showed a major peak with a molecular mass of 38,192 Da, consistent with the addition of two dimedone molecules (140 Da each) to two AR-SOH molecules. Furthermore, treatment of the modified enzyme with DTT completely reversed AR modification by H2O2 (Fig. 5D). These observations suggest that treatment with H2O2 results in the formation of two cysteine sulfenic acids (Cys-SOH) on the AR molecule.


Figure 5
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  		FIGURE 4.
Activation of aldose reductase by hydrogen peroxide. Recombinant human His tag AR was purified from Escherichia coli, reduced with 0.1 M DTT for 1 h, and then incubated with 0.1 mM H2O2 for 1 h at 37°C. Aliquots were withdrawn from the reaction mixture and the catalytic activity of the enzyme was measured with 0.15 mM NADPH and 10 mM DL-glyceraldehyde in the absence and presence of 1 µM sorbinil. The reaction was stopped by gel filtration and the modified enzyme was either reduced with 0.1 M DTT for 1 h, or treated with 0.5 mM dimedone (DD) for 30 min. In a series of control experiments, the reduced enzyme was also incubated with dimedone alone. *, p < 0.05 versus control (C) reduced enzyme; # versus the enzyme activity in the absence of DTT; and {dagger} versus activity in the absence of dimedone.

 
Modification of AR in the Ischemic Heart—To determine whether AR activation in the ischemic heart is due to Cys-SOH formation, tissue homogenates prepared from perfused (Protocol I) and ischemic (Protocol IV) hearts were incubated with 0.5 mM dimedone. As shown in Fig. 6, dimedone did not affect AR activity in perfused heart homogenates, but it inhibited the enzyme activity in the homogenates prepared from ischemic hearts, indicating that the cysteine sulfenic acids are generated in AR from the ischemic hearts. Consistent with this observation treatment with dimedone increased sorbinil sensitivity of AR in ischemic hearts, indicating that only a fraction of AR is modified/activated and residual enzyme activity, i.e. the activity not inhibited by dimedone, was due to the presence of a native (unmodified) form of the protein.

To examine the chemical nature of AR modification in the ischemic heart, tissue from the perfused (Protocol I) and ischemic (Protocol IV) hearts, or hearts subjected to coronary occlusion in situ (Protocol IX), as well as their corresponding sham controls (Protocol VIII) were homogenized in the presence of NEM (to prevent modification of sulfhydryl groups during sample processing) and the proteins were separated on two-dimensional gels. The gels were silver stained for MALDI-TOF/MS analysis, or used for Western blot analysis. Western blots of the proteins from the perfused (Fig. 7A) and sham operated (Fig. 7C) hearts showed high immunoreactivity to anti-AR antibodies. Samples from hearts subjected to either ischemia ex vivo (Fig. 7B), or coronary occlusion (Fig. 7D) showed an additional immunopositive spot (AR*) with isoelectric point (pI) slightly lower than the native enzyme. Spots corresponding to native or modified AR from the parallel silver-stained gels were excised, digested with trypsin, and analyzed by MALDI-TOF/MS analysis. For perfused hearts, 10–11 discrete peptides were identified (Fig. 8A, Table 2A). These peptides matched with rat AR and corresponded to 30–55% coverage of the protein sequence. The peptides corresponding to amino acid residues 297–305 (1078.57 Da) displayed modification of the two cysteine residues, Cys-298 and Cys-303 (located at the active site of AR) by NEM. Formation of the covalent adducts with NEM suggests that these residues were present in reduced form in the perfused hearts. Furthermore, MALDI-TOF/MS analysis of the spot corresponding to the activated AR (AR*) from the hearts subjected to global ischemia (Protocol IV) yielded 10–11 peptides with 36–42% coverage of the AR sequence. In these specimens, chemical modifications were observed in the peptides corresponding to amino acid residues 79–95 and 297–305 (2014.1 and 985.53 Da; Fig. 8B, Table 2B). MASCOT search analysis revealed that Cys-80 and Cys-92 in the peptide corresponding to amino acids 79–95, and Cys-298 and Cys-303 in the peptide corresponding to the amino acids 297–305 were oxidized to the sulfenic acids. Similar to the perfused hearts, MALDI-TOF/MS analysis of the spots excised from the sham operated hearts (Fig. 8C, Table 2C) showed that residues Cys-298 and Cys-303 in peptide 297–307 (1277.59 Da) were modified by NEM, while in one of the peptides (1179.59 Da) recovered from the hearts subjected to coronary occlusion, Cys-298 and Cys-303 were oxidized to the sulfenic acids (Fig. 8D, Table 2D). Taken together, these data indicate that cysteine residues of AR, particularly Cys-298 and Cys-303 were predominantly in the reduced form in perfused or non-ischemic hearts and that these residues were oxidized to sulfenic acids in the ischemic heart.


Figure 6
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  		FIGURE 5.
ESI+/MS spectra of hydrogen peroxide-treated aldose reductase. Recombinant human His tag AR was purified from E. coli and reduced with 0.1 M DTT, desalted, and analyzed by ESI+/MS. The de-convoluted spectrum of the protein, shown in panel A, identified the native protein (expected mass = 37,883 Da) and minor unidentified peak (37,916 Da). For modification studies, the reduced enzyme was incubated with 0.1 mM H2O2 in 10 mM Hepes (pH 7.4) for 1 h at 4 °C. Excess H2O2 was removed by gel filtration on a Sephadex G-25 column pre-equilibrated with N2-saturated 10 mM ammonium acetate. Spectrum in the panel B shows unmodified protein (37,880 Da) and modified species (37,912 and 37,944 Da). Panel C shows a de-convoluted ESI+/MS spectrum of H2O2-modified AR treated with 0.5 mM dimedone (DD) for 30 min. Peaks of both, native AR (37,880 Da) and AR modified (38,192 Da) with two dimedone molecules (140 Da each) are evident. Panel D shows the spectrum of the protein after 30 min of treatment with H2O2 followed by reduction with 0.1 M DTT. Only the native forms of the protein (37,880 and 37,916 Da) are evident.

 


Figure 7
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  		FIGURE 6.
Effect of dimedone on aldose reductase activity in perfused and ischemic heart homogenates. Perfused (Protocol I) and ischemic (Protocol IV) heart homogenates were incubated with 0.5 mM dimedone in 0.1 M potassium phosphate (pH 6.0) for 30 min at room temperature. Excess dimedone was removed by gel filtration and AR activity was measured in the absence or presence of 1 µM sorbinil. Values are expressed as mean ± S.D. (n = 3); p < 0.05, * versus the corresponding parameter in the presence of dimedone; and # versus the corresponding value of the parameter in hearts subjected to perfusion (P) alone.

 


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  		TABLE 2
MALDI-TOF/MS analysis of AR from rat heart homogenates

Modified cysteine residues are underlined. Some peptides displayed an increase in mass due to modification by NEM.

 


Figure 8
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  		FIGURE 7.
Two-dimensional electrophoresis and Western blot analysis of the proteins from non-ischemic and ischemic hearts. Rat hearts were: A, perfused ex vivo for 40 min with KH buffer alone (Protocol I); B, subjected to global ischemia for 30 min (Protocol IV); C, sham surgery (Protocol VIII); or D, coronary occlusion in situ for 15 min (Protocol IX). At the end of the protocol, the tissue was homogenized and the proteins were resolved by two-dimensional electrophoresis and probed for AR modification by Western blotting using anti-AR antibodies. Arrows indicate native (AR) and modified (AR*) forms of the protein.

 


Figure 9
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  		FIGURE 8.
Structural analysis of aldose reductase modification in the ischemic hearts. Homogenates prepared from hearts subjected to global ischemia ex vivo (Protocol IV) or coronary occlusion in situ (Protocol IX) were separated by two-dimensional electrophoresis. The gels were silver stained and the spots corresponding to the native AR and modified-AR* (identified using anti-AR antibodies) were excised and analyzed by MALDI-TOF/MS analysis. The spectra of the peptides corresponding to the protein from perfused (panel A; Protocol I), ischemic (panel B; Protocol IV), sham operated (panel C; Protocol VIII), and coronary occluded (panel D; Protocol IX) hearts are shown. Arrows indicate peptide peaks modified by NEM, or those containing sulfenic acids. The AR peptides from non-ischemic and ischemic hearts are listed in the Table 2, A–D.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study are that AR is activated in the ischemic heart and that the increase in AR activity could be attributed to the oxidation of the cysteine residues of the enzyme to sulfenic acids. Although, many investigators over several years have reported that activated and non-activated forms of AR are present in purified enzyme preparations or in diseased tissue (13, 22–26), the present study provides clear evidence for the first time that AR is intrinsically activated in the ischemic heart by a ROS-sensitive mechanism. In general, this work contributes to the growing body of evidence suggesting that protein-sulfenic acid formation is an in vivo mechanism of protein post-translational modification that regulates cellular responses to oxidative stress (39, 40).

The present study provides several lines of evidence suggesting that AR activation in the ischemic heart is due to a sulfenic acid formation. These include the observations that: 1) antioxidant interventions prevented AR activation in the ischemic heart; 2) reduction with DTT reversed AR activation; 3) human AR was activated by H2O2 with concurrent formation of sulfenic acids; 4) the peroxide-activated and the ischemic, but not the native, or the non-ischemic, enzymes were inhibited by the sulfenic acid-specific reagent, dimedone; and 5) cysteine-containing peptides of AR isolated from the hearts subjected to ischemia ex vivo or in situ displayed an increase in their molecular mass consistent with the formation of sulfenic acids. Taken together, these findings suggest that redox changes in the ischemic heart promote oxidation of the cysteine residues of AR to sulfenic acids.

Sulfenic acids are direct products of the reaction between H2O2 and cysteine thiolate anions, although other oxidants such as alkyl hydroperoxides, peroxynitrite, and hypochlorous acid could also convert reduced cysteine to a sulfenic acid (40). Metastable sulfenic acid intermediates have been identified in the catalytic cycle of multiple enzymes, including peroxiredoxin, NADH peroxidase, and methionine sulfoxide- and formylglycine-generating enzymes. Formation of cysteine oxyacids has also been linked to oxidative stress-induced transcriptional changes in bacteria due to altered binding of oxyR and OhR (39) and changes in the activity of the yeast peroxiredoxin (41) and Yap1 protein (40). Although, the role of sulfenic acids in regulating mammalian protein function and signal transduction pathways is less clear. Cysteine residues of several transcription factors (i.e. NF-{kappa}B, Fos, and Jun), or proteins involved in cell signaling or metabolism (glyceraldehyde-3-phosphate dehydrogenase, glutathione reductase, and protein-tyrosine phosphatases) could be converted into sulfur oxyacids in vitro (39). In addition, in cell culture studies, sulfenic acid formation has been suggested to mediate tumor necrosis factor-{alpha}-induced JNK (c-Jun NH2-terminal kinase) activation (42) and the inactivation of protein-tyrosine phosphatase 1B by the epidermal growth factor (43). However, direct evidence for the formation of sulfenic acids in mammalian tissue in situ is lacking. Ex vivo perfusion of the hearts with high (0.1–1 mM) concentrations of H2O2 has been shown to generate cysteine sulfenic acids in specific proteins (44), but, it is unclear whether such high concentrations of H2O2 are achieved in situ and whether under pathological or physiological conditions the generation of H2O2 in mammalian systems is high enough to oxidize protein cysteines to sulfur oxyacids. In this regard, our observation that the cysteine residues of AR are oxidized to sulfenic acids in the ischemic heart supports the view that the levels of ROS generated in the ischemic heart are sufficient to oxidize protein cysteines to sulfenic acids. Moreover, conversion of AR cysteines to oxyacids in the hearts subjected to ischemia in situ, or ex vivo indicates that this modification is not dependent on high oxygen concentrations that prevail in organs perfused ex vivo or cells maintained in culture.

Oxidation of the reactive cysteine residues to sulfonic acid (Cys-SO3H) has been reported for matrix metalloproteinase-9 that was affinity precipitated from the rat brain after focal cerebral ischemia and reperfusion (45), suggesting that ischemia-reperfusion during cerebral stroke, as well as myocardial ischemia (present study) is accompanied by an increase in ROS generation. However, unlike matrix metalloproteinases, which are extracellular proteins relatively unprotected by antioxidant systems, AR is located in the cytosol, which is rich in reduced glutathione and other antioxidants. Hence, its oxidation is somewhat surprising and the ultimate metabolic fate of the modified enzyme remains unclear. Protein sulfenic acids could be readily converted to disulfide intermediates or reduced back to cysteine by glutaredoxin, thioredoxin, or glutathione. In addition, cysteine sulfenic acids could react with glutathione to form mixed disulfides, or could be irreversibly oxidized to sulfinic or sulfonic acids. In some proteins such as protein tyrosine phosphate 1B (46, 47) sulfenic acids are shielded from further oxidation and maintained in a metastable state. Whereas the specific mechanism stabilizing the sulfenic acid derivatives of AR is not known, their persistence in the heart during ischemia suggests that the cysteine residues of AR are located in an unusual environment that not only prevents their uncontrolled oxidation to sulfinic and sulfonic acids, but also retards their conversion back to the reduced state.

The crystal structure of AR shows an ({alpha}/beta)8 protein that folds to a TIM-barrel motif (2). The active site of the enzyme contains few ionic groups. The Cys-298 and Cys-303 residues, located near the substrate binding COOH terminus of the protein exist in a hydrophobic microenvironment that is occluded from the solvent upon NADPH binding. The other cysteine residues, Cys-80 and Cys-92, are located in the protein interior (48). Hence, the enzyme architecture itself could provide a favorable influence on stabilization of Cys-SOH derivatives. In our previous work we have found that oxidation of Cys-298 is sufficient to increase AR activity and to decrease the sensitivity of the enzyme to sorbinil (27, 28, 32). Therefore, despite modification of several cysteines of AR in the ischemic heart, we suggest that the modification of Cys-298 alone could account for the changes in the kinetic properties and the inhibitor sensitivity of the enzyme. Nonetheless, other concurrent modifications may be helpful in stabilizing the activated form of the enzyme.

In addition to sulfenic acid formation, AR cysteines could also be glutathiolated (49), or S-nitrosylated (31). A glutathiolated form of AR has been detected in cells treated with NO donors (50). However, addition of glutathione to the active site cysteine inhibits AR activity (49); therefore, it appears unlikely that glutathiolation could account for ischemic activation of AR. On the other hand, nitrosylation, which leads to an increase in the catalytic activity of the enzyme (31), could in principle account for AR activation and increased sorbitol accumulation in the ischemic heart. However, the kinetic properties of the ischemia-activated enzyme are inconsistent with nitrosylation. Nitrosylation of recombinant AR by NO donors increases the Km of AR for glyceraldehyde (31), which was not the case for the activated enzyme isolated from the ischemic heart (this study). Additionally, nitrosylation is readily reversed by 1–5 mM DTT, whereas 100 mM DTT was required to fully de-activate the enzyme from the ischemic heart (5 mM DTT was ineffective (data not shown)). The high resistance of the activated protein to reduction is consistent with sulfenic acid formation which is reversed only under strong reducing conditions. That sulfenic acid formation rather than S-nitrosylation leads to ischemic activation of AR is further supported by the observation that the enzyme in ischemic (but not perfused) heart homogenates was inhibited by the sulfenic acid-specific reagent, dimedone. Nonetheless, we cannot rule out the possibility that in the ischemic heart, AR cysteines are first nitrosylated and then oxidized to sulfenic acids. Indeed, for matrix metalloproteinase-9, it has been shown that sulfonic acid formation is dependent upon NO generation (45), but in our work with nitrosylated AR in vitro we have found no evidence for the formation of sulfenic or sulfinic acids, although in the presence of ROS these could be generated in the ischemic heart. Further investigations are required to fully elucidate the role of NO and ROS in regulating AR activation.

Overwhelming evidence supports the view that ischemia increases ROS generation, and that oxidative stress is a significant component of myocardial ischemia-reperfusion injury (38, 51). This view is based on the observation that antioxidant interventions prevent ischemic injury. However, mild oxidative stress stimulates a variety of intrinsic antioxidant defenses. Several studies report that brief episodes of ischemia increase resistance to a prolonged ischemic insult and that this adaptation is triggered by redox changes in the ischemic heart (52). Nevertheless, the mechanisms by which non-lethal oxidative stress stimulates antioxidant defenses are not well understood. Whereas most long-term increases in antioxidant defenses are due to transcriptional up-regulation of antioxidant genes, post-translation modification leading to an increase in the activity of antioxidant proteins represents an additional mechanism for acutely enhancing resistance to oxidative injury. In this context, activation of AR during ischemia may represent one mechanism of acutely up-regulating antioxidant defenses. Furthermore, given our observation that inhibition of AR prevents activation of protein kinase C and NF-{kappa}B (18, 53–55) and abolishes ischemic preconditioning (9), we speculate that persistent activation of AR in the ischemic heart by oxidation of its cysteine residues to sulfenic acids may be required to establish a cellular phenotype permissive of survival signaling stimulated by cardioprotective interventions, such as ischemic preconditioning or increased NO generation. Alternatively, redox modification may be a significant mechanism of regulating glucose metabolism via AR. An increase in AR activity in the ischemic heart would be expected to decrease the amount of glucose available for glycolysis. As a result, AR activation could contribute to myocardial injury. This is consistent with the observations of Ramasamy and colleagues (13–15) suggesting that inhibition of AR prevents tissue injury in ex vivo models of low-flow ischemia. The role of glycolysis during ischemia is, however, complex and although glycolysis is needed to generate ATP in the ischemic heart, accumulation of glycolytic products causes tissue injury (56). Indeed, the cardioprotective effects of early ischemic preconditioning are associated with a decrease in glycolysis (57), which may be a reflection of increased glucose utilization by activated AR. Clearly, the role of AR in myocardial metabolism during ischemia needs to be investigated in greater detail to fully assess the functional significance of the activation of this enzyme by redox modification of its cysteine residues.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL55477, HL59378, HL65618, and ES11860. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 502-852-5966; Fax: 502-852-3663; E-mail: aruni{at}louisville.edu.

2 The abbreviations used are: AR, aldose reductase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate; DTT, dithiothreitol; KH buffer, Krebs-Henseleit buffer; MPG, N-(2-mercaptoproprionyl)glycine; NEM, N-ethylmaleimide; ROS, reactive oxygen species; MALDI-TOF/MS, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry; ESI, electrospray ionization. Back



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