INTRODUCTION

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IT IS NOW WELL ESTABLISHED that free radicals are produced in the heart during ischemia (1, 15, 26, 28, 35, 36, 40) and reperfusion (5, 7, 14, 40, 46). This free radical production has been causally associated with both electrical and contractile dysfunction on reperfusion (7, 18). Although lipid peroxidation may be involved in mediating these effects, it has been suggested that sulfhydryl groups on ion-translocating proteins may be the primary targets for free radical damage (22, 31).

The reactive, short-chain aldehyde trans-4-hydroxy-2-nonenal (HNE) is formed in biological tissues during oxidative stress. It is produced as a result of -cleavage of alkoxy or peroxy radicals. Alkoxy and peroxy radicals are formed from the decomposition of lipid hydroperoxides, which are produced when membrane -6 polyunsaturated fatty acids (e.g., linoleic acid, arachidonic acid, and docosahexaenoic acid) undergo free radical-mediated peroxidation (11, 43). HNE is also produced as a result of ischemia and reperfusion in many tissues (34), including the isolated perfused rat heart (4).

HNE has a considerably longer half-life than free radical species and is capable of inhibiting the function of key enzymes such as glyceraldehyde-3-phosphate dehydrogenase (38) and ion-translocating proteins such as the sodium pump (33). Indeed, the latter has been proposed as a potential target for oxidation during cardiac reperfusion injury (17, 22). Protein function is disrupted by HNE because of its reactivity with the amino acids cysteine, histidine, and lysine. Evidence for the potential toxicity of HNE is derived from studies in which isolated rat hearts are perfused with exogenous HNE and show a resulting disturbance of coronary flow (41), GSH depletion, and contractile impairment (20). Exposure of the rat left atrium to HNE can result in a reduction of -adrenoceptor function and eventual contractile failure (16). Because HNE is produced in the heart during oxidative stress and because it disrupts cardiac function, it is possible that at least some component of the myocardial dysfunction that characterizes ischemia and reperfusion might be attributable to the production of endogenous HNE. The location of ion exchangers, pumps, and channels within the lipid environment of the membrane may make them particularly vulnerable to HNE modification because they are close to the lipid environment in which HNE concentration is likely to be highest.

The aim of the present study was to assess the extent to which HNE-modified proteins are produced in the isolated perfused rat heart during ischemia and reperfusion and to see whether this can be attenuated by an antioxidant such as MPG. This study exploits the availability of an antibody produced and characterized by Uchida et al. (39). This antibody is specific for proteins containing adducts of HNE coupled to cysteine, histidine, or lysine and has been used previously with Western immunoblotting to assess HNE protein modification in hepatocytes (39) and of glyceraldehyde-3-phosphate dehydrogenase (38). This anti-HNE antibody has also been used for immunohistochemical demonstration of the accumulation of HNE-modified proteins in atherosclerotic lesions (37).

	MATERIALS AND METHODS

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Animals. Male Wistar rats (200-250 g, obtained from BK Universal) were used throughout this study. The rats were maintained humanely and in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).

Isolated heart preparations. Animals were anesthetized with diethyl ether and injected with 200 IU of sodium heparin via the femoral vein. Hearts were rapidly excised and placed in cold (4°C) bicarbonate buffer, and the aorta was cannulated. The hearts were then perfused with bicarbonate buffer gassed with 95% O₂-5% CO₂ at 37°C (pH 7.4). Perfusion was performed in the nonrecirculating Langendorff mode at a constant pressure equivalent to 100 cmH₂O. The bicarbonate buffer contained (in mM) 118.5 NaCl, 3.1 KCl, 1.18 KH₂PO₄, 25.0 NaHCO₃, 1.2 MgCl₂, 1.4 CaCl₂ and 10.0 glucose.

Perfusion protocols. The perfusion protocols used in this study are summarized in Fig. 1. All hearts were subjected to an initial period of 20 min of aerobic perfusion. Global zero-flow ischemia was achieved by terminating perfusion. During ischemia, hearts were maintained in a thermostatically controlled chamber filled with bicarbonate buffer at 37°C. In protocol I, biopsies (~20-mg wet wt) were cut from the left ventricle of ischemic hearts (n = 5) every 5 min up to a total duration of 40 min. Protocol II was designed to assess the effect of reperfusion (20 min) after 30 min of ischemia on HNE-induced protein modification. Because in the rat heart 30 min of ischemia may result in a mixture of stunned and lethally injured cells, shorter, sublethal periods of ischemia were used in protocol III to allow any additive effects of reperfusion to be revealed. The preischemic administration of the antioxidant N-(2-mercaptopropionyl)glycine (MPG) was examined in protocols IV and V. Antioxidant solutions were prepared immediately before use by dissolving MPG to a final concentration of 1 mM in bicarbonate buffer. Hearts were perfused with this solution during the last 5 min of the initial aerobic period. In all protocols (except protocol I, in which biopsies were taken) four hearts were used per treatment group.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Perfusion protocols used in this study. In protocol I, arrows indicate times at which ventricular biopsies (20 mg) were taken. In all other protocols, arrows indicate when hearts were taken for protein analysis or were snap-frozen for immunofluorescence studies. MPG, N-(2-mercaptopropionyl)glycine; HNE, 4-hydroxy-2-nonenal.

Protein analysis. Cardiac protein was prepared by grinding tissue under liquid nitrogen and then boiling the powder in SDS sample buffer (23). SDS-PAGE was carried out using the Bio-Rad mini protean II system on 10% polyacrylamide gel with 30 µg of protein loaded per lane.

Immunofluorescence. Four perfusion sequences (shown in Fig. 1, protocol V) were used in the immunofluorescent localization of HNE-modified proteins. In this study, ischemic duration was limited to 20 min to minimize irreversible structural damage to the myocardium that might distort tissue architecture and complicate the immunohistochemical localization. Hearts were snap-frozen using liquid nitrogen and isopentane, and left ventricular tissue was cryosectioned into 6-µm sequential sections. Pretreatment of sections and immunofluorescence was carried out as previously described (25). Normal rabbit IgG (5 µg/ml) was used in each experiment as a negative control. The specific antibody binding was detected using FITC-conjugated goat anti-rabbit IgG (Sigma Chemical). Specific fluorescent staining of cardiac tissue was identified under ultraviolet light with an Olympus BH2-RFCA microscope using an oil-immersion lens (magnification ×40) and photographed. Three sections from each heart were analyzed, and two different hearts were used for each experiment.

Statistics. Results are presented as means ± SE. Differences between groups were assessed using ANOVA, followed by a t-test. Differences were considered significant at the 95% confidence level.

	RESULTS

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Effect of ischemia on HNE-protein adduct content of isolated perfused hearts. The Western blot shown in Fig. 2 demonstrates that ischemia induces an HNE-mediated, time-dependent modification of proteins in the isolated rat heart (protocol I). Each lane on the blot represents a sample from a single heart. Modification of proteins by HNE was first detectable after 5 min of ischemia and then progressively increased to reach a maximum after ~30 min. No further increase in HNE signal intensity was apparent when ischemia was extended beyond 30 min. The antibody used in these experiments recognizes all proteins containing adducts of HNE coupled to cysteine, histidine, or lysine. It is interesting to note that of the many thousands of proteins present in these whole heart homogenates, only a small number of discrete bands can be identified, suggesting that only a relatively small number of proteins (12) appear to be susceptible to modification by HNE.

View larger version (97K): [in this window] [in a new window]   Fig. 2.   Western immunoblot showing time course of HNE-adduct formation during ischemia (protocol I). Each lane represents a protein sample from a single heart. Molecular mass protein standards are shown on right.

Time course for appearance of HNE-protein adduct content in biopsy samples. Figure 3 quantifies, via digitization of Western blots (including the one shown in Fig. 2) probed with the anti-HNE antibody, the time-dependent HNE modifications that occur in the heart during ischemia. This was achieved by taking biopsies from five different hearts every 5 min during ischemia (protocol I). The graph shows a time-dependent increase in HNE-modified protein in the biopsy samples during ischemia, which reached a maximum after 30 min. The profile of HNE modification of cardiac proteins during ischemia is sigmoidal with a half-time of maximal HNE-adduct formation of 18 min. It is important to note that the extent of protein adduct formation after various durations of ischemia was not influenced by the biopsy procedure and was quantitatively comparable whether measured in the biopsy samples (protocol I) or in whole heart homogenates (protocols II-V).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Relationship between ischemic duration and quantitative change in HNE-protein adduct formation in biopsy samples (protocol I). Data are shown as means ± SE (n = 5). A representative example of a Western blot showing time course from which these data are quantified and averaged is shown in Fig. 2. The sigmoidal curve was drawn using a least-squares computer program according to the equation y = ymax/[1+(t/x)nH], where half-time for protein HNE-adduct formation (t) was estimated to be 18 min and the Hill coefficient (nH) was estimated to be 3.40.

Effect of reperfusion on HNE-protein content in isolated perfused hearts. Figure 4 illustrates the HNE-protein content of the heart after 1) 70-min aerobic perfusion (control), 2) 30-min ischemia, and 3) 30-min ischemia plus 20-min reperfusion as described in protocol II (Fig. 1). It is evident that there is very little HNE-protein adduct produced during 70-min aerobic perfusion (296 arbitrary units) but substantial production after 20-min aerobic perfusion plus 30-min ischemia (15,356 units). It is also evident that 20 min of reperfusion after 30-min ischemia does not further increase the HNE-protein content over and above that achieved during the 30-min ischemic period alone.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of ischemia (hatched bar) and ischemia plus reperfusion (filled bar) on HNE-protein adduct content in isolated perfused hearts (protocol II). Control hearts (open bar) were aerobically perfused for 70 min, and reperfusion was for 20 min. Data are shown as means ± SE. * P < 0.05 compared with control.

Effect of short periods of ischemia with or without reperfusion on HNE-protein content of isolated perfused heart. Because it is generally assumed that it is during reperfusion that the heart may be subjected to the major oxidant stress, the lack of further HNE-adduct formation on reperfusion was surprising. We therefore investigated whether the lack of further adduct formation during reperfusion was caused by the "saturation" of the system by the cumulative stresses involved in the relatively long 30-min ischemic period (see Fig. 3). This was undertaken by investigating the effects of shorter periods of ischemia (5, 10, and 15 min), in which the ischemia-induced HNE adduct formation should be submaximal, with or without 20-min reperfusion (see protocol III, Fig. 1). Figure 5 confirms the time-dependent increase in HNE-protein adduct production with increasing durations of ischemia. It also shows that, even after short periods of ischemia, reperfusion does not further increase the production of the HNE adducts.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of reperfusion on HNE-protein adduct formation after short (5, 10, or 15 min) periods of ischemia (protocol III). Hearts were homogenized either at end of ischemia or after 20-min reperfusion. Data are plotted as means ± SE. There were no differences between ischemic and reperfusion samples at any given ischemic duration (ns).

Effect of MPG on HNE-protein adduct content of isolated heart. Figure 6 shows that the antioxidant MPG (1 mM given 5 min before the onset of ischemia, see protocol IV, Fig. 1) reduced, by ~75%, the formation of HNE-modified proteins during 30-min ischemia. This MPG-mediated attenuation in HNE-protein adduct formation during 30-min ischemia was unchanged by 20-min reperfusion in the absence of MPG and by 20-min reperfusion in the presence of MPG.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of antioxidant MPG on HNE-protein formation during ischemia and reperfusion (protocol IV). Control hearts (open bars) were analyzed after 70 min of aerobic perfusion. Hearts were rendered ischemic for 30 min (hatched bars) or made ischemic for 30 min and reperfused for 20 min (filled bars) before analysis for HNE-protein adducts. MPG (1 mM) was included either during ischemia alone or during ischemia and reperfusion as indicated. Data are shown as means ± SE. * P < 0.05 compared with respective control value in absence of MPG.

Immunolocalization of HNE-protein adducts. Figure 7 shows immunohistochemical localization of HNE-modified proteins in heart cryosections. It is evident that ischemia (Fig. 7B) caused a major increase in HNE signal compared with control aerobic tissue (Fig. 7A). The majority of the HNE adduct signal was localized in the cell membrane with little change in nonspecific labeling. Some intracellular localization was also noted, possibly associated with the myofilaments. Reperfusion (20 min, Fig. 7C) resulted in a slight decline in the HNE signal compared with that from the ischemic heart. Sections from the heart made ischemic in the presence of MPG (Fig. 7D) showed a marked attenuation in the formation of HNE-modified proteins.

View larger version (112K): [in this window] [in a new window]   Fig. 7.   Immunohistochemical analysis of HNE-modified proteins in cryosections from hearts aerobically perfused for 60 min (A), hearts subjected to 20-min ischemia (B), hearts subjected to 20-min ischemia and 20-min reperfusion (C), and hearts made ischemic for 20 min in presence of antioxidant MPG (D; protocol V).

	DISCUSSION

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Ischemia and HNE-protein adduct formation in isolated perfused hearts. Our studies demonstrate that in the isolated perfused rat heart, ischemia leads to the modification of proteins by the highly reactive lipid peroxidation product HNE. Because HNE can only be formed by the interaction of free radicals with lipids (11), it can be concluded that ischemia promotes the formation of free radicals that, in turn, lead to structural modifications of cardiac proteins. Because protein modification of this type can lead to loss of function (33, 38), this cytotoxic metabolite might represent a further factor that contributes to the loss of cellular homeostasis during ischemia. The concept that HNE may induce detrimental modifications of critical membrane proteins during ischemia is supported by reports that mechanical and electrical dysfunction can be induced by the exogenous application of pathophysiologically relevant concentrations of HNE (3, 16).

Time course of HNE protein modification. The present studies have shown that the HNE protein modifications occur in a time-dependent manner during the first 30 min of ischemia. It is possible that the time course of HNE protein modification is directly related to the rate of ischemia-induced antioxidant depletion and/or free radical production. In this connection, there is evidence in the literature for antioxidant depletion (10) and free radical formation (26, 28, 35) in the heart during a similar ischemic duration. In particular, the concentration of GSH in the heart falls rapidly and substantially within minutes of the onset of ischemia. Because GSH is a major cellular antioxidant and scavenger of free radicals, its loss may well leave the heart vulnerable to lipid peroxidation, HNE formation, and protein modification. Because GSH can also scavenge reactive, electrophilic cytotoxins such as HNE directly, its depletion may further promote the damaging effects. The enzymatic coupling of GSH to HNE is also an efficient detoxification mechanism in the heart (20, 21) that might be compromised during ischemia. It is therefore possible that the modifications of proteins by HNE during ischemia may occur as an indirect consequence of the fall in GSH concentration that accompanies ischemia. This loss of GSH during ischemia occurs because the biochemical pathway required to reduce GSSG and restore GSH is dependent on ATP, which itself becomes depleted during the first 30 min of severe ischemia (45). Depletion of the myocardial pool of GSH has been suggested to diminish the ability of the heart to recover from ischemia (44), even if the ischemic duration is short (6). Additionally, it has been shown that the GSH status of the heart is critical for the recovery of contractile function during reperfusion (8, 9). Our studies now raise the possibility that the loss of GSH during ischemia may compromise survival and recovery because of the loss of ability to limit the accumulation of cytotoxic species such as HNE that are then able to damage key cellular proteins.

Reperfusion and HNE-protein adducts. Surprisingly, our results indicate that HNE-mediated modifications that occur during ischemia are neither reversed nor exacerbated by reperfusion and that reperfusion has little or no effect on the HNE-protein content of the heart. The failure of reperfusion to reduce the tissue content of HNE-protein adducts formed during ischemia might be explained by the findings of Friguet et al. (12), who demonstrated that HNE-modified proteins are not degraded at any greater rate than native proteins and therefore could persist in tissue for some time. Although reperfusion failed to reduce tissue HNE-protein adduct content, it might have been expected to increase the content, because reperfusion is associated with a burst of oxidant stress that might be expected to promote further protein modification. In this connection, Blasig et al. (4) claimed that reperfusion resulted in increased "free" HNE release into the coronary effluent of perfused rat hearts. Although at first sight this might support the concept of increased HNE production during reperfusion, it is important to note that Blasig et al. did not measure HNE production during ischemia, and therefore the peak of HNE release observed during reperfusion may simply reflect the washout of HNE accumulated during the preceding ischemia.

Saturation of HNE-protein adduct production? As discussed in Reperfusion and HNE-protein adducts, reperfusion is known to be associated with increased free radical production (5, 14, 29) and oxidant stress. It was therefore surprising that it was during ischemia and not reperfusion that the HNE-protein modifications occurred. The rapid depletion of cellular antioxidants during ischemia may, however, leave the cell vulnerable to a low level of oxidant stress that persists during ischemia. This may lead to a relatively rapid saturation of all of the available sites for HNE modification. This would be consistent with our observation that HNE-protein adduct formation appears to saturate after ~30 min of ischemia (Fig. 3). To test whether the saturation of available HNE-modifiable sites occurs and effectively masks any reperfusion-induced HNE-protein adduct production, we undertook experiments with reperfusion after short periods of ischemia. After ischemic durations in which adduct formation was not saturated, however, reperfusion induced no further HNE-protein adduct production.

MPG and HNE-protein adduct content. Because HNE is a free radical end product that freely reacts with sulfydryl-containing compounds, we investigated the ability of the thiol-containing antioxidant MPG to inhibit the formation of HNE-modified proteins. MPG was particularly effective at inhibiting the production of the HNE-protein adducts. The efficacy of MPG may be related to its ability to terminate free radical chain reactions and/or to directly sequester free HNE. Previous studies showed that MPG (24, 27), as well as other antioxidants (19, 30), can protect the ischemic-reperfused heart. In light of our results, it is possible that the protective effects of antioxidants, when administered before ischemia and reperfusion, may involve limiting the generation of the reactive lipid peroxidation product HNE or scavenging HNE after its production during ischemia.

Immunolocalization of HNE-protein adducts. In the present studies, subcellular immunohistochemical analysis of cryosections showed that 20 min of ischemia resulted in a large increase in binding of the anti-HNE antibody. The HNE concentration at its site of production, the lipid membrane, may be particularly high [concentrations of 4-10 mM were reported by Benedetti et al. (2)], and, consequently, membrane-associated proteins may be particularly susceptible to modification. The increased HNE immunofluorescence observed during ischemia was primarily localized to the sarcolemma, suggesting that the membrane is indeed the major site of formation of HNE-modified proteins. The HNE-reactive amino acids (cysteine, histidine, and lysine) are often critical in the functional mechanism of ion transporters, and thus modification of these groups by HNE may have detrimental effects on ion homeostasis. The localization of the HNE-protein immunostaining to the plasma membrane provides additional evidence for the hypothesis that oxidant stress in the heart results in the dysfunction of important cardiac ion-translocating proteins (17, 18). Indeed, it has been shown in myocytes that a key ion translocator, the Na⁺-K⁺ pump, is particularly susceptible to oxidant stress (17, 31, 32) and that this protein can be directly modified by HNE, resulting in pump inactivation (2). HNE immunofluorescence from ischemic heart sections also showed longitudinal striations with possible colocalization with the myofibrils, suggesting possible modification of contractile proteins. Considerable evidence now suggests that the myofilaments may also be an important target for oxidant stress during ischemia-reperfusion (13, 42), and thus myofilament dysfunction may be related to the formation of HNE adducts with key contractile proteins.