Generation of superoxide in cardiomyocytes during ischemia before reperfusion

Sections of Emergency Medicine and Pulmonary and Critical Care, Department of Medicine, The University of Chicago, Chicago, Illinois 60637

	ABSTRACT

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Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O₂ of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 ± 0.1 to 2.3 ± 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 ± 0.1, as did the site I inhibitors rotenone (1.0 ± 0.1), amytal (1.1 ± 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 ± 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 ± 0.1) and decreased with exogenous SOD (1.1 ± 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.

reactive oxygen species; mitochondria

	INTRODUCTION

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REACTIVE OXYGEN SPECIES (ROS) have been implicated in the tissue injury that follows ischemia and reperfusion (6, 12, 19, 24, 37). Studies have suggested that a burst of ROS generation occurs during the first minutes after ischemic tissues are reoxygenated, leading to the conclusion that the return of O₂ to ischemic tissues is a critical event for the generation of ROS (1, 19, 34, 37). However, O₂ may still be present during ischemia, giving rise to the possibility that ROS generation may also occur before reperfusion (32). An increase in the generation of ROS during ischemia appears paradoxical because it requires the donation of an electron to O₂ while the lack of O₂ limits ATP production. In support of this hypothesis, studies have revealed oxidant injury and stunning in cardiac tissue during ischemia without reperfusion (2, 3). Preliminary findings from our laboratory have also revealed increased ROS generation during simulated ischemia of cultured cardiomyocytes before reperfusion (32). These preliminary studies suggested that increased ROS generation during ischemia was caused by residual O₂ and was associated with increased cell death during reperfusion. If this is true, ROS generation during ischemia could have important implications for ischemia-reperfusion therapies and may explain why clinical trials of antioxidants given only at reperfusion have failed to show benefit.

Multiple sources of oxidant generation could function during ischemia, although a likely source is the mitochondria (4, 29, 30, 33). Highly redox-reduced electron carriers in the respiratory chain during ischemia could directly transfer electrons to the residual molecular O₂, producing superoxide (13, 30, 33). Despite the possible importance of these ROS during ischemia, their source and handling by the ischemic cell remain unclear.

The purpose of our study was to clarify the nature of ROS generation during ischemia in terms of the source and primary species of ROS generated. Cultured chick cardiomyocytes were studied in a superfusion system that has previously demonstrated ROS generation during simulated ischemia-reperfusion (32, 34). The system allows for serial measurement of intracellular ROS using intracellular fluorescent probes while adding various inhibitors to identify the species and site of ROS generation.

	MATERIALS AND METHODS

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Cell Culture and Microscope Perfusion System

Cardiac culture preparation. The methods for preparation of embryonic ventricular cardiac myocyte preparation have been described previously (34). Heart ventricles from 10-day-old chick embryos were dissected, minced, and enzymatically dispersed with 0.025% trypsin (GIBCO, New York, NY), yielding 5-6 × 10⁵ cells/embryo. Suspended cells (0.7 × 10⁶) were pipetted onto coverslips, allowed to attach to the surface, incubated, and grown into contractile layers. Synchronous contractions were present by the third day in culture. Cultures were checked for nonmuscle cell contamination (>95% of cells stain with anti-myosin heavy chain monoclonal antibodies, CCM-52). Experiments were performed with 3- to 5-day cardiac cell cultures, at which point viability exceeded 99%.

Perfusion chamber. Coverslips with synchronously contracting cells were placed inside a Sykes-Moore chamber (1.2 ml; Bellco Glass, Vineland, NJ). The chamber and inflow tubing were maintained at 37°C. Flow rate (0.25 ml/min), pH, and O₂ tension (PO₂) of the perfusate were controlled. Hypoxic conditions were verified with an optical method of phosphorescence quenching (Oxyspot; Medical Systems, Greenvale, NY; see Ref. 18). An extracellular Pd-porphyrin dye (10 µM) bound to albumin was added to the perfusate, and the PO₂-dependent phosphorescence decay was recorded in response to pulsed excitation light. Perfusion with hypoxic medium resulted in measured PO₂ values of 3-5 Torr within the chamber during steady-state perfusion. Tubing supplying perfusate to the chamber was of low O₂ permeability, constructed of PharMed (Cole-Parmer Instrument, Chicago, IL) or stainless steel to minimize O₂ leaks.

Perfusion media composition. Standard perfusion media consisted of oxygenated balanced salt solution (BSS) with a PO₂ of 149 Torr, PCO₂ of 36 Torr, pH of 7.4, K⁺ concentration of 4.0 meq/l, and a glucose concentration of 5.6 mmol/l. Simulated ischemia consisted of BSS containing no glucose, with 2-deoxyglucose (20 mmol/l) added to inhibit glycolysis and a K⁺ concentration of 8.0 meq/l. This was bubbled with 80% N₂ gas and 20% CO₂ to produce a PO₂ of <5 Torr, a PCO₂ of 144 Torr, and a final pH of 6.8.

Video/fluorescent microscopy. Cells were imaged with an Olympus IMT-2 inverted-phase/epifluorescent microscope. Fluorescence was measured using a cooled Hamamatsu slow-scanning PC-controlled camera (Hamamatsu, Hamamatsu City, Japan) coupled with Image-One software (Image Pro Plus) for quantification of changes in emission fluorescence.

Measurement of intracellular oxidant generation. Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation. Dihydroethidine (DHE, 1-10 µmol/l; Molecular Probes) enters the cell and is oxidized by ROS, particularly superoxide, to yield fluorescent ethidium. Ethidium binds to DNA (Eth-DNA), further amplifying its fluorescence (5). Eth-DNA fluorescence is generally stable but can be decreased by hydroxyl radical attack (27). Thus increases in DHE oxidation to Eth-DNA (i.e., increases in Eth-DNA fluorescence) are suggestive of superoxide generation. To confirm the notion that DHE is specific for superoxide, prior works have conducted dose-response studies to verify the response of DHE to various radical species (32). We found that, whereas superoxide readily oxidized DHE in solution in concentrations below 100 µM, H₂O₂ had almost no effect in concentrations in excess of 1 mM. In addition, in our cardiomyocytes, DHE oxidation was markedly stimulated by addition of menadione plus DDC (known to generate superoxide), whereas menadiones plus superoxide dismutase (SOD, known to generate approximately equimolar H₂O₂) showed no DHE oxidation in cells, suggesting the increased sensitivity of DHE to superoxide. In addition, DHE oxidation will decrease rapidly in the presence of hydroxyl radical, a likely reason for the sudden drop in oxidation seen at reperfusion in many reports. These reported specificities of the Eth-DNA probe for different ROS have been verified in additional cuvette and chick cardiomyocyte experiments and have been described by other laboratories (32, 35).

Simulated Ischemia Protocols

For most experiments, cardiomyocytes were allowed to equilibrate for 1 h and then were exposed to 1 h of simulated ischemia (simultaneous hypoxia, hypercarbic acidosis, hyperkalemia, and substrate deprivation) followed by 30 min of reperfusion. Previous work has shown that this protocol yields ~50% cell death within 3 h after reperfusion via an oxidant-mediated injury mechanism (32, 34).

Inhibitors to Identify Sites of ROS Generation

The following metabolic inhibitors and chelators were used to identify sites of ROS generation: amytal (2.5 mM, a site I electron transport inhibitor), rotenone (10 µM, a site I electron transport inhibitor), myxothiazol (0.6 µM, a site III electron transport inhibitor), cyanide (2.5 mM, a site IV electron transport inhibitor), diphenyleneiodonium (DPI, 100 µM, an inhibitor of flavoprotein oxidases), nitro-L-arginine methyl ester [L-NAME, 200 µM, inhibitor of nitric oxide synthase (NOS)], allopurinol (100 µM, inhibitor of xanthine oxidase), apocynin (300 µM, an inhibitor of NADPH oxidases), bovine liver SOD (200 U/ml, dismutates superoxide to H₂O₂), and diethyldithiocarbamic acid (DDC, 1 mM, which chelates Cu in Cu,Zn-SOD, thereby inhibiting its function).

Data Analysis

Data were collected, and simple descriptive analyses were performed. An individual experiment (n) was the result of observations of a single field of ~500 cells on a coverslip. Replicates were performed on separate coverslips. Fluorescence measurements are listed as arbitrary units. Results are reported as means ± SE. For tests of significance, ANOVA and two-tailed paired t-tests were performed, with P < 0.05 considered to be significant.

	RESULTS

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Sources of ROS Generation During Ischemia

Previous studies using the oxidant probe DHE revealed that ROS generation increases during ischemia, but the sources of oxidant generation were not identified (32). In the present study, the site of ROS generation during ischemia was determined using metabolic inhibitors during ischemia in the presence of DHE. Cells demonstrated significant oxidant generation detected by Eth-DNA fluorescence during 1 h of ischemia. Fluorescence increased from 0.7 ± 0.1 to a peak of 2.3 ± 0.3 after 1 h of ischemia (n = 4, Fig. 1). Addition of myxothiazol (0.6 µM) to inhibit electron transport at site III significantly attenuated peak Eth-DNA fluorescence during 1 h of ischemia from 2.3 ± 0.3 in controls to 1.3 ± 0.1 in treated cells (P < 0.05, Fig. 1). The addition of site I inhibitors amytal (2.5 mM) or rotenone (10 µM) also attenuated peak Eth-DNA fluorescence during ischemia (to 1.1 ± 0.1, n = 3, P < 0.01 and 1.0 ± 0.1 n = 3, P < 0.01, respectively; Fig. 2). Addition of myxothiazol (0.6 µM) plus amytal (2.5 mM) attenuated Eth-DNA fluorescence compared with control cells (deceased to 1.3 ± 0.1, n = 3, P < 0.01) but failed to further attenuate Eth-DNA fluorescence compared with either myxothiazol or amytal alone (Fig. 3). Administration of the flavoprotein oxidase inhibitor DPI (100 µM), known to have nonspecific effects, is also reported to block electron transfer at mitochondrial site I (17). DPI significantly attenuated peak Eth-DNA fluorescence (0.9 ± 0.1 compared with 2.3 ± 0.3 in controls, P < 0.01; Fig. 4). By contrast, the site IV inhibitor cyanide (2.5 mM) failed to attenuate ROS production during ischemia (1.6 ± 0.1, n = 3 vs. 1.7 ± 0.1, n = 4 in controls; Fig. 5). Collectively, these findings suggest that ROS are generated during ischemia from the mitochondrial electron transport chain at a location distal to site I but proximal to site IV.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Oxidation of dihydroethidine during ischemia and its attenuation with myxothiazol. During a 1-h exposure to ischemia, cardiomyocytes demonstrate significant oxidant generation. Addition of myxothiazol (0.6 µM) to inhibit mitochondrial electron transport at site III significantly attenuated the oxidation of dihydroethidine. Eth-DNA, ethidium-DNA; a.u. arbitrary units. * P < 0.05 compared with control.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Oxidation of dihydroethidine during ischemia with mitochondrial site I inhibitors. Addition of site I inhibitors amytal (2.5 mM) or rotenone (10 µM) attenuated dihydroethidine oxidation during ischemia. * P < 0.05 compared with control.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dihydroethidine oxidation during ischemia with site I plus site III inhibitors. Addition of myxothiazol (0.6 µM) plus amytal (2.5 mM) attenuated oxidation of dihydroethidine but not significantly more than with either myxothiazol or amytal alone. * P < 0.05 compared with control.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of diphenyleneiodonium (DPI) on dihydroethidine oxidation during ischemia. Administration of the flavoprotein oxidase inhibitor diphenyleneiodonium (100 µM) significantly attenuated dihydroethidine oxidation during ischemia. * P < 0.05 compared with control.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of cyanide on dihydroethidine oxidation during ischemia. Administration of the site IV inhibitor cyanide (2.5 mM) failed to attenuate dihydroethidine oxidation during ischemia.

Studies have shown that NOS may generate superoxide under certain conditions (36). To test for oxidant generation from NOS during ischemia, experiments were performed with the NOS inhibitor L-NAME (200 µM), which failed to attenuate oxidant generation (1.8 ± 0.1, n = 5 vs. 1.7 ± 0.1, n = 5 in controls; Fig. 6). To determine if NADPH functions as an electron donor for oxidant generation, the NADPH oxidase inhibitor apocynin (300 µM) was studied. Apocynin failed to attenuate peak Eth-DNA fluorescence (1.7 ± 0.1, n = 3 vs. 1.7 ± 0.1, n = 5 in controls, Fig. 6). To test for oxidant generation from xanthine oxidase, the inhibitor allopurinol (100 µM) was added, which also failed to attenuate the peak Eth-DNA fluorescence (1.5 ± 0.1, n = 3 vs. 1.5 ± 0.1, n = 3 in controls; Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of nitro-L-arginine methyl ester (L-NAME) and apocynin on dihydroethidine oxidation during ischemia. Nitric oxide synthase inhibitor L-NAME (200 µM) failed to attenuate dihydroethidine oxidation. NADPH oxidase inhibitor apocynin (300 µM) also failed to attenuate dihydroethidine oxidation during ischemia.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of allopurinol on oxidation of dihydroethidine during ischemia. Xanthine oxidase inhibitor allopurinol (100 µM) failed to attenuate dihydroethidine oxidation during ischemia.

Determining Initial Species of ROS Generated During Ischemia

To test whether superoxide was the original ROS species generated, SOD, which degrades superoxide to H₂O₂, was added to the perfusate to attenuate the superoxide signal. Addition of exogenous SOD (200 U/ml) caused an attenuation of peak fluorescence during ischemia (1.1 ± 0.01, n = 3, P < 0.001; Fig. 8). In separate studies, the Cu chelator DDC (1 mM) was used to inhibit cytosolic Cu,Zn-SOD. In the presence of DDC, the degradation of superoxide in the cytosol would be suppressed, leading to an increase in Eth-DNA fluorescence if superoxide were the oxidant species generated initially. When compared with control cells (peak Eth-DNA fluorescence of 1.7 ± 0.1, n = 6), cell groups treated with DDC (1 mM) showed an increase in peak Eth-DNA fluorescence (2.7 ± 0.01, n = 3, P < 0.001; Fig. 8). These results suggest that superoxide generated from the mitochondrial electron transport chain enters the cytosol during ischemia, where it is degraded by Cu,Zn-SOD to H₂O₂.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effects of increasing or decreasing superoxide dismutase (SOD) activity during ischemia. When exogenous SOD (200 U/ml), which degrades superoxide to H2O2, was added to ischemic cells, the oxidation of dihydroethidine was attenuated. By contrast, the Cu chelator diethyldithiocarbamic acid (DDC, 1 mM), which inhibits cytosolic Cu,Zn-SOD, resulted in an increase in dihydroethidine oxidation during ischemia. * P < 0.05 compared with control.

Discussion

Our results extend previous work on the generation and metabolism of ROS during ischemia by demonstrating mitochondria function as an important source of superoxide anion generation before reperfusion. Other potential sources of ROS, such as NOS, NADPH oxidases, and xanthine oxidase, appear to contribute little to ROS generation in this model of ischemia before reperfusion.

Oxidant Generation During Ischemia

Studies have suggested that a "burst" of ROS generation occurs when molecular O₂ is returned to ischemic cells (1, 3, 12, 19, 37). However, few have suggested that ROS are generated before reperfusion because the ischemic conditions include a lowered PO₂ that might limit production of oxyradicals. However, our results suggest otherwise by demonstrating oxidant generation despite a low PO₂. Our initial study of oxidant generation during ischemia demonstrated evidence for this concept (32). ROS generation during ischemia was attributed to residual O₂ levels by showing that scavenging of the residual O₂ (from 4 Torr down to <1 Torr) paradoxically resulted in less oxidation and less cell death. The present study extends our understanding of ROS generation during ischemia by identifying the intracellular source and species of ROS generated under ischemic conditions. Although oxidant generation during ischemia may seem paradoxical, increasing evidence supports this concept. Although ischemia is associated with lowered O₂ levels, it is not normally associated with anoxia. Moreover, ischemia must be accompanied by progressive tissue hypoxia, creating an opportunity for significant ROS generation. Animal studies of tissue ischemia rarely show PO₂ levels <4 Torr, and our cell system creates an environment in that range during ischemia. Therefore, sufficient molecular O₂ is still available for the creation of superoxide during ischemia.

Mitochondria as a Source of Oxidants During Ischemia

Our data, along with the work of others (7, 9, 25), implicate the mitochondrial electron transport chain as an important source of free radicals in isolated cells. Mitochondria have been reported to generate superoxide and may release these radicals into the extramitochondrial space (22). Under normal physiological conditions, it is estimated that 2-5% of O₂ utilized by intact mitochondria is reduced by electrons that escape the electron carriers of the respiratory chain (4). However, during ischemia-reperfusion, highly reductive redox conditions increase the likelihood of electron transfer to O₂ (7, 23). The principal product is superoxide anion, which is consistent with our observed increases in oxidation of DHE, which is sensitive to oxidation by superoxide anions (32, 35).

The site of ROS generation along the electron transport chain is suggested by our results with specific mitchondrial inhibitors. Site I (amytal, rotenone) inhibition attenuated DHE oxidation, indicating that mitochondrial electron transport is involved. The site III inhibitor (myxothiazol) also attenuated ROS, but cyanide, a site IV inhibitor, failed to attenuate the oxidant signal during ischemia. All three inhibitors block mitochondrial electron transport, but only those acting upstream of the ubisemiquinone site were able to attenuate the oxidant signal, indicating that the response is not a nonspecific response to a blockage of mitochondrial phosphorylation or electron transport. These findings suggest that ubisemiquinone functions as the primary source of ROS generation during ischemia in our model. No additional effect was seen when both amytal and myxothiazol were used together, which suggests that the majority of ROS originates from site III. Although inhibitors are never completely specific, our results are consistent with work by others who have described two potential sites of superoxide generation, including the reduced flavin mononucleotide of NADH dehydrogenase in complex I (31) and the ubisemiquinone site with the cytochrome b-c₁ segment of complex III (29).

Alternative Sources for Oxidant Generation During Ischemia

Other possible sources of oxidant generation in intact hearts during ischemia include phospholipases, autooxidation of catacholamines, neutrophils, cytokines, and the inflammatory cascade (9, 13). The ROS generation seen in the present study must have originated within cardiomyocytes themselves, as our cell culture system did not contain endothelial cells, inflammatory cells, or humoral mediators found in intact organs. Within myocytes, mitochondria have most often been implicated as sources of ROS (1, 8, 13, 24). Alternative sources, such as xanthine oxidase, are active in endothelial cells and could contribute to oxidants in the intact heart, but there is little evidence that this enzyme is present in cardiomyocytes, and there is clear-cut evidence that it does not exist in chick cardiomyocytes (10, 14). Moreover, we tested for this possibility with allopurinol, which failed to attenuate ROS generation and which makes oxidant generation by that system unlikely (10). NADH-dependent superoxide formation originating from a myocardial microsomal electron transport chain has also been described (11, 21, 28). However, that system would not explain our results, given that mitochondrial inhibitors attenuated ROS generation in our study. Moreover, ROS generation by that system was shown to decrease at low PO₂, making it less likely to be a source during ischemia (20). Nevertheless, such systems could still participate in ROS generation at reoxygenation, a possibility that was not evaluated in the present study. Several other intracellular sources of oxidant generation could exist during ischemia, such as the mixed-function oxygenases. However, we would not expect these sources to be inhibited by the three mitochondrial inhibitors as seen in our cells, and so contributions from these sources would be minimal. Another potential source of oxidants, NOS, is expressed in cardiomyocytes, (15, 16) and under certain conditions can become a source of superoxide generation (26, 36). Pou et al. (26) demonstrated that L-NAME is an effective inhibitor of superoxide generation from NOS I; thus, we selected L-NAME to test NOS inhibition. Because L-NAME showed no effect on ROS generation during ischemia, it strengthens the notion that NOS does not contribute significantly to superoxide generation in our cells during ischemia. Additionally, we find that cyanide (which is also known to inhibit the heme portion of NOS) failed to attenuate ROS generation in our model, making the enzymatic generation of nitric oxide or superoxide unlikely from NOS in our cells during ischemia. Some investigators have suggested that nonenzymatic generation of nitric oxide could occur during ischemia (38). However, our results show a nearly complete attenuation of the oxidation signal with mitochondrial inhibitors and therefore do not support a major role for nonenzymatic generation during ischemia. Although we cannot rule out a small contribution of nitric oxide in the oxidant stress seen during ischemia, our data suggest that superoxide is the initial species, and a significant contribution by nitric oxide seems less likely.

Superoxide as Primary Species of Initial ROS

Our observation that Eth-DNA fluorescence increased with DDC, which acts to chelate Cu ions and inactivate cytosolic Cu,Zn-SOD, further suggests that superoxide is the specific radical species responsible for oxidation of our fluorescent probe. This conclusion is supported by the observation that SOD administered exogenously also attenuated the ROS signal, because SOD should dismutate superoxide to H₂O₂ and attenuate its signal. However, these data do raise the question of why such an efficient enzyme as native SOD does not completely metabolize the extra superoxide generated during ischemia. It seems possible that ischemia may lead to the inhibition of SOD, as ischemia seems to act in a similar fashion as DDC with respect to superoxide.

Clinical Implications

The finding that oxidant generation increases during ischemia provides an alternative explanation, other than ATP depletion, as to why lengthening ischemia increases cell death. Progressive ROS generation during ischemia would also explain why clinical trials using antioxidants have failed to show clear benefit when treatments are administered after the ischemic event. It would further suggest a mechanism for the observation that animal and human studies have required pretreatment with an antioxidant to show protection, as pretreatment with an antioxidant would make the agent available active during ischemic ROS generation. Additional understanding of the role of ischemic oxidant generation may be vital to the development of appropriate treatment strategies for ischemia and reperfusion.

	ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-32646, HL-35440, and HL-03459.

	FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. B. Becker, Section of Emergency Medicine, MC5068, The Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: lbecker{at}medicine.bsd.uchicago.edu).

Received 18 May 1999; accepted in final form 27 July 1999.

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