Published online before print September 5, 2008, doi: 10.1161/CIRCRESAHA.108.180869
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© 2008 American Heart Association, Inc.
Cellular Biology |
Reactive Oxygen Species Production in Energized Cardiac Mitochondria During Hypoxia/Reoxygenation
Modulation by Nitric Oxide
From the Cardiovascular Research Laboratory, Departments of Medicine (Cardiology) and Physiology, David Geffen School of Medicine, University of California, Los Angeles.
Correspondence to Paavo Korge, PhD, Department of Physiology, 3641 MRL Bldg, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095. E-mail pkorge{at}mednet.ucla.edu
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Abstract |
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Mitochondria are an important source of reactive oxygen species (ROS), implicated in ischemia/reperfusion injury. When isolated from ischemic myocardium, mitochondria demonstrate increased ROS production as a result of damage to electron transport complexes. To investigate the mechanisms, we studied effects of hypoxia/reoxygenation on ROS production by isolated energized heart mitochondria. ROS production, tracked using Fe2+-catalyzed, H2O2-dependent H2DCF oxidation or Amplex Red, was similar during normoxia and hypoxia but markedly increased during reoxygenation, in proportion to the duration of hypoxia. In contrast, if mitochondria were rapidly converted from normoxia to near-anoxia ([O2], <1 µmol/L), the increase in H2DCF oxidation rate during reoxygenation was markedly blunted. To elicit the robust increase in H2DCF oxidation rate during reoxygenation, hypoxia had to be severe enough to cause partial, but not complete, respiratory chain inhibition (as shown by partial dissipation of membrane potential and increased NADH autofluorescence). Consistent with its cardioprotective actions, nitric oxide (
O) abrogated increased H2DCF oxidation under these conditions, as well as attenuating ROS-induced increases in matrix [Fe2+] and aconitase inhibition caused by antimycin. Collectively, these results suggest that (1) hypoxia that is sufficient to cause partial respiratory inhibition is more damaging to mitochondria than near-anoxia; and (2) O suppresses ROS-induced damage to electron transport complexes, probably by forming O-Fe2+ complexes in the presence of glutathione, which inhibit hydroxyl radical formation.
Key Words: mitochondria • reactive oxygen species • hypoxia/reoxygenation • nitric oxide
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Introduction |
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Reactive oxygen species (ROS) generated during prolonged ischemia and subsequent reperfusion are known to contribute to ischemia/reperfusion injury (reviewed elsewhere1), although, paradoxically, transient ROS generation is also an essential trigger for cardioprotection by ischemic (IP) and pharmacological preconditioning (reviewed elsewhere2). In cardiac cells, mitochondria are the major source of ROS, although intracellular NADPH oxidase, xanthine oxidase, monoamine oxidase, etc, may become important ROS sources under pathophysiological conditions. Mitochondria energized with physiologically relevant NADH-related substrates produce very little ROS, unless other factors, such as respiratory chain inhibitors (reviewed elsewhere3) or mitochondrial ATP-sensitive K channel activators,4 are also present. Experiments with isolated cardiac myocytes have demonstrated that hypoxia increases ROS production and implicate mitochondria as the major source.5,6 ROS are known to damage electron transport complexes,7 potentially leading to a positive-feedback cycle during ischemia/reperfusion or hypoxia/reoxygenation, in which increased ROS toxicity impairs respiration, leading to further increases in ROS production, further respiratory dysfunction, and so forth. If this hypothesis is correct, then anoxia should be less effective in supporting this positive-feedback cycle, because ROS production requires the presence of some O2. To test the importance of hypoxia versus near-anoxia on ROS production, we compared ROS production by isolated energized cardiac mitochondria exposed to graded levels of hypoxia, followed by reoxygenation. We find that ROS production, measured from Fe2+-dependent H2DCF oxidation rate, is markedly increased after reoxygenation preceded by hypoxia, but not near-anoxia ([O2], <1 µmol/L).
A second goal of this study was to test how nitric oxide (O) impacts hypoxia-induced ROS production, because O plays an important role in redox-based cell signaling and is cardioprotective against ischemia/reperfusion injury.8 Both ROS and O interact with thiol groups, and O also binds Fe2+ with high affinity, preventing its participation in hydroxyl radical formation from H2O2 by the Fenton reaction. Consistent with this idea, we find that O markedly suppresses reoxygenation-induced H2DCF oxidation, ROS-induced matrix Fe2+ increase, and aconitase inactivation, which may contribute to its cardioprotective actions.
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Materials and Methods |
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This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Washington DC, 1996). All procedures were approved by UCLA Animal Research Committee. All measurements were carried out using a Fiber Optic Spectrofluorometer (Ocean Optics) in a partially open continuously stirred cuvette at room temperature (22 to 24°C). Mitochondria were isolated from rabbit hearts as described previously9 and added (0.5 to 1.0 mg/mL) to incubation buffer containing 100 mmol/L KCl, 10 mmol/L Hepes, pH 7.4 with Tris, followed by addition of 2.5 mmol/L potassium phosphate (Pi) and substrates as indicated. In some experiments, ADP was added in combination with hexokinase, glucose, and MgCl2 to ensure a continuous ADP load to mitochondria, similarly to that in active cardiac cells.
O2 delivery was regulated by adjusting stirring speed (settings 1 to 10, corresponding to 60 to 1100 rpm). The effect of stirring speed on the rate of O2 diffusion into buffer, and how this was balanced by O2 consumption by respiring mitochondria to regulate buffer [O2] levels, is shown in the online data supplement, available at http://circres. ahajournals.org. Alternatively, O2 delivery could be increased rapidly and significantly by injecting a small amount of compressed air into the buffer.
Mitochondria O2 consumption was measured continuously by monitoring buffer O2 content via a fiber optic oxygen sensor FOXY-AL300 (Ocean Optics). The O2 sensor responded to O2 changes at the level of >1 µmol/L (=0.1 kPa=0.75 mm Hg) (see the online data supplement).
Mitochondrial membrane potential (
m) was estimated using tetramethylrhodamine methyl ester (TMRM) (200 nmol/L) in the buffer solution.9
ROS production was monitored from reduced dichlorofluorescin (H2DCF) oxidation (excitation/emission, 490/525 nm) after incubating mitochondria with H2DCF diacetate (10 µmol/L) and washing away extramitochondrial dye. Alternatively, 
production was measured using MitoSOX red (excitation/emission, 510/580 nm).10 H2O2 release from mitochondria was measured using Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen) (excitation/emission, 560/590 nm) (see Figure IV in the online data supplement).
Mitochondrial NADH autofluorescence was recorded at excitation/emission (340/460 nm) wavelengths. The NADH signal was calibrated by making mitochondria anoxic with N2 to fully reduce NAD and subsequently adding O2 and FCCP to fully oxidize NAD.
Mitochondrial iron uptake or release of bound iron in the matrix was determined by monitoring quenching of Phen Green or calcein fluorescence by chelatable iron.11 Mitochondria were incubated with 10 µmol/L Phen Green FL or 5 µmol/L calcein-acetoxymethyl ester (calcein-AM) for 15 minutes at room temperature and washed, and fluorescence was measured at 490 nm excitation/520 nm emission. "Dequenching" was accomplished with 200 µmol/L 1,10-phenanthroline.
Aconitase activity was determined by an increase in NADPH fluorescence after adding reaction mixture and permeabilizing inner membrane with alamethicin, as described.12
Changes in buffer O level released from diethylamine NONOate sodium salt (DETA-NONO) or S-nitroso-N-acetylpenicillamine were monitored electrochemically using a O electrode (World Precision Instruments, Sarasota, Fla).
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Results |
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Graded Hypoxia in Isolated Mitochondria
We hypothesized that when hypoxia becomes severe enough to limit electron transport rate at complex IV (ie, near the Kd of cytochrome c oxidase for O2), increased
production at complexes I and III may result.3 To create a controllable level of hypoxia in a range sufficient to cause respiratory inhibition, we carefully balanced O2 supply rate against O2 consumption rate by isolated mitochondria in a cuvette. Figure 1A shows simultaneous traces of buffer [O2] and TMRM fluorescence measuring 
m in isolated mitochondria energized with malate and glutamate. When added to a cuvette equilibrated with room air (pO2=160 mm Hg or [O2]=220 µmol/L), mitochondria gradually depleted [O2] to very low levels. m remained fully polarized, consistent with the ability of mitochondria to downregulate O2 consumption to match supply at low [O2].13 However, when the speed of the stirring bar was lowered from 7 to 6.5 to reduce O2 diffusion into the cuvette, m depolarized slightly and stabilized at a new level. Further reductions in stirring speed caused graded m depolarization, which reversed immediately when the stirring speed was increased back to 7. Replacing air with N2, on the other hand, depolarized m rapidly and completely.
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To document that hypoxia sufficient to depolarize m was attributable to respiratory inhibition, we also measured NADH autofluorescence (Figure 1B). NADH fluorescence increased significantly when O2 was depleted to the level causing partial m depolarization. Further decreases in O2 supply by decreasing the stirring speed increased NADH fluorescence reversibly. The maximum value of NADH fluorescence was ascertained by replacing air with N2, and the minimum value by adding FCCP in the presence of O2 (Figure 1B).
These findings demonstrate that the level of hypoxia to which isolated mitochondria are subjected can be finely regulated over a range causing graded respiratory inhibition, as manifested by partial m depolarization and increased NADH levels.
ROS Production During Hypoxia/Reoxygenation Versus Anoxia/Reoxygenation
Our next goal was to examine how graded hypoxia versus anoxia affects ROS production by isolated mitochondria. As described in detail in the online data supplement, we found that and subsequent H2O2 production in isolated mitochondria is severely underestimated by H2DCF oxidation rate unless Fe2+ is added, so that its rapid uptake into the matrix by iron transporters generates 
H radicals and/or other reactive intermediates associated with Fenton chemistry to catalyze H2DCF oxidation. Using this method, we compared how substrates and [O2] affected mitochondrial ROS production. In Figure 2A, isolated mitochondria loaded with H2DCF were energized with malate/glutamate and incubated at the indicated stirring speeds to regulate O2 supply. The amount of mitochondria and stirring speed were selected to allow a gradual decrease in buffer [O2] to a few micromolar (see the online data supplement for calibration). Addition of 5 µmol/L Fe2+ to hypoxic mitochondria increased H2DCF oxidation rate, similar to normoxic mitochondria (supplemental Figure IC). On average, the H2DCF oxidation rate after 5 µmol/L Fe2+ was quantitatively similar for normoxic and hypoxic mitochondria (Figure 2D). In contrast to hypoxia, however, when 5 µmol/L Fe2+ was added at the same time that mitochondria were reoxygenated with compressed air, H2DCF oxidation rate markedly accelerated (Figure 2B and 2D). O2 consumption rate after reoxygenation was also decreased by 43±18% (n=7, P<0.005), indicating that the hypoxia/reoxygenation had caused persistent impairment of respiratory chain activity.
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Similar findings were obtained using Amplex Red to monitor mitochondrial H2O2 release into the buffer: H2O2 release was inhibited by decreasing and accelerated by increasing O2 supply, especially on reoxygenation. These changes were small compared to H2O2-dependent H2DCF oxidation in the matrix (supplemental Figure IVA), probably because the site of H2O2 formation is primarily in the matrix. It is also possible that, unlike liver mitochondria, catalase in the matrix of heart mitochondria obscures the ability of extramitochondrial probes such as Amplex Red to detect H2O2 release, as noted previously for diazoxide-induced H2O2 release.4
On the other hand, if buffer O2 was depleted with N2 before adding mitochondria, addition of 5 µmol/L Fe2+ initiated slow H2DCF oxidation (Figure 2C), possibly because of the small amount of O2 added with the mitochondria or to the reaction of thiyl radicals with H2DCF in the absence of O2 (supplemental Figure III). With full oxygenation, H2DCF oxidation rate increased modestly but to a much lesser extent than in mitochondria exposed to hypoxia/reoxygenation (Figure 2D). These findings indicate that hypoxia at a level causing partial respiratory inhibition primes mitochondria to produce excessive ROS on reoxygenation, whereas total anoxia does not.
We also studied ROS production by succinate-energized mitochondria, which consume O2 at a higher rate and can therefore deplete buffer oxygen more rapidly and completely, even in state 2. Figure 3A shows that when isolated mitochondria energized with succinate were added to buffer equilibrated with room air, they depleted O2 much more rapidly and completely than an equivalent amount of mitochondria energized with complex I substrates (compare the rates of O2 depletion in Figure 3A to Figures 1A, and 2A, and 2
B). The final level of O2 depletion was below the O2 electrode sensitivity (<1 µmol/L), as seen at the end of the trace in Figure 3A when room air was replaced with N2. At this near-anoxic level, H2DCF oxidation rate increased modestly. As shown in the online data supplement, these changes are attributable to H2DCF oxidation by thiyl radicals under anoxic conditions, which is masked in the presence of O2.14 Consistent with this interpretation, the increase in H2DCF oxidation rate was inhibited by transient reoxygenation with an air bolus and then resumed after buffer O2 was again depleted. Under these conditions, addition of Fe2+ had little effect on H2DCF oxidation rate (Figure 3A). Moreover, when Fe2+ was added at the time of reoxygenation, the increase in H2DCF oxidation rate was also very modest (Figure 3B). The average changes in H2DCF oxidation rate are summarized in Figure 3D. Note that compared to the same amount of identically H2DCF-loaded isolated mitochondria energized with complex I substrates, the increase in H2DCF oxidation rate in succinate-energized isolated mitochondria after reoxygenation in the presence of Fe2+ was markedly reduced (compare to Figure 2D). In all of the succinate experiments, the standard amount of mitochondria was sufficient to rapidly deplete extramitochondrial O2 level below the sensitivity of the O2 electrode (near-anoxia), resulting in rapid m depolarization (data not shown). Thus, the time window during which isolated mitochondria were exposed to a level of hypoxia sufficient to partially inhibit respiratory chain activity, but not yet at the near-anoxia level associated with complete m dissipation, was relatively short. After reoxygenation, mitochondrial O2 consumption rate showed no signs of respiratory chain inhibition, averaging 90±3% of the prehypoxic rate.
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We postulated that the lesser ROS production on reoxygenation in succinate-energized mitochondria compared to complex I–energized mitochondria might be attributable to their higher rate of O2 consumption causing more severe O2 depletion. To test this possibility, we added half the amount of succinate-energized mitochondria to the cuvette, to avoid severe O2 depletion and marked m dissipation (Figure 3C). The thiyl radical-related increase in H2DCF oxidation rate was avoided (consistent with hypoxic rather than near-anoxic conditions), and addition of Fe2+ at the time of reoxygenation now markedly accelerated H2DCF oxidation rate (Figure 3C and 3D) to a level comparable to complex I–energized mitochondria (Figure 2D), when corrected for the lesser amount of mitochondria.
Together, these findings demonstrate that hypoxia sufficient to cause partial respiratory chain inhibition results in significant Fe2+-dependent ROS production on reoxygenation, whereas exposure to an equivalent period of anoxia or near-anoxia does not have this effect.
Nitric Oxide Inhibits Fe2+-H2O2–Induced H2DCF Oxidation After Hypoxia/Reoxygenation
O interacts with ROS signaling and, when given at the time of reperfusion, is known to protect the heart from ischemia/reperfusion injury (reviewed elsewhere15). O also protects cells against damaging effects of ROS,16 and 1 possible mechanism may be inhibition of the Fenton reaction17 caused by O binding to Fe2+ coordination sites.18 We therefore tested the effects of O on hypoxia-induced ROS production. In Figure 4A, complex I–energized mitochondria depleted buffer [O2] to a low level, after which, they were challenged with reoxygenation and 3 Fe2+ pulses, which significantly and progressively increased in H2DCF oxidation rate. When 10 µmol/L DETA-NONO was added just before reoxygenation, however, the increase in Fe2+-dependent H2DCF oxidation was markedly suppressed (Figure 4B). The middle O electrode trace in Figure 4B documents that DETA-NONO spontaneously released O, which was accompanied by transient inhibition of mitochondria O2 consumption in the upper trace. The finding that the third Fe2+ pulse was delivered after buffer O level had decreased to back to 0, but H2DCF oxidation rate remained low, may also indicate some suppression of ROS production by O at the level of respiratory complexes. Figure 4C summarizes the average increase in H2DCF oxidation rate after the first, second, and third Fe2+ additions, illustrating the marked suppression by pretreatment with DETA-NONO.
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Nitric Oxide Attenuates ROS-Induced Increases in Matrix [Fe2+] and Depression of Aconitase Activity
In isolated heart, a substantial portion of ischemia/reperfusion damage is thought to be related to free radical production, catalyzed by Fe2+ with open coordination sites (required for Fenton-based H generation19).1 In calcein-loaded mitochondria exposed to hypoxia-reoxygenation, however, we did not detect significantly increased calcein quenching, as an indicator of increased matrix Fe2+. To test the hypothesis that O inhibits the Fenton reaction17 by binding to Fe2+ coordination sites exposed by ROS,18 we needed a more robust method to generate ROS and, so, used antimycin to increase H2O2 production in energized mitochondria.
Figure 5A shows that when energized calcein-loaded mitochondria were exposed to antimycin to cause a sustained increase in H2O2 production (see supplemental Figure IVB), calcein fluorescence was progressively quenched. The quenching was attributable to iron release, because it was reversed with the iron chelator phenanthroline. In Figure 5B, the O donor DETA-NONO was added after antimycin. Whereas O (middle trace) remained elevated, the quenching of calcein fluorescence ceased and then subsequently resumed at a slower rate after O levels had returned to 0 and O2 consumption increased. These changes are consistent with O binding to Fe2+ coordination sites, preventing interaction with calcein and subsequent quenching. Because evidence indicates [4Fe-4S] clusters in aconitase (which are required for aconitase activity) are a major source of chelatable iron released exposure to H2O2 in vitro,20 as well as during ischemia/reperfusion,21 we examined the effects of anti-mycin on aconitase activity. Figure 5D shows that anti-mycin–induced ROS significantly inhibited aconitase activity, which was attenuated by DETA-NONO. These results show that O binding to iron can suppress the ability of endogenously generated H2O2 to oxidize [4Fe-4S] clusters, release iron, and inhibit aconitase activity.
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Discussion |
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In this study, we examined the effects of hypoxia/reoxygenation and near-anoxia/reoxygenation on ROS production by isolated mitochondria and characterized a mechanism by which
O protects against ROS-induced damage to electron transport complexes, possibly by forming dinitrosyl-dithiolato–Fe complexes in the presence of Fe2+ and matrix glutathione (GSH).22 Our major conclusions are as follows: (1) matrix ROS production is similar during hypoxia as during normoxia, despite decreased O2 availability in the former situation; (2) following hypoxia, reoxygenation results in a marked increase in ROS production, which is associated with reduced O2 consumption reflecting electron transport dysfunction; (3) for reoxygenation to cause a marked increase in ROS production, the degree of preceding hypoxia must be severe enough to cause partial respiratory inhibition, as reflected by modest m dissipation and increased NADH autofluorescence; (4) in contrast, following anoxia or near-anoxia severe enough to induce major m dissipation, reoxygenation does not increase ROS to nearly the same extent, and O2 consumption returns to preanoxic levels, indicating preserved electron transport function; and (5) the increase in Fe2+-H2O2–dependent H2DCF oxidation after hypoxia/reoxygenation is persistently attenuated by O, most likely as a result of O binding to free coordination sites of iron, with subsequent inhibition of hydroxyl radical formation by the Fenton reaction.
The role of partial, in contrast to nearly complete, m dissipation in accelerating production on reoxygenation deserves further comment. In succinate-energized mitochondria, in which production by reverse electron transport requires a maintained proton gradient, m dissipation inhibits reverse electron transport, thereby suppressing production. In complex I–energized mitochondria, however, reverse electron transport should not play a significant role in generation,3 so full m dissipation during near-anoxia may not be the protective mechanism. It is more likely that production is just too severely limited by the near absence of O2 to impair electron transport.
In general, 2 conditions are required for "primary" ROS () production by the respiratory chain: reduced electron transport centers (in complexes I or III) and the presence of O2 to allow 1 electron reduction to . A sustained O2 supply to mitochondria with inhibited respiratory chain promotes a large increase in ROS production, which can further damage electron transport.7 Thus, our findings are consistent with a scenario in which partial respiratory inhibition during hypoxia maintains significant ROS production in the matrix despite reduced O2 availability, causing progressive and persistent electron transport dysfunction, such that when reoxygenation delivers O2 in excess, ROS production markedly accelerates. In general these results support conclusions by Schumacker and colleagues23 that were made by using cultured cardiomyocytes and novel redox-sensitive fluorescence resonance energy transfer probes. It could be argued that in our experimental setup, hypoxia is not really uniform, because isolated mitochondria are intermittently exposed to high O2 when they swirl near the air-water interface at the top of the cuvette. However, this same process would have occurred in succinate-energized mitochondria, yet they did not show the marked increase in H2DCF oxidation on reoxygenation, unless the concentration of mitochondria in the cuvette was decreased. This suggests that O2 gradients in the cuvette are unlikely to account for our findings, although we cannot exclude the possibility that ROS generation via succinate-mediated reverse electron transport is inhibited because of severe m depolarization.
Our observation that anoxia or near-anoxia did not result in comparably high ROS production or persistent respiratory suppression on reoxygenation suggests that the ongoing ROS production during hypoxia plays a key role in impairing the electron transport chain. Complex I is the most sensitive to ischemia/reperfusion injury24 and also contains at least nine Fe/S clusters that are used to transfer electrons between active sites. Interestingly, these clusters are not sensitive to exogenous H2O2,25 suggesting that generation of in the immediate vicinity of Fe-S clusters in complex I may be required to disrupt function during ischemia.7 A highly localized interaction might also account for our inability to detect matrix iron release with calcein during relatively brief hypoxia/reoxygenation, in contrast to antimycin–induced ROS production into the matrix, which released iron from aconitase Fe/S clusters and inhibited aconitase activity. Regarding Fe release, antimycin may better mimic the sustained ROS production during prolonged hypoxia/reoxygenation.
It is important to distinguish the putative deleterious effects of ROS after prolonged ischemia/reperfusion from the protective effects of transient mitochondrial ROS generation during ischemic and pharmacological preconditioning.2 In the latter case, stimulation of mitochondrial ROS production, typically triggered by interventions that open mitochondrial ATP-sensitive K channels, activates downstream signaling via protein kinase pathways to protect mitochondria from injury during prolonged ischemia/reperfusion or hypoxia/reoxygenation.26 In triggering cardioprotection, one protective action of ROS signaling is to prevent subsequent excessive ROS production during prolonged ischemia/reperfusion.6 The mechanisms by which ROS-mediated protein kinase signaling impacts hypoxia/reoxygenation-induced ROS production in isolated mitochondria will be an interesting area for future investigation using the approaches developed in the present study.
Effects of O
O administered before ischemia triggers cardioprotection by activating mito-KATP channels via protein kinase G, stimulating mitochondrial ROS production to activate downstream cardioprotective pathways (reviewed elsewhere26). However, O administered at the time of reperfusion also protects the heart from injury.15 We found that an O bolus markedly suppressed the hypoxia-reoxygenation induced increase in H2DCF oxidation rate. This effect could be explained by high-affinity binding of O to Fe2+22 limiting Fe2+ oxidation by Fenton reaction.18 Binding of O to released Fe2+, or iron in [4Fe-4S] clusters exposed to H2O2, provides simplest explanation why O attenuated ROS-induced matrix iron increase and aconitase inhibition. It may seem surprising that O protects cells exposed not only to exogenous H2O2 but also to enzymatic systems generating ,16,17 because O can react with to generate peroxynitrite radicals. However, peroxynitrite rapidly reacts with GSH inside cells and mitochondria. Because relatively low [GSH] (IC50=10 µmol/L) is sufficient to prevent inhibition of complexes I to III caused by 200 µmol/L peroxynitrite,27 5 to 10 mmol/L glutathione in the matrix should effectively avoid peroxynitrite toxicity. In addition, the high catalytic activity of manganese superoxide dismutase in the matrix that keeps levels low can also limit peroxynitrite formation. Because peroxynitrite is capable of directly inducing H2DCF oxidation28 and Fe2+/H2O2-induced oxidation was inhibited by O, we assume that relatively little peroxynitrite was generated. In our experiments the inhibitory effect of O on H2DCF oxidation rate persisted after extramitochondrial O returned to 0 levels (Figure 5A), suggesting that a longer-lasting interaction, such as matrix protein S-nitrosation, may also be involved. Recent data indicates that reversible inhibition of complex I by S-nitrosation reduces ROS generation at reperfusion, with less oxidative inactivation of respiratory chain, aconitase activity, and inhibition of PTP opening.29,30 S-Nitrosation of mitochondrial thiols by O also blocks thiol oxidation, inhibiting cell death induced by GSH depletion.31 Recently, it was reported that O can directly activate mitochondrial protein kinase C
2, which inhibits permeability transition pore opening.32
Caveats in Using H2DCF As an ROS Indicator
Despite well-established experimental documentation that Fe2+ or Fe2+-containing catalysts are required to measure H2O2-induced H2DCF oxidation,28 recent analysis of literature suggests that in most studies, the role of the catalyst has been ignored,33 leading to serious underestimation of matrix ROS production. However, mitochondria have a Fe2+ carrier in the inner membrane,34 making it possible to increase matrix [Fe2+] simply by adding a known amount Fe2+. Fe2+ uptake into the matrix had no direct effect on production but promoted H2DCF oxidation by H2O2 with greatly increased sensitivity. As shown in the online data supplement and documented recently by others,14 in the absence of Fe2+, ROS production generates thiyl radicals from glutathione or cysteine, which do not oxidize H2DCF in the presence of O2 but do in its absence. In intact cells, H2DCF oxidation is also promoted by cytochrome c release, independent of ROS production.35 These technical limitations make it difficult to prove unequivocally in intact cells that mitochondria are the source of ROS, especially during hypoxia/reoxygenation and emphasize the importance of isolated mitochondria studies.
Conclusions
We have further elucidated the source of ROS production during ischemia/reperfusion by showing that in isolated mitochondria exposed to a degree of hypoxia sufficient to cause partial, but not complete, respiratory inhibition, electron transport chain is persistently depressed by ongoing matrix ROS generation. As a result, during reoxygenation, ROS production markedly accelerates, causing further suppression of electron transport, which promotes greater ROS production, etc, in a vicious cycle. O inhibits this scenario, potentially contributing to its cardioprotective effects when administered at the time of reoxygenation in intact heart.
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Acknowledgments |
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We thank Tan Duong for technical assistance.
Sources of Funding
This work was supported by NIH/National Heart, Lung, and Blood Institute grants P50 HL080111 and RO1 HL071870 and by the Laubisch and Kawata Endowments.
Disclosures
None.
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Footnotes |
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This manuscript was sent to Joseph Loscalzo, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received July 16, 2007; resubmission received June 5, 2008; revised resubmission received August 14, 2008; accepted August 21, 2008.
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