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Am J Physiol Heart Circ Physiol 284: H566-H574, 2003. First published October 31, 2002; doi:10.1152/ajpheart.00711.2002
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Vol. 284, Issue 2, H566-H574, February 2003

Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion

Leo G. Kevin1, Amadou K. S. Camara1, Matthias L. Riess1,2, Enis Novalija1,2, and David F. Stowe1,2,3,4

Anesthesiology Research Laboratories, Departments of 1 Anesthesiology and 2 Physiology, and 3 Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee 53226; and 4 Research Service, Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Reactive oxygen species (ROS) are believed to be involved in triggering cardiac ischemic preconditioning (IPC). Decreased formation of ROS on reperfusion after prolonged ischemia may in part underlie protection by IPC. In heart models, these contentions have been based either on the effect of ROS scavengers to abrogate IPC-induced preservation or on a measurement of oxidation products on reperfusion. Using spectrophotofluorometry at the left ventricular wall and the fluorescent probe dihydroethidium (DHE), we measured intracellular ROS superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) continuously in isolated guinea pig heart and tested the effect of IPC and the O<UP><SUB>2</SUB><SUP>−</SUP></UP>· scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on O<UP><SUB>2</SUB><SUP>−</SUP></UP>· formation throughout the phases of preconditioning (PC), 30-min ischemia and 60-min reperfusion (I/R). IPC was evidenced by improved contractile function and reduced infarction; MnTBAP abrogated these effects. Brief PC pulses increased O<UP><SUB>2</SUB><SUP>−</SUP></UP>· during the ischemic but not the reperfusion phase. O<UP><SUB>2</SUB><SUP>−</SUP></UP>· increased by 35% within 1 min of ischemia, increased further to 95% after 20 min of ischemia, and decreased slowly on reperfusion. In the IPC group, O<UP><SUB>2</SUB><SUP>−</SUP></UP>· was not elevated over 35% during index ischemia and was not increased at all on reperfusion; these effects were abrogated by MnTBAP. Our results directly demonstrate how intracellular ROS increase in intact hearts during IPC and I/R and clarify the role of ROS in triggering and mediating IPC.

cardiac injury; fluorescent dyes


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INTRODUCTION
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BRIEF EXPOSURE OF THE HEART to ischemia leads to a state of increased resistance to the effects of subsequent ischemia and reperfusion (I/R). This is termed ischemic preconditioning (IPC) (23) and has been demonstrated in all animal species studied (25); there are also reports of its occurrence in humans (20, 24). There is indirect evidence for involvement of reactive oxygen species (ROS) in the triggering mechanism of IPC because protection was abrogated when ROS scavengers were administered with preconditioning pulses of ischemia (1, 4, 11, 13, 28, 39); moreover, it was demonstrated that pharmacological generation of endogenous ROS (4) or administration of exogenous ROS (43, 52) can similarly lead to preconditioning. Thus ROS generated during brief I/R, in quantities below those that damage the heart, appear to activate mediators that trigger preconditioning and promote cardioprotection. Other studies show the effect of IPC to decrease release of ROS during index I/R in coronary effluent during reperfusion (12) or indirectly to decrease lipid peroxidation, also at the time of reperfusion (41).

It is well known that prolonged ("index") I/R leads to generation of ROS that play a central role in subsequent tissue damage and infarction (9). Therefore, it is possible that ROS generated briefly during the preconditioning phase mediate protection in part by reducing ROS generation during index I/R. Although it was previously held that ROS formation occurred primarily or solely at return of O2 supply at reperfusion (42), there is now convincing evidence that significant ROS generation occurs not only on reperfusion but also during ischemia, as shown in isolated cardiomyocytes (6) and in intact dogs with aspirates of stationary coronary perfusate (27). We recently showed that ischemic and pharmacological preconditioning attenuated changes in NADH (33) and reduced mitochondrial Ca2+ overload (34) during ischemia in isolated hearts. These studies suggest that preconditioning leads to improved mitochondrial energetics during the period of index ischemia that may determine global cardiac outcome after reperfusion.

A role for ROS in triggering IPC is implied by the effect of ROS scavengers given during the preconditioning phase to reverse protection afforded by IPC (1, 4, 11, 13, 28, 39). The principal cause of ROS formation during ischemic myocardial injury is believed to be mitochondrial electron transport chain (ETC) dysfunction (6). We hypothesized first that ROS generated during the ischemic pulses, and not during the reperfusion phases, is the initial trigger and that a threshold of ROS is necessary to elicit IPC. Second, we hypothesized that IPC is mediated in part by improved mitochondrial bioenergetics as demonstrated by less ROS generation, not only on reperfusion but also during index ischemia. To test these hypotheses, we developed a technique for measuring cellular ROS generation continuously in the left ventricle of intact hearts by spectrophotofluorometry using dihydroethidium (DHE), which is converted to fluorescent ethidium (Eth) by ROS.


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Langendorff heart preparation. The investigation conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health No. 85-23, Revised 1996). Approval was obtained from the Medical College of Wisconsin animal studies committee. Our Langendorff preparation was described in detail previously (2, 3, 26, 33, 34, 36, 48). Guinea pig hearts (n = 50) were perfused at constant pressure (55 mmHg) at 37°C with an oxygenated Krebs-Ringer (KR) solution of the following composition (in mM): 138 Na+, 4.5 K+, 1.2 Mg2+, 2.5 Ca2+, 134 Cl-, 14.5 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 1.2 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.1 probenecid, and 0.05 EDTA with 5 U/l insulin.

Left ventricular pressure (LVP) was measured isovolumetrically with a transducer connected to a saline-filled latex balloon placed in the left ventricle through an incision in the left atrium. The measured characteristics of LVP included diastolic and systolic LVP. Coronary inflow (CF) was measured by an ultrasonic flowmeter (T106X; Transonic, Ithaca, NY). Atrial and ventricular bipolar leads were used to measure spontaneous heart rate (HR). Coronary inflow and coronary venous Na+, K+, Ca2+, PO2, pH, and PCO2 were measured off-line with an intermittently self-calibrating analyzer (ABL 505; Radiometer, Copenhagen, Denmark). Coronary sinus PO2 (PvO2) was also measured continuously on-line with a Clark electrode (model 203B; Instech, Plymouth Meeting, PA). Percent oxygen extraction was calculated as 100 × (PaO2 - PvO2/PaO2), where PaO2 is arterial PO2 and PvO2 is venous PO2. Myocardial O2 consumption (MVO2) was calculated as CF/heart weight (g) · (PaO2 - PvO2) · 24 ml O2/µl at 760 mmHg. Cardiac efficiency was calculated as developed LVP × HR/MVO2.

Global ischemia was achieved by clamping the aortic inflow line. If ventricular fibrillation occurred on reperfusion a bolus of lidocaine (250 µg) was given immediately. At the end of 120-min reperfusion, hearts were removed, cut into six transverse sections, and stained with 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M KH2PO4 buffer (pH 7.4, 38°C) for 10 min. Infarct size was expressed as a percentage of total ventricular weight (2, 26).

Eth fluorescence to assess ROS in intact hearts. Oxidation of the fluorescent dye DHE (Molecular Probes, Eugene, OR) to Eth was used to measure ROS formation. DHE enters cells and, when oxidized by ROS, with a relative selectivity for O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (7, 46), DHE is converted to Eth; in this form, it intercalates with DNA and causes the nucleus to exhibit red fluorescence. DHE and Eth are retained within cells with minimal leakage (19). Fluorescent intensity (FI) of Eth was measured in a light-blocking Faraday cage as we described in detail previously for measurements of cytosolic Ca2+ (2, 3, 36, 48), mitochondrial Ca2+ (34), Na+ (2, 48), and NADH (33, 48) in isolated, beating hearts. Briefly, the distal end of a trifurcated fiber-optic cable (optical surface area 3.85 mm2) was placed against the left ventricular free wall through a hole in the tissue bath. Netting was applied around the heart for optimal contact without impeding relaxation. The fiber-optic cable was connected to a modified spectrophotofluorometer (SLM Aminco-Bowman II; Spectronic Instruments, Urbana, IL). Fluorescent emissions at F590 (bandwidth 4 nm) were amplified by a photomultiplier tube (700 V) and recorded after excitation with a 150-W xenon arc lamp filtered through a 540-nm monochromator (bandwidth 4 nm). The excitation wavelength penetrates the whole 4-5 mm of the guinea pig left ventricle.

In preliminary experiments (n = 4), background FI (no DHE) was determined after initial perfusion and equilibration and for each experimental protocol. All subsequent recorded values of Eth FI were adjusted for the minimal change in background fluorescence with any maneuver or drug. DHE was initially dissolved in 1 ml of DMSO containing 16% (wt/vol) Pluronic I-127 (Sigma). The perfusate contained probenecid (100 µM) to help retard cellular leakage of DHE. Hearts were loaded with 10 µM DHE in KR solution for 25 min followed by washout of residual DHE with standard KR solution for 15 min. Loading of DHE was found to increase diastolic LVP ~8% and to increase coronary flow ~10%; the effect on diastolic LVP was partly (but incompletely) reversed by washout. Flow returned to baseline values on washout. DHE loading increased FI from 0.03 ± 0.01 arbitrary units (a.u.) before loading to 2.7 ± 0.2 a.u. after washout. Each washout value was adjusted to 0 a.u. to normalize FI values for all experiments.

In preliminary experiments (n = 8), the specificity of Eth FI for ROS and the effects of possible sources of artifact including movement and induced changes in LVP, pH, and flow were examined. A rate increase to 375 beats/min by isoproterenol and a decrease to 125 beats/min by labetalol did not effect Eth FI, nor did pH in the range 6.2-8.0 or mechanically altered flow between 5 and 20 ml · g-1 · min-1. Brief pharmacological arrest induced with adenosine or lidocaine had no effect on FI. Endogenous generation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· with the mitochondrial ETC inhibitors rotenone (10 µM) and antimycin A (10 µM) was found to increase Eth FI by 21% and 88% from baseline, respectively. Exogenous administration of H2O2 (10-100 µM) did not affect Eth FI, whereas H2O2 caused significant dose-dependent increases (3-36%) in fluorescent intensity of the dye 2',7'-dichlorofluorescin (DCF). DCF is believed to have a higher specificity for H2O2 and ·OH than for O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (22, 45).

Protocol. There were four experimental groups (n = 8 animals/group) and one time-control group (n = 6) (Fig. 1). Each experiment lasted 190 min after a 30-min equilibration period, a 25-min DHE loading period, and a 15-min washout period. The untreated (time control) group was not subject to ischemia; the ischemia-control group underwent only 30-min index ischemia and 120-min reperfusion. Cardiac preconditioning consisted of two 5-min pulses of global ischemia with an intervening 5-min perfusion period and a 20-min perfusion period before the index ischemia. In the IPC+manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) group, the ROS scavenger MnTBAP (20 µM; OxisResearch, Portland, OR) was given from 5 min before the first IPC pulse, during the intervening perfusion period, and for 5 min after the conclusion of the second IPC pulse before the index ischemia. In the MnTBAP group, MnTBAP was given continuously for 25 min without IPC before the index ischemia. Eth FI was sampled for 100 ms every 12 s during the entire experimental protocol for a total fluorescence recording time of 95 s. At restitution of flow after ischemia, increases in the extracellular-to-intracellular volume ratio were hypothesized to alter FI, introducing a potential source of artifact. Eth FI in this preparation reflects an aggregate of intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in cells underneath the probe. Increases in volume of the extracellular component could therefore decrease this signal. Eth FI values were therefore corrected for initial reperfusion flow using the formula Eth FI (a.u.)/CF/heart weight (g).


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  		Fig. 1.   Experimental protocols. Dihydroethidium (DHE) loading and washout required 25 and 15 min, respectively. Time 0 indicates the end of this period. Ischemic preconditioning (IPC) was elicited by two 5-min pulses of global ischemia separated by 5-min perfusion. IPC was followed by 20-min perfusion before "index" ischemia. All hearts (except the time-control group) underwent 30-min global index ischemia and 120-min reperfusion. The reactive oxygen species (ROS) scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) was infused for 5 min before and continued until 5 min after the end of the IPC pulses and was infused during the intervening perfusion period (IPC+MnTBAP group).

Statistical analysis. All data are expressed as means ± SE. Among-group data were compared with two-way analysis of variance at the following time points: baseline (0 min), during and after the preconditioning stimuli, during ischemia at 5-min intervals, during reperfusion at 5-min intervals to 15 min of reperfusion, and at 60 and 120 min of reperfusion. Post hoc Student-Newman-Keuls tests were utilized where differences were found (Prisma version 3.0a; GraphPad Software, San Diego, CA). The groups compared were IPC+ MnTBAP, MnTBAP, and IPC vs. ischemia-control and all ischemia groups vs. time control. Differences among means were considered statistically significant when P < 0.05.


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Changes in mechanical function and infarct size after IPC. Baseline values (0 min) and values immediately before index ischemia (40 min) were similar for each group. Figures 2 and 3 and Table 1 show that IPC was manifested by improved developed and diastolic LVP, CF, and MVO2 on reperfusion, and reduced infarct size. These effects of IPC were blocked completely by MnTBAP. MnTBAP alone before ischemia did not produce values different from those in the ischemia control group for any of these variables.


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  		Fig. 2.   Developed (systolic - diastolic) left ventricular pressure (LVP) for each group from time 0. In the 2 groups exposed to IPC pulses (IPC and IPC + MnTBAP), developed LVP returned to preischemia values between the IPC pulses and after the 2nd pulse (before index ischemia). On reperfusion after index ischemia, the IPC group had a better return of developed LVP than the ischemia control group, the IPC + MnTBAP group, or the MnTBAP group.



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  		Fig. 3.   Ventricular myocardial infarct size after global ischemia for all groups expressed as % of total ventricular weight. Infarct size in the IPC group was less than in the other ischemia groups, which did not differ from each other.


                              
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  		Table 1.   Coronary flow, myocardial oxygen consumption, and diastolic LVP before, during, and after preconditioning stimuli and on reperfusion after index ischemia

Changes in ROS generation during and after IPC. Figure 4 shows ROS generation, primarily O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (7), assessed by Eth FI for IPC, IPC+MnTBAP, ischemia control, and time control groups. ROS did not change significantly throughout the study in the time control group. ROS increased significantly during the brief ischemic pulses (~35% increase over baseline) and returned immediately to baseline levels during the intervening perfusion periods. In the presence of MnTBAP, the increases in ROS during the brief ischemic pulses were markedly attenuated but not abolished. At initiation of the longer index ischemia, ROS increased within 12 s in all groups. ROS levels were similar during the first 20 min of ischemia in each ischemia group and similar to the levels found during the two brief ischemic pulses. In the ischemia control group, there was a marked rise in ROS generation during the last 10 min of ischemia. This occurred similarly in the IPC+MnTBAP and MnTBAP groups. In the IPC group, the increase in ROS was significantly attenuated compared with the ischemia control group at 25 min and 30 min of ischemia.


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  		Fig. 4.   Fluorescence intensity (FI) in arbitrary units (a.u.), normalized to zero at baseline, of the oxidation product of DHE ethidium (Eth), an indicator of intracellular ROS, primarily superoxide. ETH was measured spectrofluorometrically (excitation wavelength 540 nm and emission wavelength 590 nm) in intact hearts via a fiber-optic probe placed against the left ventricular free wall. MnTBAP group data are not shown because they were not different from those of the ischemia-control group. Sampling rate was 5 recordings/min, but to enhance clarity this figure shows only 1.6 recordings/min. Intracellular ROS generation occurred during IPC pulses but was attenuated in the presence of MnTBAP. During later ischemia and throughout reperfusion, ROS generation was markedly less in the IPC group than in the other ischemia groups.

On reperfusion in the IPC group, ROS levels returned abruptly to near time control levels. On reperfusion in the other groups, ROS declined briefly and then increased and gradually declined but remained markedly elevated throughout reperfusion with respect to the time control group and the IPC group. For the MnTBAP alone group (not shown), values were indistinguishable from those for the ischemia control and IPC + MnTBAP groups. To control for changes in tissue volume underneath the fiber-optic probe caused by rapid restitution of coronary flow at early reperfusion and possible consequent effects on extracellular to intracellular volume ratios, FI values were corrected for flow. Figure 5 shows that the brief apparent decline in myocardial cell ROS on initial reperfusion does not likely represent a true temporary reduction in ROS generation on initial reperfusion.


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  		Fig. 5.   FI of Eth during the first 10 min of reperfusion after 30-min ischemia, corrected for changes in coronary flow (CF). The early decline and subsequent rise in FI on reperfusion seen in the ischemia-control and IPC+MnTBAP groups (Fig. 4) was abolished, but differences between the IPC group and the other ischemia groups remained.


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This is the first study in the intact, beating heart to directly assess intracellular ROS formation continuously during IPC pulses and to examine the effects of IPC on ROS formation throughout index I/R. It was demonstrated previously that IPC (12, 41) and pharmacological preconditioning (26) cause a reduction in ROS release during reperfusion after index ischemia. We show additionally in isolated hearts that reduced ROS formation also occurs during ischemia and that the preconditioning effect is likely triggered by the burst of ROS formed during the brief ischemic pulses rather than during the reperfusion phases. Moreover, we show the effects of an ROS scavenger to attenuate, but not block, the rise in ROS during preconditioning ischemic pulses, suggesting that a threshold level of ROS is necessary for preconditioning.

ROS scavenging abrogated IPC-protective effects on cardiac function and infarction and restored ROS formation during index I/R to the ischemia control (unpreconditioned) values. Our study indicates that IPC delays the onset of irreversible myocardial dysfunction as evidenced by the delayed and reduced rise in ROS formation during the last 10 min of ischemia. In concert with our other studies using the same preparation (33, 34), this study suggests a primary role of IPC in preserving mitochondrial bioenergetics during ischemia.

Evidence for role of ROS in IPC. Existing studies in isolated hearts or intact animals have either deduced a role for ROS in IPC from the observed effects of ROS scavengers to abrogate improvement in cardiac functional and structural variables induced by IPC (1, 4, 11, 13, 28, 39) or have used techniques that allow for measurement of ROS only during reperfusion (12, 41). In the latter measurement, however, it was not possible to know whether collected samples represented ROS generated during reperfusion only or included a flushout of the ROS probe accumulated during the ischemic period. The latter possibility was supported by the work of Zweier and Kuppusamy (53). They used electron paramagnetic resonance spectroscopy in isolated rat hearts to provide evidence that the time constant of enzymatic clearance of administered ROS (a nitroxide) during ischemia was unaltered during ischemia but that a slower clearance of the nitroxide occurred because of loss of the washout effect present in the perfused heart. This suggested that ROS accumulation in the ischemic heart was due both to increased production and to reduced clearance of ROS. Thus measurement of ROS in coronary effluent at reperfusion may include ROS accumulated in the cells and interstitium during ischemia and ROS formed at reperfusion and therefore may overestimate ROS formed on reperfusion.

Our method of continuous ROS measurement in the intact heart permits the study of ROS formation synchronous with global cardiac function and metabolism throughout I/R. We found an increase in ROS production in hearts during brief ischemia (the preconditioning pulses) and during the 30-min index ischemia. These findings are supported by reports that oxidant injury occurs in cardiac tissue during ischemia without reperfusion (10) and by work in isolated cardiomyocytes that indicates significant ROS production during simulated ischemia (6, 46). The latter group reported an approximately twofold increase in Eth oxidation at the end of 30-min ischemia (6). O'Neill et al. (27) used an intact feline model in which samples of coronary venous blood were taken at intervals during ischemia. They reported a severalfold increase in ·OH formation estimated by hydroxylation of phenylalanine. Using a similar measurement technique, Sun et al. (38) reported a large increase in ·OH within 1 min of ischemia in open-chest dogs. Importantly, with regard to possible mechanistic insights into the triggering of IPC, we found that brief ischemia caused an increase in ROS formation whereas reperfusion after brief ischemia did not cause an increase in ROS. This agrees with findings in isolated cardiomyocytes subjected to IPC (44). Our observations, moreover, suggest that ROS released during the ischemic pulses, and not on reperfusion, leads to activation of cardioprotective pathways during the intervening perfusion period. Nonetheless, it is possible that O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, produced during ischemia, is converted to downstream products such as H2O2 and ·OH on reperfusion and that it is these species that initiate preconditioning. In support of this possibility, investigations in cardiomyocytes suggest that dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· is a requisite of IPC (44).

Use of ROS scavengers in IPC. MnTBAP is a cell-permeant superoxide dismutase mimetic that is reported to dismutate O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and, to a lesser extent, H2O2 (14). We found that 20 µM MnTBAP did not completely obliterate the rise in FI during the brief ischemic pulses. We believe this is because MnTBAP cannot be delivered continuously to the myocardium during ischemia. As ROS generation continues during ischemia, a ROS scavenger present in the heart at the beginning of ischemia is likely to be rapidly consumed and not replaced. Although our signal was initially suppressed, it began to rise after a brief delay. To confirm this theory, we found that higher doses of MnTBAP further delayed the increase in FI during brief ischemia (unpublished observations). Despite the incomplete scavenging of ROS, however, the achieved decrease in Eth FI was adequate to prevent preconditioning.

ROS are constitutively formed under basal conditions (2-5% of available O2 is consumed in this manner), but this low level does not trigger preconditioning. It appears that a threshold level of ROS generation is required to trigger preconditioning without tissue injury but the additional ROS formed during later index ischemia (the last 10 min) is sufficient to cause greater injury. In rabbit hearts, ROS scavengers successfully blocked IPC induced by a single preconditioning cycle but not by multiple cycles (4). Other investigators also found that scavengers failed to block IPC induced by three (32) or four (21) cycles of I/R. This suggested to us that ROS may not be scavenged sufficiently, so the quantity of ROS produced over several ischemic pulses might be sufficient to attain a preconditioning threshold despite the use of scavengers. This study also points out that a failure to adequately scavenge ROS during ischemia is related to the difficulty in delivering scavenger drugs to the myocardium during prolonged ischemia. This may account for the failure of clinical trials to demonstrate a benefit of scavengers to protect the heart from I/R injury (16).

Complete washout of MnTBAP was confirmed by the return of Eth FI to baseline levels before the initiation of ischemia. This demonstrates that the presence of the scavenger only during the preconditioning phase was sufficient to block IPC and to reverse the reduction of ROS generation during I/R. This finding, and the results obtained during preconditioning pulses, render unlikely the possibility that any residual scavenger would be present in the heart throughout I/R and be responsible for these effects.

Mechanisms of ROS in IPC. An essential role for ROS in the triggering mechanism of preconditioning, especially IPC, appears to be accepted. Exogenous administration of H2O2 can induce a preconditioned state in cardiomyocytes (44) and intact hearts (43), and a similar effect can be achieved by endogenous ROS formation by the xanthine/xanthine oxidase reaction (4, 52) or by using the ETC inhibitor menadione (49). Furthermore, scavenging of ROS has been shown to abrogate IPC in most (4, 11, 13, 28, 39), but not all (32) studies. Although there is evidence to link ROS with other factors involved in IPC, the mechanism by which ROS induce preconditioning is unknown. ROS have been shown to activate transcription factors (13), ATP-sensitive potassium (KATP) channels (40, 50), protein kinase C (50), and mitogen-activated protein kinases (8), each of which has been implicated in preconditioning (5, 13, 18). Nonetheless, neither the signaling sequence nor the mechanism by which proposed mediators lead to a state of protection has been elucidated.

Our reperfusion results are consistent with those of Park et al. (30), who reported that IPC attenuated ROS production by isolated mitochondria, and those of Crestanello et al. (12) and our laboratory (26), in which IPC and pharmacological preconditioning, respectively, attenuated ROS release in isolated hearts at reperfusion. Most important is our observation that IPC decreased ROS production during the index ischemia as well as during reperfusion. This supports and extends the results of Vanden Hoek et al. (45), who showed reduced DCF fluorescence intensity in isolated cardiomyocytes after hypoxic preconditioning and subsequent simulated ischemia and reoxygenation. In light of our finding of reduced ROS formation during the latter period of 30-min ischemia after IPC, it is noteworthy that we found, using a similar model, more normalized NADH levels during the latter period of 30-min ischemia after IPC (33) and reduced mitochondrial Ca2+ loading during 30-min ischemia after pharmacological preconditioning (34). Together, these results indicate that mitochondrial bioenergetics are better preserved during ischemia after preconditioning and suggest that this is responsible for improved global cardiac function and reduced infarction at reperfusion. In addition, our studies suggest that decreased production of mitochondrial ROS, rather than increased antioxidant systems, is primarily responsible for decreased ROS formation after IPC.

The mechanism by which IPC leads to improved mitochondrial function during I/R requires further study. The mitochondrial (m)KATP channel is a regulator of mitochondrial bioenergetics and may have a role in preconditioning (18). Activation of the mKATP channel may partially uncouple the mitochondrion; this has been proposed to optimize ETC function (37) and may be a mechanism to reduce ROS formation. It has been suggested that the mKATP channel regulates mitochondrial ROS production to effect preconditioning (17, 29, 45). In one study, the mKATP channel opener nicorandil enhanced postischemic supply of high-energy phosphates and decreased oxidant injury (35); both factors are consistent with improved ETC function and with evidence that the mitochondrion is the most likely source of ROS during ischemia (44, 47). Interestingly, although the mKATP channel may regulate ROS production by the ETC, there is also evidence that ROS directly activate the mKATP channel (50, 51), suggesting a model for IPC that includes a positive-feedback mechanism involving ROS.

Possible limitations. Certain limitations to our method of ROS measurement must be considered. This model lacks blood-borne sources of ROS, particularly neutrophils, that could contribute to either IPC or I/R injury in the heart (15). Furthermore, the area of the probe on the surface of the left ventricular free wall is small and we cannot distinguish ROS produced by cardiomyocytes from those produced by other cells underlying the probe, including vascular cells and occasional macrophages. Cardiomyocytes exist in vast excess over other cell types, however. During late reperfusion, infarction of cells will lead to loss of nuclei (in which Eth is contained), and therefore the fluorescent signal might decrease. Nonetheless, infarction occurred less in the IPC group so that the real difference between this and the other groups could be greater than reported here.

It is important to note that Benov et al. (7) demonstrated that although DHE is oxidized by O<UP><SUB>2</SUB><SUP>−</SUP></UP>· to produce fluorescent Eth, DHE can also catalyze the dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. Therefore, the ratio of Eth produced per O<UP><SUB>2</SUB><SUP>−</SUP></UP>· may decrease as O<UP><SUB>2</SUB><SUP>−</SUP></UP>· increases and Eth fluorescence may not necessarily increase linearly with increases in O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. Eth has relative selectivity for O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, so changes in other ROS, such as ·OH, may exhibit different trends during the I/R period. Indeed, our preliminary results with the fluorescent probe DCF suggest that this may be the case. Further study of downstream products of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· is particularly required because it is possible that that H2O2 or ·OH is the species that triggers preconditioning pathways (44). Moreover, ·OH radicals have been reported to decrease Eth FI in isolated cells (46), likely because of a direct interaction of ·OH with the Eth-DNA intercalation complex (31), and therefore might contribute to the decreased Eth FI we observed in early reperfusion and also reported by Vanden Hoek and co-workers (6) during reoxygenation of anoxic myocytes. In support of this possibility, preliminary results (data not shown) with DCF, which is relatively sensitive to H2O2 and ·OH rather than to O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, demonstrate a prompt increase in fluorescence during the first minutes of reperfusion, coincident with the early decline in Eth FI. During later reperfusion, DCF FI declines, similar to Eth FI, confirming a decline in formation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and its downstream products. An increase in tissue volume underneath the probe could also attenuate Eth FI. This might occur if changes in the intracellular-to-extracellular volume ratio occurred during reperfusion of previously ischemic tissue. Indeed, the initial decline and subsequent increase in measured O<UP><SUB>2</SUB><SUP>−</SUP></UP>· during early reperfusion after index ischemia were abolished when Eth FI was corrected for the change in coronary flow.

In conclusion, we have introduced a novel technique for real-time measurement of ROS generation in the intact, beating heart. Our results directly demonstrate not only the role of a threshold formation of ROS in triggering and mediating IPC but also the effects of ROS formed only during brief preconditioning pulses on reducing ROS during subsequent index I/R. Our study supports a key role for improved mitochondrial energetics in effecting cardioprotection induced by IPC.


    ACKNOWLEDGEMENTS

The authors thank the following for valuable contributions to this study: James Heisner, Dr. Ming Tao Jiang, Samhita S. Rhodes, Peter Katz, Jeff Fitzgerald, and Anita Tredeau.


    FOOTNOTES

The study was supported in part by National Institutes of Health Grants HL-58691 and GM-8204-06, by American Heart Association Grant 0020503Z, and by the Department of Veterans Affairs.

Portions of this work have appeared in abstract form (Kevin LG, Riess ML, Chen Q, and Stowe DF. Anesthesiology 96: A80, 2002).

Address for reprint requests and other correspondence: D. F. Stowe, M4280, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226 (E-mail: dfstowe{at}mcw.edu).

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

First published October 31, 2002;10.1152/ajpheart.00711.2002

Received 13 August 2002; accepted in final form 15 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(2):H566-H574


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