INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE LATE PHASE OF ISCHEMIC preconditioning (PC) is a delayed cardioprotective adaptation that develops 24-72 h after a sublethal ischemic stress and confers a relative resistance to myocardial stunning (6, 8, 34, 39), infarction (3, 26, 36, 47), and arrhythmias (47). Considerable evidence suggests that the oxidative stress incurred during the initial ischemic challenge is important in the development of this response. It is well known that brief episodes of myocardial ischemia and reperfusion are associated with generation of reactive oxygen species (ROS) (7, 9, 48), such as superoxide radical (O·), H₂O₂, and hydroxyl radical (·OH). Using conscious pigs, we (35) have previously shown that administration of a "broad spectrum" antioxidant therapy [superoxide dismutase (SOD) + catalase + N-2-mercaptopropionyl glycine (MPG)] during the PC ischemia completely abolished the protective effect of late PC against myocardial stunning 24 h later, supporting the concept that the development of this cardioprotective phenomenon is triggered by the formation of ROS (O·, H₂O₂, and/or ·OH) during the initial ischemic insult. However, this study could not identify the exact species responsible for triggering the late PC effect because a combination of antioxidants was used. Furthermore, the mechanism for the generation of ·OH during ischemic PC is unknown. At least two pathways have been identified, which can lead to ·OH formation in biological systems (4). One is the reaction of H₂O₂ with Fe²⁺ (Fenton reaction). A second mechanism for ·OH formation in vivo involves the diffusion rate-limited reaction of NO with O· to form peroxynitrite (ONOO), which in turn decomposes to form ·OH or an oxidant with similar reactivity (4). It is unknown whether the ·OH involved in the genesis of late PC is derived from H₂O₂ or ONOO. Furthermore, it is unknown whether ROS are sufficient to induce late PC in vivo.

The present study had two aims. First, in an effort to identify the oxygen species that is responsible for triggering the development of late PC against myocardial stunning, we examined whether separate administration of the O· scavenger SOD, the H₂O₂ detoxifying enzyme catalase or the ONOO and ·OH scavenger MPG blocks the development of late PC against stunning. In addition, to further corroborate the role of ROS in triggering late PC, we examined whether exogenous ROS, given in lieu of ischemia, can mimic the beneficial effect of the late phase of ischemic PC against stunning. The study was conducted in conscious rabbits to obviate the confounding effects of factors associated with open-chest preparations, such as anesthesia, surgical trauma, fluctuations in temperature, elevated catecholamines, and cytokine release, which could interfere with ROS formation (19), with myocardial stunning (5, 19, 40), or with PC (16).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine and with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, Revised 1986).

Experimental preparation. The experimental preparation has been described in detail previously (6, 8, 24, 25, 36, 37, 45). Briefly, New Zealand White male rabbits (2.0-2.5 kg wt and 3-4 mo old) were instrumented under sterile conditions with a balloon occluder around a major branch of the left coronary artery, a 10-MHz pulsed Doppler ultrasonic crystal in the center of the region to be rendered ischemic, and bipolar electrocardiogram (ECG) leads on the chest wall. Before being assigned to the experiments, the animals were allowed to recover for a minimum of 10 days after surgery. Throughout the occlusion/reperfusion protocol, rabbits were kept in a cage in a quiet, dimly lit room. Left ventricular (LV) systolic wall thickening (WTh), range gate depth, and the ECG were continuously recorded on a thermal array chart recorder (model TA6000, Gould; Valley View, OH). Arterial blood pressure was monitored during drug administration. No sedative or antiarrhythmic agents were given at any time.

Experimental protocol. The experimental protocol consisted of three consecutive days of coronary artery occlusions (days 1, 2, and 3, respectively). On each day, the rabbits were subjected to a sequence of six 4-min coronary occlusion/4-min reperfusion cycles (Fig. 1).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Experimental protocol. All groups underwent a sequence of six 4-min coronary occlusion and 4-min reperfusion cycles, followed by a 5-h observation period for 3 consecutive days (days 1, 2, and 3). O, occlusion; R, reperfusion; PC, preconditioning; SOD, superoxide dismutase; MPG, N-2-mercaptopropionylglycine; CuOOH, cumene hydroperoxide.

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Exogenous ROS infusion. To examine the role of exogenous ROS in triggering late PC against myocardial stunning, rabbits were subjected to an intracoronary infusion of an ROS-generating solution on day 0 (24 h before the first coronary occlusion) (Fig. 1). Chronically instrumented rabbits were lightly anesthetized with pentobarbital sodium (20 mg/kg iv), intubated, and ventilated. The left carotid artery was dissected, and a 5-Fr sheath was cannulated. Under fluoroscopic guidance, a modified 3-Fr pediatric pig tail catheter (serving as a guide catheter) was placed near the left coronary ostium. A modified drug delivery catheter (1.5-Fr) together with a guide wire was advanced via the pig tail catheter into the left coronary artery and positioned ~1 cm past the left ostium. After the guide wire was withdrawn, 1 ml of contrast medium (50% diluted Isovue-370; Bracco Diagnostics) was injected to ensure that the catheter was in the correct position. The rabbits then received an intracoronary infusion of vehicle (normal saline) or exogenous ROS generating solution [cumene hydroperoxide (CuOOH)]. WTh, arterial blood pressure, and ECG were monitored continuously to ensure that no ischemic events took place during this procedure. Group V (control) underwent on day 0 cardiac catheterization and received an intracoronary infusion of vehicle for 30 min at 0.2 ml/min. Group VI (CuOOH) underwent on day 0 cardiac catheterization and received an intracoronary infusion of CuOOH (0.24 µmol/min), calculated to give a coronary blood concentration of ~12 µM based on an estimated coronary flood flow of 20 ml/min) for 30 min of 0.2 ml/min. CuOOH (Sigma) was diluted in normal saline. All solutions were filtered through a 0.2-µm filter (Millipore) to ensure sterility before infusion.

Measurement of regional myocardial function and ischemic zone size. Regional myocardial function was assessed as systolic thickening fraction using the pulsed Doppler probe, as previously described (6, 8, 34, 35). The total deficit of systolic WTh (an integrative assessment of the overall severity of myocardial stunning) was calculated by measuring the area comprised between the systolic WTh-versus-time line and the baseline (100% line) during the 5-h recovery phase after the sixth reperfusion (6, 8, 24, 25, 37, 39). In all animals, measurements were averaged from at least 10 beats at baseline and from at least 5 beats at all subsequent time points. The size of the ischemic-reperfused zone was determined by postmortem perfusion of 5% Phthalo blue dye as previously described (24, 26, 36, 37).

Statistical analysis. Data are reported as means ± SE. For intragroup comparisons, hemodynamic variables and WTh were analyzed by a two-way repeated-measures analysis of variance (ANOVA) (time and day) to determine whether there was a main effect of time, a main effect of day, or a day-by-time interaction. If the global tests showed a significant main effect or interaction, post hoc contrasts between different time points on the same day or between different days at the same time point were performed with Student's t-tests for paired data, and the resulting P values were adjusted according to the Bonferroni correction. For intergroup comparisons, continuous variables were analyzed by either a one-way or a two-way repeated-measures (time and group) ANOVA, as appropriate, followed by unpaired Student's t-tests with the Bonferroni correction. All statistical analyses were performed using SAS software (27). Two-way ANOVA was performed using the general linear models procedure (27).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exclusions. A total of 47 conscious rabbits were used (7 for the pilot studies and 40 for the studies of late PC). Of the 40 rabbits instrumented for the studies of late PC, 7 were assigned to each of groups I-IV and VI and 5 were assigned to group V. One rabbit in group I was excluded due to ventricular fibrillation during the fourth coronary occlusion on day 1, one in group III due to malfunction of the WTh probe and one in group IV due to persistent dyskinesis after the sixth reperfusion on day 1 (tetrazolium staining demonstrated a myocardial infarction, probably due to malfunction of the occluder); therefore, six rabbits in groups I, III, and IV completed the protocol and were included in the analysis. Two rabbits in group II were excluded, one due to ventricular fibrillation during the fifth occlusion on day 1 and one due to failure of the balloon occluder on day 2; therefore, five rabbits in group II completed the protocol and were included for the analysis. One rabbit in group V was excluded due to ventricular fibrillation during the fifth occlusion on day 1; therefore four rabbits were completed the protocol. All seven rabbits in group VI completed the protocol.

Postmortem analysis. Postmortem analysis showed that the size of the occluded-reperfused vascular bed was similar in the six groups: 1.20 ± 0.10 g (23.2 ± 2.2% of LV weight) in group I, 1.35 ± 0.24 g (21.4 ± 3.1% of LV weight) in group II, 1.15 ± 0.09 g (20.0 ± 1.5% of LV weight) in group III, 1.30 ± 0.20 g (20.6 ± 2.22% of LV weight) in group IV, 1.21 ± 0.1 g (21.4 ± 1.1% of LV weight) in group V, and 1.11 ± 0.09 g (21.1 ± 1.3% of LV weight) in group VI. Tissue staining with triphenyltetrazolium chloride confirmed the absence of infarction in all animals, indicating that the injury associated with the six 4-min occlusion/4-min reperfusion cycles was completely reversible. In all rabbits, the ultrasonic crystal was found to be at least 3 mm from the boundaries of the ischemic-reperfused region.

Pilot studies. To determine the dose of catalase to be used, one rabbit received an infusion of catalase at 2,125 U · kg¹ · min¹ starting 5 min before the first occlusion and continuing until 5 min after the sixth reperfusion plus one bolus (78,703 U/kg) at the beginning of the infusion. This is the same dose previously used in conscious pigs (35). Because this dose of catalase did not block the development of late PC against stunning, we used a dose that was 10 times higher. Five rabbits were used to determine the dose of MPG. The first rabbit received MPG 1.67 mg · kg¹ · min¹ iv starting 1 h before the first occlusion and ending 1 h after the sixth reperfusion, which is the same dose previously used in conscious pigs (35). This dose significantly attenuated myocardial stunning on day 1 and prevented the development of late PC against stunning on day 2. To determine whether lower doses of MPG could also exert such an effect, two rabbits received MPG 0.83 mg · kg¹ · min¹ iv and two rabbits 0.42 mg · kg¹ · min¹ iv. Both of these doses also effectively attenuated stunning and prevented the development of late PC. Thus we selected a dose of MPG of 0.42 mg · kg¹ · min¹ for this study and included the last two rabbits, which received this dose for the data analysis of group IV.

Plasma levels of SOD, catalase, and MPG. Plasma SOD activity reached ~300 U/ml after the first bolus and was maintained at ~200 U/ml throughout the sequence of occlusion-reperfusion cycles (Fig. 2). Plasma catalase activity reached 15,000 U/ml after the bolus injection and increased gradually to 25,000 U/ml with the subsequent infusion (Fig. 2). Thus in SOD- and catalase-treated rabbits the plasma activity of SOD and catalase was maintained at high levels during the sequence of six occlusion-reperfusion cycles (Fig. 2). Plasma MPG levels exceeded 40 nmol/ml before the first occlusion and remained stable throughout the infusion period, which covered the six occlusion/reperfusion cycles and 1 h thereafter (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Plasma levels of SOD in group II (SOD + PC, n = 5) and catalase in group III (catalase + PC, n = 6). Plasma samples were obtained at baseline (S#1), 1 min after the first bolus injection (4 min before the first coronary occlusion, S#2), 2 min into the second reperfusion (S#3), at the end of the infusion (5 min after the sixth reperfusion, S#4), and 30 min after the sixth reperfusion (S#5). Data are means ± SE.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Plasma concentration of MPG in group IV (MPG + PC, n = 6). Plasma samples were obtained at baseline (S#1), 1 min before the first coronary occlusion (S#2), immediately after the sixth reperfusion (S#3), at the end of the infusion (1 h after the sixth reperfusion; S#4), and 2 h after the sixth reperfusion (S#5). Data are means ± SE.

Hemodynamic variables. Heart rate and mean arterial pressure are summarized in Tables 1 and 2. No significant change in heart rate was observed throughout the six occlusion/reperfusion cycles and the ensuing 5-h reperfusion interval (Table 1). Administration of SOD (group II), catalase (group III), or MPG (group IV) on day 1 and the intracoronary infusion of CuOOH on day 0 did not alter heart rate (Table 1) or systemic arterial pressure (Table 2).

Regional myocardial function. The measurements of baseline systolic thickening fraction on days 1, 2, and 3 are summarized in the legends of Figs. 4-9; there were no significant differences among the six groups on the same day or among different days within the same group. Compared with baseline (pretreatment) values, the measurements of WTh obtained after treatment (immediately before the first coronary occlusion [preocclusion values]) were virtually unchanged in all groups (Figs. 4-7), indicating that SOD, catalase, and MPG had no significant effect on regional myocardial function. The changes in thickening fraction associated with coronary occlusion and reperfusion were expressed as a percentage of preocclusion measurements and are illustrated in Figs. 4-9.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Systolic thickening fraction in the ischemic-reperfused region in group I (PC, n = 6) before vehicle infusion (baseline), 1 min before the first occlusion [preocclusion (pre-O)], 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the sixth occlusion. O/R, occlusion/reperfusion. Thickening fraction is expressed as a percentage of pre-O values. Baseline thickening fraction averaged 36.7 ± 2.4% on day 1, 35.7 ± 2.9% on day 2, and 35.4 ± 2.3% on day 3. Data are means ± SE.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Systolic thickening fraction in ischemic-reperfused region in group II (SOD + PC, n = 5) before administration of SOD (baseline), 1 min before the first occlusion (pre-O), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the sixth occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (PC group) are also shown (thick dashed line without symbols, n = 6). Thickening fraction is expressed as a percentage of preocclusion values. Baseline thickening fraction averaged 43.8 ± 4.3% on day 1, 42.1 ± 3.5% on day 2, and 41.6 ± 4.5% on day 3. Data are means ± SE.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Systolic thickening fraction in ischemic-reperfused region in group III (catalase + PC, n = 6) before administration of catalase (baseline), 1 min pre-O, 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the sixth occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (PC group) are also shown (thick dashed line without symbols, n = 6). Thickening fraction is expressed as a percentage of preocclusion values. Baseline thickening fraction averaged 46.2 ± 5.4% on day 1, 45.1 ± 5.5% on day 2, and 46.6 ± 5.8% on day 3. Data are means ± SE.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Systolic thickening fraction in ischemic-reperfused region in group IV (MPG + PC, n = 6) before administration of MPG (baseline), 1 min pre-O, 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the sixth occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (PC group) are also shown (thick dashed line without symbols, n = 6). Thickening fraction is expressed as a percentage of preocclusion values. Baseline thickening fraction averaged 35.2 ± 3.5% on day 1, 35.0 ± 3.4% on day 2, and 34.1 ± 3.7% on day 3. Data are means ± SE.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Systolic thickening fraction in ischemic-reperfused region in group V (control, n = 4) 5 min before the first occlusion (baseline), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the sixth occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (PC group) are also shown (thick dashed line without symbols, n = 6). Thickening fraction is expressed as a percentage of preocclusion values. Baseline thickening fraction averaged 42.3 ± 5.2% on day 1, 40.1 ± 3.9% on day 2, and 40.9 ± 5.6% on day 3. Data are means ± SE.

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Systolic thickening fraction in ischemic-reperfused region in group VI (CuOOH, n = 7) 5 min before the first occlusion (baseline), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval following the sixth occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (PC group) are also shown (thick dashed line without symbols, n = 6). Thickening fraction is expressed as a percentage of preocclusion values. Baseline thickening fraction averaged 45.6 ± 3.8% on day 1, 43.9 ± 3.7% on day 2, and 45.6 ± 3.4% on day 3. Data are means ± SE.

Group I (PC). On day 1, thickening fraction remained significantly (P < 0.05) depressed for at least 3 h after the sixth reperfusion and recovered by 5 h (Fig. 4), indicating that the sequence of six 4-min occlusion and 4-min reperfusion cycles resulted in severe myocardial stunning. On days 2 and 3, however, the recovery of WTh was markedly improved after the 6 occlusion/reperfusion cycles compared with day 1 (Fig. 4). The total deficit of WTh after the sixth reperfusion was 54% and 47% less on days 2 and 3, respectively, compared with day 1 (P < 0.01) (Fig. 10). Thus, as expected (6, 8, 25, 45), myocardial stunning was attenuated markedly, and to a similar extent, on days 2 and 3 compared with day 1, indicating a late PC effect.

View larger version (36K): [in this window] [in a new window]   Fig. 10.   Total deficit of wall thickening (WTh) after the sixth reperfusion on days 1-3 in the PC (n = 6), SOD + PC (n = 5), catalase + PC (n = 6), MPG + PC (n = 6), control (n = 4), and CuOOH (n = 7) groups (groups I-VI), respectively. Left, values of total deficit of WTh in individual rabbits. Right, mean ± SE values of total deficit of WTh. Total deficit of WTh was measured in arbitrary units, as described in the text.

Groups II (SOD treated) and III (catalase treated). On day 1, thickening fraction at 5 min after the sixth reflow averaged 22.1 ± 5.8% of preocclusion values in group II (Fig. 5) and 27.4 ± 4.6% in group III (Fig. 6). Over the ensuing 5 h, the recovery of WTh (Figs. 5 and 6) and the total deficit of WTh (Fig. 10) in both groups were similar to those observed in the control group, indicating that neither SOD nor catalase alone had any appreciable effect on the severity of myocardial stunning on day 1. In both groups, the recovery of WTh on days 2 and 3 was significantly improved compared with day 1 (Figs. 5 and 6), and the total deficit of WTh was comparable to that observed on days 2 and 3 in group I (Fig. 10), indicating that the six occlusion/reperfusion cycles on day 1 elicited a late PC effect despite the presence of SOD or catalase. Thus neither SOD nor catalase alone blocked the development of late PC against myocardial stunning.

Group IV (MPG treated). On day 1, the recovery of WTh after the sixth reflow was considerably faster than in group I (Fig. 7). Statistical analysis demonstrated that thickening fraction was significantly greater than that in group I at 5 min (P < 0.01), 15 min (P < 0.05), 30 min (P < 0.01), 1 h (P < 0.05), 2 h (P < 0.05), and 3 h (P < 0.01) after the sixth reperfusion. The total deficit of WTh after the sixth reperfusion was 53% less in the MPG-treated group compared with the untreated group (group I, P < 0.01) (Fig. 10). Thus administration of MPG alone significantly attenuated the severity of myocardial stunning.

Group V (control for CuOOH). These rabbits received an intracoronary infusion of vehicle on day 0. On day 1, the recovery of WTh during the 5 h of reperfusion (Fig. 8) was similar to that observed on day 1 in group I, so that the total deficit of WTh after the sixth reperfusion did not differ from that observed in group I (Fig. 10). On days 2 and 3, the recovery of WTh was significantly improved compared with day 1 (Fig. 8), and the total deficit of WTh was comparable to that observed on days 2 and 3 in group I (Fig. 10). Thus the procedure of cardiac catheterization and infusion of vehicle did not initiate a late PC effect 24 h later.

Group VI (CuOOH). These rabbits received an intracoronary infusion of CuOOH on day 0 to determine whether exogenous ROS are sufficient to initiate the development of late PC against myocardial stunning. Although on day 1 the extent of paradoxical wall thinning was similar to that noted in rabbits of groups I and V, the recovery of WTh after the sixth reperfusion was markedly faster than in groups I and V, and this improvement was sustained throughout the entire reperfusion interval (Fig. 9). The total deficit of WTh was 55% and 54% less than that observed on day 1 in groups I and V, respectively (P < 0.05), and similar to that observed in groups I and V on days 2 and 3 (Fig. 10). On days 2 and 3, there was no further improvement in either the recovery of WTh (Fig. 9) or the total deficit of WTh (Fig. 10) compared with day 1. Thus intracoronary infusion of CuOOH 24 h before the sequence of six 4-min occlusion/reperfusion cycles resulted in an attenuation of myocardial stunning on day 1 that was essentially equivalent to that effected by ischemic PC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present investigation demonstrates that in conscious rabbits, 1) administration of either SOD or catalase has no significant effect on the development of late PC against myocardial stunning; 2) in contrast, administration of the ONOO and ·OH scavenger MPG effectively prevents the development of late PC against stunning, suggesting that ONOO and/or ·OH play an essential role in triggering this phenomenon; and 3) conversely, intracoronary infusion of an ROS generating solution (CuOOH), given in lieu of ischemia, triggers the development of late PC against myocardial stunning. Because high doses of catalase were ineffective in preventing late PC, the data suggest that this phenomenon is triggered by ONOO or by ·OH derived from ONOO rather than from H₂O₂ generated via the Fenton reaction.

The conscious rabbit model used in this study is characterized by stable baseline systolic WTh for several weeks after surgical instrumentation, reproducible degrees of myocardial stunning, and consistent development of late PC against stunning (6, 8, 25). The use of a conscious animal preparation was felt to be particularly important for the present study because factors associated with open-chest preparations, such as surgical trauma, fluctuations in body temperature, abnormal hemodynamic conditions, elevated catecholamine levels, and elevated cytokine levels may elicit excessive free radical formation. Indeed, it has been shown (19) that generation of ROS after brief ischemia-reperfusion is greatly exaggerated in open-chest models compared with conscious animal preparations. Because the primary objective of this study was to examine the role of ROS in triggering late PC, these factors would have confounded the results of the present investigation. Thus we felt it was extremely important to avoid these variables by using conscious animals.

The dose of SOD used in this study was based on our previous study in conscious pigs (35) and was higher than that used by Tanaka et al. (38), who reported that infusion of SOD at a rate of 250 U · kg¹ · min¹ without bolus injection (total dose 15,000 U/kg) prevented the early phase of ischemic PC against infarction in open-chest rabbits. In the present study, SOD was infused at a rate of 270 U · kg¹ · min¹ with two boluses (total dose 28,080 U/kg) and the plasma activity of SOD was substantially elevated throughout the sequence of six occlusion/reperfusion cycles (Fig. 2). The dose of catalase was 10 times higher than that previously used in conscious pigs (35), resulting in substantial elevations in plasma catalase activity (Fig. 2). Previous studies (7, 9, 23, 29, 33, 48) have documented that the burst of ROS generation subsides within few minutes of reperfusion, that is, before the infusion of SOD and catalase was discontinued in this study. Thus it seems unlikely that the failure of SOD and catalase to block late PC can be ascribed to insufficient dosages. MPG is a thiol compound and is an extremely potent scavenger of ·OH, reacting with this radical at nearly diffusion-controlled rates (rate constant, 8.1 × 10⁹ M¹ · S¹ by pulse radiolysis) (7). The ability of MPG to scavenge ·OH in vivo has been demonstrated (29, 33). In addition, MPG is a potent scavenger of ONOO by virtue of its thiol group (10, 11). A recent study by Altug et al. (1) in isolated rat hearts has shown that MPG produces a concentration-dependent inhibition of ONOO and blocks the early phase of ischemic PC against arrhythmias. Because of its small size, MPG readily traverses the cell membrane (41) and thus may scavenge ·OH and ONOO both in the intracellular and in the extracellular space.

In a previous study in conscious pigs, we (35) have demonstrated that administration of a combination of antioxidants (SOD + catalase + MPG) completely prevented the development of late PC against myocardial stunning, suggesting that ROS generated during the first ischemic insult play an important role in triggering the development of late PC. However, because that study employed a "broad spectrum" antioxidant therapy (an O· scavenger plus an H₂O₂ detoxifying enzyme plus an ·OH scavenger), we could not identify the species involved. In the present study, we expanded these observations and administered the three antioxidants separately. The finding that ONOO and/or ·OH trigger the development of late PC not only confirms an important role of ROS in the genesis of this phenomenon but also sheds new light on the nature of the molecular species that act as the triggers of late PC.

It is well known that in the presence of Fe²⁺ or other transition metals, ·OH can be generated from H₂O₂ via the Fenton reaction (21, 44). Because MPG has been reported to scavenge H₂O₂ (22, 42), it could be argued that the ability of this agent to block PC reflects removal of H₂O₂ rather than ·OH. Although we cannot rule out the possibility that the abrogation of late PC by MPG resulted from scavenging of intracellular H₂O₂, we feel this is not likely because H₂O₂ is a water-soluble reactive species that can readily cross biological membranes (32). Hence, in the presence of exogenous catalase, as in this study, H₂O₂ can be easily metabolized to O₂ and H₂O because of its hydrophilic properties. If ·OH were generated from H₂O₂ via the Fenton reaction to trigger late PC, scavenging H₂O₂ by administration of catalase should have blocked the generation of ·OH, thereby preventing the development of late PC against stunning. The finding that administration of high doses of catalase failed to block the development of late PC suggests that the ·OH responsible for triggering late PC is unlikely to be generated from H₂O₂ via the Fenton reaction. Another species that could contribute to enhanced ·OH generation and/or oxidative stress during ischemia and reperfusion is nitric oxide (NO) (13-15, 20, 46). O· and NO react rapidly to form ONOO, which then protonates and decomposes to generate ·OH or some oxidant with similar reactivity (4, 12, 17). Previous studies (6) in conscious rabbits have demonstrated that inhibition of NO production with N-nitro-L-arginine blocks the development of late PC against stunning, suggesting that this phenomenon is triggered either by NO itself or by one of its byproducts (such as ONOO and/or ·OH). On the basis of the present results, it would appear that NO initiates late PC against stunning by reacting with O· to form secondary oxidant species, such as ONOO and/or ·OH.

It could be argued that if late PC is triggered by ONOO or its byproduct ·OH, then scavenging O· with SOD should prevent the formation of ONOO and therefore the development of late PC. However, because O· is generated mainly within the intracellular space (32) and because exogenous SOD cannot readily enter this compartment, it is conceivable that O· may not be easily scavenged by the administration of SOD, particularly because it appears that considerable O· formation occurs in the hydrophobic interior of cellular membranes (2). To address this issue, it will be necessary to use specific intracellular or hydrophobic O· scavengers, which at the moment are not available.

If the ROS hypothesis of late PC is valid, then exposure to exogenous ROS should reproduce this phenotypic shift. This has never been examined. The present study demonstrates that intracoronary infusion of CuOOH, in the absence of ischemia, induces a significant protection against myocardial stunning 24 h later, which is indistinguishable from that observed during the late phase of ischemic PC. The ability of CuOOH to induce a late PC effect cannot be ascribed to myocardial ischemia secondary to a decrease in blood flow, because no reduction in myocardial blood flow was observed during the intracoronary infusion of CuOOH at the dose used for this study. CuOOH has been used to generate ROS in cultured cells (43) and has been reported (18) to induce lipid peroxidation in isolated rat hearts. As with any ROS-generating system, the precise identity of the oxidant species generated by CuOOH is not clear. In vitro studies using the spin trap 5,5-dimethyl-pyrroline-N-oxide (DMPO) have shown that in the presence of oxidized glutathione and Ni²⁺, CuOOH generates DMPO/·OH (30) and that CuOOH enhances the yield of ·OH resulting from the incubation of Ni²⁺ with cysteine in an aerobic environment (31). Regardless of the exact species generated by CuOOH, the ability of this compound to generate ROS has been abundantly demonstrated (28, 31).

In conclusion, the present study expands our understanding of the role of ROS in late PC. The observations reported herein demonstrate that ROS play an important role in triggering the development of late PC against myocardial stunning in conscious rabbits, thereby expanding previous data, which were obtained in conscious pigs (35). Furthermore, this study demonstrates that MPG alone can block the development of late PC, implicating ONOO and/or ·OH as the oxidant species responsible for the initiation of this phenomenon. Finally, the present data demonstrate that administration of an ROS-generating solution (CuOOH) reproduces the phenotype of late PC, indicating that ROS are not only necessary but also sufficient to trigger this defensive adaptation of the heart to stress.