INTRODUCTION

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ISCHEMIC PRECONDITIONING (PC) elicits an early (1, 7, 8-10, 26, 28) and a late (2, 3, 5, 18, 22, 23, 28, 31, 32, 34, 39) phase of protection against ischemic injury. Although much research has focused on the early phase of ischemic PC, the mechanism for the late phase remains poorly understood (4). We (5) have previously found that N-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide (NO) synthase, blocks late PC against myocardial stunning, indicating that NO triggers this form of delayed myocardial adaptation. However, it is unknown whether NO also triggers late PC against infarction. Myocardial stunning and infarction represent two very different types of injury, and the effects of PC on one cannot be extrapolated to the other. For example, in dogs the early phase of PC confers powerful protection against myocardial infarction (1, 26) but apparently fails to protect against the stunning induced by a 10- or 15-min coronary occlusion (4, 24, 27). Conversely, in conscious pigs a sequence of ten 2-min coronary occlusions elicits a late PC effect against stunning (31, 32, 34) but not against infarction (28). These examples of a dissociation between the effects of PC on stunning and on infarction underscore the notion that, at least in certain experimental conditions, different mechanisms may be involved in the PC protection against reversible and irreversible ischemic injury (4).

Accordingly, the goal of the present study was to explore the role of NO in the development of the late phase of PC against myocardial infarction. Because the very existence of a delayed protection against cell death is somewhat controversial (16, 28, 33), we first determined whether a late PC effect against infarction does occur in our conscious rabbit model. To this end, we used the same PC protocol (six 4-min occlusion/4-min reperfusion cycles) that has previously been found to induce a potent and reproducible late PC effect against myocardial stunning in this preparation (5, 22). We then investigated whether administration of L-NNA during the PC ischemia blocks the delayed protection against infarction. The study was conducted in conscious animals to obviate the potentially confounding influence of factors associated with open-chest preparations, such as anesthesia, surgical trauma, abnormal hemodynamics, elevated catecholamine levels, fluctuations in body temperature, exaggerated formation of reactive oxygen species (ROS), release of cytokines, etc., which could interfere with NO-mediated signaling, myocardial infarction, and/or ischemic PC (7, 14, 9). The results demonstrate, for the first time, that generation of NO triggers late PC against infarction.

	MATERIALS AND METHODS

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The conscious rabbit model of myocardial ischemia has been described in detail previously (5, 22) and is briefly summarized here.

Experimental preparation. New Zealand White male rabbits (wt 2.4 ± 0.1 kg) 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, bipolar pacing leads in the left atrial appendage, and bipolar electrocardiogram (ECG) leads on the chest wall. The chest wound was closed in layers, and a small tube was left in the thorax for 3 days to evacuate air and fluids postoperatively. Gentamicin was administered before surgery and on the first and second postoperative days (0.7 mg/kg im each day). Rabbits were allowed to recover for a minimum of 10 days after surgery.

Experimental protocol. Throughout the experiments, rabbits were kept in a cage in a quiet, dimly lit room. Left ventricular (LV) systolic wall thickening (WTh), range gate depth, and ECG were continuously recorded on a thermal array chart recorder. Regional myocardial function was assessed as systolic thickening fraction using the pulsed Doppler probe, as previously described (5, 22). All rabbits were subjected to a 30-min coronary artery occlusion followed by 3 days of reperfusion. The performance of successful coronary occlusion was verified by observing the development of S-T segment elevation and changes in the QRS complex on the ECG and the appearance of paradoxical wall thinning on the ultrasonic crystal recordings. Diazepam was administered 20 min before the onset of ischemia (4 mg/kg iv) to relieve the stress caused by the coronary occlusion. No antiarrhythmic agents were given at any time. Rabbits were assigned to four groups (Fig. 1). Group I (control group) underwent the 30-min occlusion with no PC protocol and no drug treatment. Group II (PC group) underwent a sequence of six 4-min coronary occlusions interspersed with 4 min of reperfusion 24 h before the 30-min coronary occlusion. Group III (L-NNA-treated group) underwent the same protocol as group II except that the rabbits received an intravenous infusion of L-NNA at a rate of 1.3 mg · kg¹ · min¹ for 10 min starting 20 min before and ending 10 min before the first coronary occlusion (total dose 13 mg/kg). To maintain constant heart rate, atrial pacing was initiated after the administration of L-NNA and continued until 5 h after reperfusion at rates similar to the baseline (pretreatment) values. L-NNA (Sigma Chemical, St. Louis, MO) was dissolved in normal saline (total volume infused 20 ml), and the solution was filtered through a 0.2-µm Millipore filter to ensure sterility. In group IV (L-NNA-pretreated group), rabbits received the same dose of L-NNA but did not undergo the sequence of six occlusion-reperfusion cycles; these animals were subjected to the 30-min coronary occlusion 24 h later. Atrial pacing at rates similar to pretreatment heart rates was started after administration of L-NNA and continued for 6 h.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Experimental protocol. Four groups of rabbits were studied. On day 2, all groups underwent 30-min coronary occlusion followed by 3 days of reperfusion. On day 1, rabbits in group I (n = 10, control group) received only an infusion of normal saline (20 ml). Rabbits in group II (n = 10, PC group) received same saline infusion on day 1 and underwent a sequence of six 4-min coronary occlusion (O)-4-min reperfusion (R) cycles. Rabbits in group III [n = 11, N-nitro-L-arginine (L-NNA)-treated group] underwent same sequence of O-R cycles on day 1; in addition, they received an intravenous infusion of L-NNA (1.3 mg · kg1 · min1) starting 20 min and ending 10 min before 1st coronary occlusion (total dose 13 mg/kg). Rabbits in group IV (n = 9, L-NNA-pretreated group) received same dose of L-NNA on day 1 but did not undergo coronary occlusion.

Postmortem tissue analysis. At the conclusion of the study, the rabbits were given heparin (1,000 U iv), after which they were anesthetized with pentobarbital sodium (50 mg/kg iv) and euthanized with KCl. The heart was excised, and the size of the occluded-reperfused coronary vascular bed was determined by tying the coronary artery at the site of the previous occlusion and by perfusing the aortic root for 2 min with a 5% solution of phthalo blue dye in normal saline at a pressure of 70 mmHg using a Langendorff apparatus. The heart was then cut into six or seven transverse slices, which were incubated for 10 min at 37°C in a 1% solution of triphenyltetrazolium chloride in phosphate buffer (pH = 7.4). All atrial and right ventricular tissues were excised, after which the slices were weighed, fixed in a 10% formaldehyde solution, and photographed (Nikon AF N6006). Transparencies were projected onto a paper screen at a 10-fold magnification, and the borders of the infarcted, ischemic-reperfused, and nonischemic regions were traced. The corresponding areas were measured by computerized planimetry (Adobe Photoshop, version 4.0), and from these measurements infarct size was calculated as a percentage of the region at risk.

Statistical analysis. Data are reported as means ± SE. Heart rate and thickening fraction were analyzed by a two-way repeated-measures analysis of variance (ANOVA; time and group). Infarct sizes and risk region sizes were analyzed with a one-way ANOVA followed by Student's t-tests for unpaired data with the Bonferroni correction.

	RESULTS

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Exclusions. Of the 49 rabbits instrumented for this study, 13 were assigned to the control group (group I), 12 to the PC group (group II), 13 to the L-NNA-treated group (group III), and 11 to the L-NNA-pretreated group (group IV). Of the 13 rabbits assigned to the control group, 2 died of ventricular fibrillation during coronary occlusion and 1 was excluded because of failure of the balloon occluder. Of the 12 rabbits assigned to the PC group, 2 died of ventricular fibrillation during the 30-min coronary occlusion. Of the 13 rabbits assigned to the L-NNA-treated group, 1 was excluded because of ventricular fibrillation during the 30-min occlusion and 1 because of a technical problem with the postmortem perfusion. Of the 11 rabbits assigned to the L-NNA-pretreated group, 2 were excluded because of malfunction of the occluder. Therefore, a total of 10 rabbits completed the protocol in the control group, 10 in the PC group, 11 in the L-NNA-treated group, and 9 in the L-NNA-pretreated group.

Hemodynamic variables. It has previously been shown that the dose of L-NNA used in this study does not alter systemic arterial pressure or systolic thickening fraction in conscious rabbits (5). In that investigation, however, L-NNA induced a sustained decrease in heart rate. Consequently, in this study we elected to pace the heart at a rate similar to the baseline heart rate throughout the six coronary occlusion-reperfusion cycles and the first 5 h of reperfusion in group III and for equivalent periods of time in group IV (Fig. 1). As a result, there were no appreciable differences in heart rate between groups II and III on the first day of the protocol [occlusion 1: 263 ± 6 and 260 ± 7 beats/min (bpm), respectively; occlusion 6: 260 ± 5 and 259 ± 6 bpm, respectively]. On the second day of the protocol, the heart rate did not differ among the four groups before, during, or after the 30-min coronary occlusion (preocclusion: 243 ± 8, 253 ± 6, 245 ± 9, and 232 ± 8 bpm, respectively, in groups I-IV; 15 min of occlusion: 246 ± 5, 252 ± 8, 248 ± 9, and 238 ± 7 bpm, respectively; 1 h of reperfusion: 251 ± 8, 258 ± 7, 232 ± 11, and 236 ± 7 bpm, respectively). As expected (see Ref. 5), in groups III and IV systolic thickening fraction was not altered by L-NNA (data not shown).

Region at risk and infarct size. There were no significant differences among groups I-IV with respect to LV weight (4.7 ± 0.4, 4.3 ± 0.2, 4.7 ± 0.3, and 3.8 ± 0.1 g, respectively) or weight of the region at risk [0.8 ± 0.1 g (17.0 ± 1.7% of LV wt), 0.7 ± 0.1 g (15.8 ± 1.2% of LV wt), 0.7 ± 0.1 g (14.8 ± 1.2% of LV wt), and 0.6 ± 0.1 g (15.4 ± 1.5% of LV wt), respectively]. The average infarct size was 50% smaller in group II compared with control animals (group I) (28.6 ± 3.2 and 56.9 ± 5.9% of region at risk, respectively; P < 0.05; Fig. 2), indicating a late PC effect against myocardial infarction. In group III, however, infarct size (48.6 ± 3.8% of the region at risk) was significantly greater than in group II (P < 0.05) and not significantly smaller than in controls (Fig. 2), indicating that L-NNA abrogated the late PC effect against infarction. In group IV, infarct size (47.8 ± 5.3% of region at risk) did not differ significantly from that in controls (Fig. 2), indicating that pretreatment with L-NNA, in itself, did not affect the extent of necrosis. In all four groups, the size of the infarction was positively and linearly related to the size of the region at risk (r = 0.83, 0.85, 0.86, and 0.91 in groups I-IV, respectively). The regression line, however, was shifted to the right in group II (y = 0.43x  0.087) compared with group I (y = 0.54x 0.016), indicating that, for any given size of the region at risk, the resulting infarction was smaller in preconditioned than in control rabbits. In groups III and IV, the regression lines were similar to that of group I (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Myocardial infarct size in groups I-IV. Infarct size is expressed as percentage of region at risk of infarction. , Individual rabbits; , means ± SE; n, number of rabbits.

Regional myocardial function. Because of Doppler probe malfunction, complete measurements of WTh for 3 days after reperfusion could be obtained only in 5 of 10 rabbits in group I and 9 of 10 rabbits in group II. On the second day of the protocol, baseline systolic thickening fraction averaged 39.7 ± 2.5% in group I and 35.7 ± 5.1% in group II [P = nonsignificant (NS)]. After release of the 30-min occlusion, control rabbits (group I) exhibited essentially no recovery of WTh, even at 3 days (Fig. 3). In preconditioned rabbits (group II), the recovery of WTh was significantly (P < 0.05) improved compared with controls at 5 h and 1, 2, and 3 days after reperfusion (Fig. 3). The total deficit of WTh over the 3-day reperfusion period (an integrative assessment of the overall severity of contractile dysfunction in this time interval; see Refs. 5, 22) was decreased by 18% in group II vs. group I (P < 0.05; Fig. 3). However, the effect of late PC on WTh was highly heterogeneous, with some rabbits in group II exhibiting no recovery (persistent dyskinesis) for 3 days and others resuming active WTh as early as 1 h after reperfusion. This variability is manifested in the large standard errors bracketing the means in Fig. 3. In groups III and IV, the recovery of WTh was indistinguishable from that in the control group (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Systolic thickening fraction in ischemic-reperfused region in groups I and II. Measurements were obtained at baseline, 15 min into 30-min occlusion (Occl), and 30 min and 1, 3, 5, 24, 48, and 72 h after reperfusion. Thickening fraction is expressed as percentage of baseline values. Total deficit of wall thickening (WTh; inset) was calculated by measuring area between systolic WTh vs. time line and baseline (100% line) during 3-day reperfusion period after 30-min occlusion (see Refs. 5, 22). PC, preconditioning. Data are means ± SE.

	DISCUSSION

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Although NO has been shown to elicit a late PC effect against myocardial stunning (5), no previous study has examined the role of NO as a possible trigger of the late phase of ischemic PC against infarction. With regard to the role of NO in the early phase of ischemic PC, the available data are conflicting. Some studies have concluded that NO production induces early PC against arrhythmias (35, 36) and infarction (15), whereas others (6, 12, 21, 37, 38) found no evidence to support this concept. The reason(s) for these discrepant results is unknown.

The present study demonstrates that the NO synthase inhibitor L-NNA blocks the development of late PC against myocardial infarction in conscious rabbits, indicating that formation of NO during the PC ischemia is necessary to trigger the development of the cardioprotection observed 24 h later when the heart is subjected to a lethal ischemic insult. The abrogation of protection against infarction cannot be attributed to an inherent deleterious effect of L-NNA pretreatment, because administration of L-NNA 24 h before the 30-min coronary occlusion in group IV had no influence on infarct size. These results show, for the first time, that NO plays a key role in eliciting the late phase of ischemic PC against myocardial infarction. This is also the first study to examine the role of NO in ischemic PC against myocardial infarction (early or late) in a conscious animal model.

We used the same dose of L-NNA that was previously found to prevent late PC against stunning in conscious rabbits (5). This dose of L-NNA blunts acetylcholine-induced vasodilation without raising arterial pressure (i.e., without causing systemic vasoconstriction) (5), suggesting that it is sufficient to block increased synthesis of NO (such as that which occurs in response to ischemia-reperfusion) without affecting basal endothelial release of NO. Previous studies have shown that administration of L-NNA causes a decrease in heart rate without changes in arterial pressure in conscious rabbits (5, 20) and in conscious dogs (29), an effect that is thought to reflect the central regulatory function of NO on sympathetic and parasympathetic tone (20). To avoid any potential interference caused by decreases in heart rate, in the present investigation all rabbits treated with L-NNA were paced at rates similar to their baseline (pretreatment) heart rates.

In this study, the same ischemic PC protocol previously found to induce late PC against stunning (5, 22) effected a 50% reduction in infarct size 24 h later. Although this reduction in infarct size is less than that generally observed during the early phase of PC (1, 7-10, 26, 28), it is still substantial. Thus, in the conscious rabbit, the late phase of ischemic PC confers powerful protection not only against stunning, but also against cell death. These results are consonant with those previously reported by Yang et al. (39) in conscious rabbits preconditioned 24 h earlier with four 5-min coronary occlusions, although the reduction in infarct size observed in that study (32%) was less than that noted in the present investigation. In open-chest rabbits preconditioned 24 h earlier with four 5-min occlusions, reductions in infarct size ranging between 35 and 45% have been reported (2, 3, 23), although in one study (33) no protection was observed. We (28) have previously failed to detect an infarct-limiting effect during the late phase of ischemic PC in conscious pigs subjected to ten 2-min coronary occlusions followed 24 h later by a 40-min occlusion. The reason(s) for the apparent discrepancy between those previous results and our present observations probably relates to the differences in species and PC protocols. Further studies will be necessary to determine whether late PC against infarction is dependent on the species used.

The measurements of WTh demonstrate that the reduction in cell death afforded by late PC in group II was associated with an enhanced recovery of contractile function (Fig. 3). Although the average improvement observed in the first 3 days of reperfusion was modest (because of a marked variability among rabbits), it is plausible that a greater benefit would have been detected over the subsequent days and weeks, because it has been shown that full recovery of surviving, stunned myocardium after a lethal ischemic insult requires up to 2 wk (19). To our knowledge, this is the first study to demonstrate that the beneficial effects of ischemic PC (early or late) on myocardial infarction are translated into a functional improvement.

The source of increased NO formation during the PC stimulus is likely to be the constitutively expressed endothelial (type III) isoform of NO synthase, which has been identified not only in endothelial cells but also in cardiac myocytes (13, 17). Ischemic PC could stimulate synthesis of NO by this enzyme by at least three mechanisms: ischemia-induced increases in intracellular calcium and NADPH, increased shear stress during the repeated reactive hyperemias, and/or release of bradykinin with activation of endothelial B₂ receptors (25). Regardless of the source of NO, we propose that formation of this radical during the PC ischemia leads to the synthesis of cardioprotective proteins that subsequently render the heart more resistant to infarction. NO exerts a plethora of actions that could culminate in upregulation of gene transcription; for example, several transcription factors (such as nuclear factor-B, activator protein-1, adenosine 3',5'-cyclic monophosphate response element binding protein), enzymes, receptors, G proteins, protein kinases, protein phosphatases, and ion channels are known to be affected by NO (reviewed in Ref. 13). NO could also modulate gene expression through the formation of ONOO and/or secondary ROS, which in turn could act via activation of protein kinase C (PKC; Ref. 11) or a cis-acting regulatory element (antioxidant responsive element) that mediates cellular responses to oxidative stress (30). Further studies will be necessary to elucidate the mechanism whereby NO induces late PC against infarction and also to determine whether this phenomenon is triggered by NO itself or by one of its reactive byproducts (such as ONOO; Ref. 13).

Previous studies have implicated adenosine receptors (3) and PKC (2) in late PC against infarction. These prior results and our present data are not mutually exclusive. Because NO can cause formation of ROS (13), and because both adenosine (8) and ROS (11) may activate PKC, it is possible that PKC serves as a common transduction element in the signaling pathways that lead to late PC. At present, it is unclear whether adenosine acts independently of NO and whether both of these stimuli are needed to achieve the threshold necessary to induce late PC against infarction.

In conclusion, this study expands our understanding of the complex cellular functions of NO by demonstrating that inhibition of NO production prevents the development of late PC against myocardial infarction in conscious rabbits. We propose that NO serves as a key signal in the cellular events responsible for the delayed myocardial adaptation to brief ischemic stresses.