The COX-2/PGI2 receptor axis plays an obligatory role in mediating the cardioprotection conferred by the late phase of ischemic preconditioning

Abstract

Background: Pharmacologic studies with cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by COX-2. However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Furthermore, the specific prostaglandin (PG) receptors responsible for the salubrious actions of COX-2-derived prostanoids remain unclear.

Objective: To determine the role of COX-2 and prostacyclin receptor (IP) in late PC by gene deletion.

Methods: COX-2 knockout (KO) mice (COX-2(-/-)), prostacyclin receptor KO (IP(-/-)) mice, and respective wildtype (WT, COX-2(+/+) and IP(+/+)) mice underwent sham surgery or PC with six 4-min coronary occlusion (O)/4-min R cycles 24 h before a 30-min O/24 h R.

Results: There were no significant differences in infarct size (IS) between non-preconditioned (non-PC) COX-2(+/+), COX-2(-/-), IP(+/+), and IP(-/-) mice, indicating that neither COX-2 nor IP modulates IS in the absence of PC. When COX-2(-/-) or IP(-/-) mice were preconditioned, IS was not reduced, indicating that the protection of late PC was completely abrogated by deletion of either the COX-2 or the IP gene. Administration of the IP selective antagonist, RO3244794 to C57BL6/J (B6) mice 30 min prior to the 30-min O had no effect on IS. When B6 mice were preconditioned 24 h prior to the 30-min O, IS was markedly reduced; however, the protection of late PC was completely abrogated by pretreatment of RO3244794.

Conclusions: This is the first study to demonstrate that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, and that the IP, downstream of the COX-2/prostanoid pathway, is a key mediator of the late PC. These results provide unequivocal molecular genetic evidence for an essential role of the COX-2/PGI2 receptor axis in the cardioprotection afforded by the late PC.

Figure 1. Experimental protocols.

Fourteen groups of mice including were studied for infarct size analysis in three phases. In Phase I (panel A), on day1, COX-2^+/+ and COX-2^−/− mice were subjected to either PC or sham surgery. On day 2, all mice were subjected to a 30-min LAD occlusion followed by 24 h of reperfusion. In Phase II (panel B), in addition to the day 2 protocol of Phase I, RO3244794 or vehicle was administered 30 min prior to the induction of acute MI on day 2. In Phase III (panel C), on day 1, IP^+/+ and IP^−/− mice were subjected either to PC or sham surgery. On day 2, all mice were subjected to a 30-min LAD occlusion followed by 24 h of reperfusion. All animals were sacrificed after 24 h of reperfusion to measure infarct size. The open square (□) indicates the reperfusion or no ischemia protocol. The solid black square (▪) indicates the occlusion protocol. (n = 6–16 each group).

Figure 2. Representative examples of a heart from each group.

The infarcted region was delineated by perfusing the aortic root with 2,3,5-triphenyltetrazolium chloride (TTC); the region at risk was delineated by perfusing the aortic root with phthalo blue after tying the previously occluded artery. As a result of this procedure, the nonischemic portion of the left ventricle (LV) was stained dark blue and viable tissue within the region at risk was stained bright red (TTC positive), whereas infarcted tissue was light yellow or white (TTC negative). Phase I (panel A). Non-preconditioned COX-2^+/+ and COX-2^−/− mice have similarly large infarct sizes. PC 24 h prior to MI results in a significant reduction in infarct size in COX-2^+/+ but not COX-2^−/− mice. Phase II (panel B). Non-preconditioned B6 mice in naïve, vehicle-treated, and RO3244794-treated groups have similar infarct sizes. PC results in significantly smaller infarct sizes in naïve and vehicle-treated mice but not in RO3244794-treated mice. Phase III (panel C). Non-preconditioned IP^+/+ and IP^−/− mice have similar infarct sizes. PC results in a significant reduction in infarct size in IP^+/+ but not IP^−/− mice. Scale at bottom is in mm. Note the large, confluent areas of infarction spanning most of the thickness of the LV wall, with thin rims of viable subendocardial tissue. This pattern was characteristic of all 7 nonpreconditioned groups (groups I, III, V–VII, XI and XIII) and all 3 PC groups (PC in COX-2^−/− [group IV] and IP^−/− mice [group XIV] or pretreated with the RO compound [group X]). In contrast, mice subjected to the PC protocol exhibited small, sporadic areas of infarction, a pattern that was characteristic of all 3 PC alone WT mice (groups II, VIII and XIII) and of the mice with PC + vehicle (group IX).

Figure 3. Myocardial infarct size in groups I–XIV.

Infarct size is expressed as a percentage of the region at risk of infarction. Data are expressed as means ± SEM. Phase I (panel A). COX-2^−/− mice did not exhibit the infarct-sparing effects of late PC. Phase II (panel B). RO3244794-treated mice did not exhibit the infarct-sparing effects of late PC. Phase III (panel C). IP^−/− mice did not exhibit the infarct-sparing effects of late PC. (*) Marks a significant infarct size reduction in preconditioned mice compared with non-PC mice; P<0.05. ○, Individual mice; •, mean ± SE for respective group.

Figure 4. Pilot study.

Effect of RO3244794 and IP^−/− on iloprost-induced hypotension. Heart rate and mean arterial blood pressure (MAP) are shown as the changes of percentage of baselines in Figs. 4B and 4C, respectively. Data are expressed as means ± SEM. A) Experimental protocol for hemodynamic studies. B) Effect on heart rate (HR). There was no statistic significant difference in HR among the three groups (including the absolutely numbers). C) Effect on arterial blood pressure. Iloprost resulted in a significant drop in main arterial pressure (MAP); pretreatment with RO3244794 abolished the effect of iloprost on MAP, and iloprost had no effect on MAP in IP^−/− mice.