The cardioprotection of the late phase of ischemic preconditioning is enhanced by postconditioning via a COX-2-mediated mechanism in conscious rats

Abstract

The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), postconditioned at the onset of reperfusion following occlusion, or preconditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of PGE(2)) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and PGE(2) content. PGE(2) content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity.

Fig. 1

Experimental protocols for the experiments of infarct size. PC, preconditioning; PostC, postconditioning; ″, seconds; ′, minutes; O, occlusion; R, reperfusion.

Fig. 2

Experimental protocols for the experiments of cyclooxygenase-2 (COX-2).

Fig. 3

Infarct size after a 45-min coronary occlusion and 24 h of reperfusion. ○, Individual hearts; ●, group means ± SE.

Fig. 4

Relationship between the size of the region at risk and size of myocardial infarction in rats exposed to a 45-min coronary occlusion. Both individual values and the regression lines obtained by linear regression analysis are shown for groups I-V. In all groups, infarct size was positively and linearly related to the size of region at risk. Analysis of covariance (ANCOVA) demonstrated that the regression lines for groups II and IV were significantly shifted downward and to the right compared with that for group I (P < 0.05 for each comparison), indicating that for any given size of the region at risk, infarct size was smaller in rats that received either late PC alone or late PC + PostC. In group V, the line was similar to that for group I, indicating that COX-2 inhibition abrogated the additive protective effect of the combined interventions.

Fig. 5

Infarct size after a 60-min coronary occlusion and 24 h of reperfusion. ○, Individual hearts; ●, group means ± SE.

Fig. 6

Relationship between the size of the region at risk and size of myocardial infarction in rats exposed to a 60-min coronary occlusion. Both individual values and the regression lines obtained by linear regression analysis are shown for groups VI–X. In all groups, infarct size was positively and linearly related to the size of region at risk. ANCOVA demonstrated that the regression line for group IX was significantly shifted downward and to the right compared with that for groups VI–VIII (P < 0.05 for each comparison), indicating that for any given size of region at risk, infarct size was smaller in rats that received the combined interventions of late PC and PostC. In group X, the line was significantly different from that in group IX, indicating that COX-2 inhibition abrogated the additive protective effect of the combined interventions.

Fig. 7

Expression of COX-2 protein in rat myocardium. A: representative immunoblots of COX-2 expression in the ischemic-reperfused region. The Ponceau S staining reflects equal protein loading. B: densitometric analysis of COX-2 signals. In all samples, densitometric measurements of COX-2 immunoreactivity were expressed as percentages of the average value measured in control rats. PC, 12 cycles of 2-min ischemia/2-min reperfusion applied 24 h prior to occlusion (late PC); PostC, 20 cycles of 10-s ischemia/10-s reperfusion at the onset of reperfusion following occlusion; Celecoxib, 3 mg/kg ip celecoxib administered 10 min before reperfusion. Data were normalized to the Ponceau S signals and are expressed as means ± SE.

Fig. 8

Content of PGE₂ in rat myocardium. PGE₂ was extracted from the ischemic-reperfused and nonischemic regions and expressed as picograms per milligram of protein. Groups were as described in Fig. 7. Data are means ± SE.