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Recent studies have demonstrated that cyclooxygenase-2 (COX-2) is an essential mediator of the cardioprotective effects of the late phase of ischemic preconditioning (PC) in rabbits. The goal of this study was to determine whether COX-2 also plays an essential role in late PC in the mouse. B6129F2/J mice under-went a 30-min coronary occlusion followed by 24 h of reperfusion. Administration of the COX-2 selective inhibitor, NS-398, 30 min prior to the 30-min occlusion (5 mg/kg i.p.) had no appreciable effect on infarct size compared with untreated controls (58.8 ± 2.1 %, vs. 58.8 ± 4.3 % of the risk region, respectively). When mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h prior to the 30-min occlusion, infarct size was markedly reduced (19.3 ± 3.4 %), indicating a late PC effect. The protective effect of late PC was completely abrogated by administration of NS-398 30 min before the 30-min coronary occlusion (67.7 ± 3.0 %), but not by administration of vehicle alone (23.6 ± 3.7 %). These results indicate that COX-2 mediates the late phase of ischemic PC in the mouse and imply that the role of this enzyme in cardioprotection is not species-specific.
One of the most important and still unresolved issues pertaining to the mechanism of the late phase of ischemic preconditioning (PC) is the nature of the protein(s) that is/are responsible for enhancing myocardial tolerance to ischemia 24–72 h after a sublethal ischemic stress. Among the many candidates that have been proposed, current evidence supports a role for the inducible isoform of NO synthase (iNOS) (1–3, 6) as an essential mediator of protection. Recently, however, we have demonstrated that induction of another stress-responsive protein, cyclooxygenase-2 (COX-2), is also necessary for the protection of late PC to occur (5). In this study, we found that in conscious rabbits ischemic PC increases the expression and activity of COX-2 24 h later and that inhibition of COX-2 activity obliterates the cardioprotective effects of late PC (5), indicating that COX-2 is a co-mediator of late PC (together with iNOS) in the rabbit. However, it remains unknown whether COX-2 is also necessary for late PC in other species.
To address this issue, in the present study we examined the role of COX-2 in late PC in the mouse. We have previously found that iNOS is a necessary mediator of the cardioprotective effects of the late phase of ischemic PC in this species (3). We reasoned that interrogating the role of COX-2 in the mouse would be important not only because it would enable one to determine whether the participation of COX-2 in late PC is unique to the rabbit, but also because it would provide conceptually useful information that would be pertinent to the increasing utilization of genetically-engineered mice for studies of myocardial ischemia and cardiovascular pathophysiology in general.
The study was performed in male B6129F2/J mice, 25–35 g (age, 8–16 wk) purchased from Jackson Laboratory (Bar Harbor, ME). All mice were maintained in microisolator cages under specific pathogen-free conditions in a room with a temperature of 24 °C, 55–65 % relative humidity, and a 12-h light-dark cycle.
The experimental preparation has been described in detail (3, 4). Briefly, mice were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and ventilated by using carefully selected parameters (3, 4). After administration of antibiotics, the chest was opened through a midline sternotomy, and a nontraumatic balloon occluder was implanted around the mid-left anterior descending coronary artery by using an 8–0 nylon suture. To prevent hypotension, blood from a donor mouse was given during surgery. Rectal temperature was carefully maintained between 36.7 and 37.3 °C throughout the experiment.
The coronary occlusion/reperfusion protocols have been described in detail (3, 4). In all groups, myocardial infarction was produced by a 30-min coronary occlusion followed by 24 h of reperfusion (3, 4). Mice were assigned to six groups (Fig. 1). Group I (control group) underwent the 30-min occlusion with no prior PC or any other intervention. Group IV (late PC group) underwent a sequence of six 4-min occlusion/4-min reperfusion cycles on day 1; 24 h later (day 2), the 8–0 nylon suture (which had been left in place after the first surgery) was used to produce the 30-min coronary occlusion (3, 4). Mice in group II (vehicle without PC) were given vehicle (50 % DMSO solution [5 μl/g, i.p.]) 30 min before coronary artery occlusion. Group III (NS-398 without PC) was given the selective COX-2 inhibitor, NS-398 (5 mg/kg i.p.), 30 min prior to the 30-min coronary occlusion, with no prior PC or any other intervention (NS-398 [Cayman Chemicals] was dissolved in 50 % DMSO [v/v = 50 %] in normal saline). Groups V (vehicle + PC) and VI (NS-398 + PC) were subjected to the same protocol as group IV except that the mice were given vehicle (50 % DMSO solution [5 μl/g, i.p.]) or NS-398 (5 mg/kg i.p.), respectively, 30 min before the 30-min coronary occlusion on day 2.
At the conclusion of the study, the occluded/reperfused vascular bed and the infarct were identified by postmortem perfusion of the heart with triphenyltetrazolium chloride and phthalo blue dye (3, 4) (Fig. 2). Infarct size was calculated by using computerized videoplanimetry (3, 4).
Data are reported as means ± SEM. Measurements were analyzed with a one-way or a two-way repeated-measures ANOVA, as appropriate, followed by unpaired StudentÕs t-tests with the Bonferroni correction. The relationship between infarct size and risk region size was compared among groups using an ANCOVA, with size of the risk region as the covariate. The correlation between infarct size and risk region size was assessed by linear regression analysis using the least-squares method.
A total of 108 mice were used. Reasons for exclusion are summarized in Table 1.
By experimental design (3, 4), rectal temperature remained within a narrow, physiologic range (36.7–37.3 °C) in all groups (Table 1). Five minutes before the 30-min coronary occlusion, the average heart rate in groups I–VI ranged from 476 to 628 beats/min (P = NS). Heart rate did not differ significantly among the six groups at any time during the 30-min occlusion or the ensuing reperfusion (Table 2).
Examples of the infarcts observed in groups I, IV, and VI are illustrated in Fig. 2. There were no significant differences among the six groups with respect to body weight, LV weight, or weight of the region at risk (Table 3). In group I (control group, n = 10), infarct size averaged 58.8 ± 2.1 % of the region at risk (Fig. 3). Administration of either NS-398 (group III) or vehicle (group II) had no discernible effect on infarct size in the absence of ischemic PC (Fig. 3). As expected, ischemic PC elicited, 24 h later, a significant reduction in infarct size in group IV (19.3 ± 3.4 % of the risk region; P < 0.05 vs. group I). This cardioprotective effect was completely abrogated when NS-398 was administered 30 min prior to the 30-min coronary occlusion (on day 2) (group VI; 67.7 ± 3.0 % of the risk region) (Fig. 3). Administration of the vehicle for NS-398 had no effect (groups II and V, 57.1 ± 3.1 and 23.6 ± 3.7 %, respectively, of the risk region) (Fig. 3). In groups I, II, III, and VI, the size of the infarction was positively and linearly related to the size of the region at risk (r = 0.93, 0.97, 0.83, and 0.78, respectively). As expected, the regression line was significantly shifted to the right in groups IV and V (late PC group and late PC + vehicle group, respectively) compared with group I (control) (Fig. 4), indicating that for any given size of the region at risk, the resulting infarction was smaller in preconditioned mice. In contrast, in the late PC + NS-398 group (group VI), the regression line was indistinguishable from that in control mice (Fig. 4), indicating that the protective effects of late PC were completely abrogated.
This study demonstrates that administration of a COX-2 selective inhibitor, NS-398, results in complete loss of the infarct-sparing protection conferred by the late phase of ischemic PC in mice, indicating that COX-2 is an essential mediator of late PC in this species.
NS-398 was chosen to interrogate COX-2 because it is highly selective for COX-2 vs. COX-1 (IC50 for COX-1 and COX-2: 16.8 and 0.1 μM, respectively) (7). The obliteration of late PC by NS-398 cannot be ascribed to an inherent detrimental influence of this drug on ischemic cell death, since NS-398 had no effect on infarct size in the absence of ischemic PC (a finding consonant with the notion that COX-2, the target of NS-398, is induced in the heart by stresses such as ischemia (5, 7)). Furthermore, the absence of late PC in NS-398-treated mice cannot be explained by unfavorable differences in determinants of infarct size, such as heart rate, body temperature, or region at risk, for these variables were similar among all groups (Tables 2 and and3).3). Since the experimental protocol used herein results in normal arterial oxygenation and pH and in normal arterial pressure (3, 4), spurious deviations of these variables from the normal range are unlikely to account for the observed differences in infarct size.
A previous study has identified COX-2 as an essential mediator of late PC in rabbits (5). The present investigation expands and corroborates these findings by demonstrating that COX-2 also plays an indispensable role in late PC in the mouse. The data reported herein support the concept that the role of COX-2 in late PC is not species specific but rather represents a general mechanism of delayed cardiac adaptation to stress. Given the increasing utilization of genetically-engineered mice, the notion that COX-2 mediates the cardioprotection of late PC in this species has important implications for future studies of ischemic PC utilizing gene-targeted or transgenic animals.
Many important questions remain to be addressed regarding the involvement of COX-2 in delayed cardioprotection and the mechanism for COX-2 upregulation. Nevertheless, the present data indicate that, in addition to iNOS (1–3, 6), at least one additional gene, COX-2, plays a crucial role in late PC in the mouse. Our results suggest that late PC is a multigenic phenomenon with greater complexity than heretofore suspected.
We thank Ning Dai and Xiao-Ping Zhu for technical assistance. This study was supported in part by National Institutes of Health Grants R01 HL-43151 and HL-55757 (R.B.), American Heart Association Ohio Valley Affiliate Grants KY-9804557 and 9920595V (Y.G.), American Heart Association Ohio Valley Affiliate Grant-in Aid 9951533V (X.L.T.) and the Jewish Hospital Research Foundation, Louisville, KY.