Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism

Abstract

The goal of this study was to test the hypothesis that the cardioprotective effects of the late phase of ischemic preconditioning (PC) can be mimicked by treatment with NO donors. In phase I (studies of myocardial stunning), conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In group I (controls, n=6), the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 54% on days 2 and 3 compared with day 1 (P<0.05), indicating a late PC effect against myocardial stunning. When rabbits were given the NO donors diethylenetriamine/NO (DETA/NO, 0.1 mg/kg i.v., 4 times [group II, n=5]) or S-nitroso-N-acetylpenicillamine (SNAP, 2.5 microg x kg(-1) x min(-1) i.v. for 75 minutes [group III, n=51) 24 hours before the first sequence of occlusion/reperfusion cycles, the deficit of WTh on day 1 was 60% (group II) and 54% (group III) less than that observed in controls (P<0.05 for both). In both groups II and III, there was no further improvement in the deficit of WTh on days 2 and 3 compared with day 1. The protective effect of DETA/NO was completely abrogated when this agent was given in conjunction with the ONOO- and .OH scavenger mercaptopropionyl glycine (MPG) (group IV, n=5). In phase II (studies of myocardial infarction), conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. When rabbits were preconditioned 24 hours earlier with six 4-minute occlusion/4-minute reperfusion cycles, infarct size was reduced by 43% (33.2+/-2.7% versus 58.3+/-4.1% of the region at risk in controls, P<0.05), indicating a late PC effect against myocardial infarction. When rabbits were pretreated with DETA/NO (group VII, n=8) or SNAP (group IX, n=7) 24 hours before the 30-minute occlusion, infarct size was reduced by a similar degree (29.3+/-3.6% and 32.0+/-3.3% of the region at risk, respectively; P<0.05 versus controls). The degree of protection could not be increased by doubling the dose of DETA/NO (group VIII, n=5). Coadministration of MPG completely abrogated the infarct-sparing action of DETA/NO (group X, n=7). Taken together, these results demonstrate that in conscious rabbits the administration of 2 structurally unrelated NO donors induces protection 24 hours later against both reversible (stunning) and irreversible (infarction) ischemia/reperfusion injury and that the magnitude of this protection is indistinguishable from that observed during the late phase of ischemic PC. The fact that the late phase of ischemic PC can be mimicked by NO donors provides direct evidence that NO in itself is sufficient to elicit this cardioprotective mechanism. The fact that NO donor-induced late PC was abrogated by MPG indicates that the mechanism whereby NO induces this phenomenon involves the generation of oxidant species, possibly ONOO- and/or .OH. Since a relatively brief treatment with hemodynamically inactive doses of NO donors can induce long-lasting protective effects, these agents could be useful for preconditioning the heart in patients.

Figure 1

Experimental protocol for the studies of myocardial stunning (phase I). Four groups of rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfu-sion cycles (where O indicates occlusion and R indicates reperfusion) followed by a 5-hour observation period for 3 consecutive days (days 1, 2, and 3). Twenty-four hours before the first coronary occlusion (day 0), rabbits in group I (n=6, control group) received no pretreatment; rabbits in group II (n=5, DETA/NO group) received 4 intravenous bolus doses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4 mg/kg); rabbits in group III (n=5, SNAP group) received an intravenous infusion of SNAP (2.5 µg • kg⁻¹ • min⁻¹) for 75 minutes (total, 187.5 µg/kg); and rabbits in group IV (n=5, DETA/ NO+MPG group) received the same dose of DETA/NO given to group II and an IV infusion of MPG (1.7 mg • kg^•1 min⁻¹) starting 1 hour before the first DETA/NO bolus and continuing until 3 hours after the fourth bolus.

Figure 2

Experimental protocol for the studies of myocardial infarction (phase II). On day 1, 6 groups of rabbits were subjected to a 30-minute coronary occlusion followed by 3 days of reperfusion (where O indicates occlusion and R indicates reperfusion). Twenty-four hours earlier (day 0), rabbits in group V (n=10, control group) received neither ischemic PC nor drug pretreatment; rabbits in group VI (n=10, ischemic PC group) underwent six 4-minute coronary occlusion/4-minute reperfusion cycles; rabbits in group VII (n=8, DETA/NO group) received 4 intravenous bolus doses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4 mg/kg); rabbits in group VIII (n=5, DETA/NO high-dose group) received 8 intravenous bolus doses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.8 mg/kg) instead of 4 bolus doses; rabbits in group IX (n=7, SNAP group) received an intravenous infusion of SNAP (2.5 µg kg⁻¹ • min⁻¹) for 75 minutes (total, 187.5 µg/kg); and rabbits in group X (n=7, DETA/NO+MPG group) received the same dose of DETA/NO given to group VII and an IV infusion of MPG (1.7 mg • kg⁻¹ • min⁻¹) starting 1 hour before the first DETA/NO bolus and continuing until 3 hours after the fourth bolus.

Figure 3

Effect of MPG on the response of mean arterial pressure to intravenous bolus injections of DETA/NO. Increasing bolus doses of DETA/NO (0.25, 0.5, 1.0, and 1.5 mg/kg) were administered intravenously at 25-minute intervals before and 60 minutes after the beginning of a continuous infusion of MPG (1.7 mg • kg⁻¹ • min⁻¹). Thus, after the beginning of MPG infusion, the 0.25 mg/kg dose was given at 60 minutes into the infusion and the 1.5 mg/kg dose was given at 135 minutes into the infusion. This is the same time interval that was used for DETA/NO administration in groups IV and X. The response to DETA/NO during infusion of MPG was similar to that before MPG (n=6). Data are mean±SEM.

Figure 4

Systolic thickening fraction in the ischemic/reperfused region in group I (control group) 5 minutes before the first occlusion (baseline), 3 minutes into each coronary occlusion (O), 3 minutes into each reperfusion (R), and at selected times during the 5-hour reperfusion interval following the sixth occlusion. Measurements taken on day 1 are represented by the dashed line with open circles, measurements taken on day 2 are represented by the continuous line with solid circles, and measurements taken on day 3 are represented by the interrupted line with solid triangles (n=6 for all 3 days). Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.

Figure 5

Systolic thickening fraction in the ischemic/reperfused region in group II (DETA/NO group) 5 minutes before the first occlusion (baseline), 3 minutes into each coronary occlusion (O), 3 minutes into each reperfusion (R), and at selected times during the 5-hour reperfusion interval following the sixth occlusion. Measurements taken on day 1 are represented by the dashed line with open circles, measurements taken on day 2 are represented by the continuous line with solid circles, and measurements taken on day 3 are represented by the interrupted line with solid triangles (n=5 for all 3 days). To facilitate comparisons, the data pertaining to day 1 of group I (control group) are also shown (thick interrupted line without symbols, n=6). Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.

Figure 6

Systolic thickening fraction in the ischemic/reperfused region in group III (SNAP group) 5 minutes before the first occlusion (baseline), 3 minutes into each coronary occlusion (O), 3 minutes into each reperfusion (R), and at selected times during the 5-hour reperfusion interval following the sixth occlusion. Measurements taken on day 1 are represented by the dashed line with open circles, measurements taken on day 2 are represented by the continuous line with solid circles, and measurements taken on day 3 are represented by the interrupted line with solid triangles (n=5 for all 3 days). To facilitate comparisons, the data pertaining to day 1 of group I (control) are also shown (thick interrupted line without symbols, n=6). Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.

Figure 7

Systolic thickening fraction in the ischemic/reperfused region in group IV (DETA/NO+MPG group) 5 minutes before the first occlusion (baseline), 3 minutes into each coronary occlusion (O), 3 minutes into each reperfusion (R), and at selected times during the 5-hour reperfusion interval following the sixth occlusion. Measurements taken on day 1 are represented by the dashed line with open circles, measurements taken on day 2 are represented by the continuous line with solid circles, and measurements taken on day 3 are represented by the interrupted line with solid triangles (n=5 for all 3 days). To facilitate comparisons, the data pertaining to day 1 of group I (control) are also shown (thick interrupted line without symbols, n=6). Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.

Figure 8

Total deficit of WTh after the sixth reperfusion on days 1,2, and 3 in the control (n=6), DETA/NO (n=5), SNAP (n=5), and DETA/ NO+MPG groups (groups I, II, III, and IV, respectively). The values of total deficit of WTh in individual rabbits are illustrated in the left panel; the mean±SEM values of total deficit of WTh are depicted in the right panel. The total deficit of WTh was measured in arbitrary units, as described in the text.

Figure 9

Myocardial infarct size in groups V (n=10, control group), VI (n=10, ischemic PC group), VII (n=8, DETA/NO group), VIII (n=5, DETA/NO high dose group), IX (n=7, SNAP group), and X (n=7, DETA/NO+MPG group). Infarct size is expressed as a percentage of the region at risk of infarction. Open circles represent individual rabbits; solid circles, mean±SEM. *P<0.05 vs group V (control group); §P<0.05 vs group VII (DETA/NO group).

Figure 10

Relationship between size of the region at risk and size of myocardial infarction. The figure illustrates both individual values and the regression lines obtained by linear regression analysis for the control group (group V, n=10), the ischemic PC group (group VI, n=10), the NO donor-pretreated rabbits (groups VII, VIII, and IX combined, n=20), and the DETA/ NO+MPG group (group X, n=7). In all groups, infarct size was positively and linearly related to risk region size. The linear regression equations were as follows: control group, y=0.74x-0.09 (r=0.93); PC group, y=0.33x (r=0.79); NO donor group, y=0.39x-0.06 (r=0.66); and DETA/NO+MPG group, y=0.66x-0.09 (r=0.90). ANCOVA demonstrated that the regression lines for the ischemic PC and NO donor groups were significantly different from that for the control group (P<0.05 for each comparison), indicating that for any given risk region size, infarct size was smaller in the ischemic PC and NO donor-pretreated rabbits compared with control rabbits; in contrast, the line for the DETA/NO+MPG group was similar to that for the control group.

Figure 11

Systolic thickening fraction in the ischemic/reper-fused region in the control rabbits (group V, n=7), in the ischemic PC rabbits (group VI, n=7), in NO donor-pretreated rabbits (groups VII, VIII, and IX combined, n=9), and in the DETA/ NO+MPG rabbits (group X, n=5) 5 minutes before the 30-minute occlusion (baseline), 15 minutes into the 30-minute coronary occlusion (Occl), and at selected times during the 72-hour reperfusion interval. Because of Doppler probe malfunction, complete measurements of thickening fraction could not be obtained in all of the rabbits in groups V to X. Thickening fraction is expressed as a percentage of baseline values. The total deficit of WTh after infarction is depicted in the inset. The total deficit of WTh was calculated by measuring the area between the systolic WTh-vs-time line and the baseline (100% line) during the 3-day reperfusion period after the 30-minute occlusion (see text). Data are mean±SEM. *P<0.05 between group V and group VI; §P<0.05 between group V and NO donor-pretreated group. In the inset, *P<0.05 vs the control group (group V).