Hypercholesterolemia abrogates late preconditioning via a tetrahydrobiopterin-dependent mechanism in conscious rabbits

Abstract

Background: Although the late phase of ischemic preconditioning (PC) is known to confer cardioprotection in healthy animal models, it is unknown whether this phenomenon exists in the presence of hypercholesterolemia. The goal of this study was to determine whether the infarct-sparing effect of late PC is affected by hypercholesterolemia and, if so, whether a tetrahydrobiopterin (BH4)-dependent mechanism is responsible for the loss of late PC.

Methods and results: Conscious rabbits fed a normal diet or a 1% cholesterol diet for 6 weeks were subjected to ischemic PC (six 4-minute coronary occlusion/4-minute reperfusion cycles) and, 24 hours later, underwent a 30-minute occlusion followed by 3 days of reperfusion. A total of 125 rabbits were used. In normocholesterolemic rabbits, ischemic PC reduced infarct size, an effect that was abrogated by administration of the BH4 synthesis inhibitor N-acetylserotonin (15 mg/kg IV) before the 30-minute occlusion. In hypercholesterolemic rabbits, however, ischemic PC failed to reduce infarct size. Myocardial BH4 levels in the ischemic zone increased 24 hours after ischemic PC in normocholesterolemic rabbits but not in hypercholesterolemic rabbits. In addition, in normocholesterolemic rabbits, pretreatment with N-acetylserotonin completely abolished the ischemic PC-induced increase in myocardial BH4 levels.

Conclusions: This study demonstrates that (1) hypercholesterolemia abrogates both the infarct-sparing effect of late PC and the concomitant upregulation of myocardial BH4, and (2) inhibition of myocardial BH4 synthesis in the absence of hypercholesterolemia is sufficient to abolish the infarct-sparing effect of late PC. The results support the concept that hypercholesterolemia abrogates late PC by preventing the upregulation of BH4, an essential cofactor for inducible nitric oxide synthase.

Figure 1

Experimental protocol.

Figure 2

Total plasma cholesterol levels over the course of the study. Rabbits were fed a 0% cholesterol diet (normal diet) or a 1% cholesterol-enriched diet for 6 weeks. Blood samples were taken at 0 (baseline), 2, 4, and 6 weeks into the diet feeding.

Figure 3

Change in mean arterial pressure in response to a bolus intravenous injection of 0.01, 0.10, or 1.0 μg/kg of bradykinin (left) and acetylcholine (middle panel), and 1, 10, or 100 μg/kg of nitroglycerin (right panel) at baseline (before cholesterol diet) and after 6 weeks of a 1% cholesterol-enriched diet (just before sacrifice; n=8).

Figure 4

Myocardial infarct size after 30-minute coronary occlusion followed by 3 days of reperfusion in groups I (normocholesterolemic control group), II (normocholesterolemic PC group), III (hypercholesterolemic control group), and IV (hypercholesterolemic PC group). Groups I and III received no PC, whereas groups II and IV were preconditioned with a sequence of six 4-minute occlusion/reperfusion cycles 24 hours before the 30-minute occlusion. Infarct size is expressed as a percentage of the region at risk of infarction. Open circles indicate individual rabbits; solid circles, mean±SEM; NC, normocholesterolemia; and HC, hypercholesterolemia.

Figure 5

BH₄ levels in the anterior wall or ischemic/reperfused zone (solid bar) and in the posterior wall or nonischemic zone (hatched bar) of control (group V), age-matched normocholesterolemic (group VI [NC-PC]), or hypercholesterolemic (group VII [HC-PC]) rabbits. Both normocholesterolemic and hypercholesterolemic rabbits were preconditioned with a sequence of six 4-minute coronary occlusion/reperfusion cycles 24 hours earlier. NC indicates normocholesterolemia; HC, hypercholesterolemia.

Figure 6

Myocardial infarct size after 30-minute coronary occlusion followed by 3 days of reperfusion in groups VIII through XII. Groups VIII and XI received no PC, whereas groups IX, X, and XII were subjected to ischemic PC 24 hours earlier. Rabbits received an intravenous bolus of vehicle (groups VIII [control group] and IX [PC+vehicle group]) or of the BH₄ synthesis inhibitor NAS (15 mg/kg over 5 minutes) 30 minutes before the 30-minute coronary occlusion on day 2 (groups X [PC+NAS group] and XI [NAS group]) or 30 minutes before the ischemic PC protocol on day 1 (group XII [NAS Pre group]). Infarct size is expressed as a percentage of the region at risk of infarction. Open circles indicate individual rabbits; solid circles, mean±SEM.

Figure 7

BH₄ levels in the ischemic/reperfused zone (solid bar) and in the nonischemic zone (hatched bar) of normocholesterolemic rabbits that received vehicle (group XIII) or NAS (group XIV) 30 minutes before being euthanized on day 2 or that received NAS pretreatment (group XV) 30 minutes before the ischemic PC protocol on day 1 and that were euthanized on day 2. All rabbits were preconditioned with a sequence of six 4-minute coronary occlusion/reperfusion cycles 24 hours earlier.