3',4'-Dihydroxyflavonol reduces infarct size and injury associated with myocardial ischaemia and reperfusion in sheep

Abstract

1 The antioxidant properties of flavonols in vivo and their potential benefits in myocardial ischaemia/reperfusion (I/R) injury have been little investigated. We evaluated the ability of a synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF) to scavenge superoxide in post-I/R myocardium and to prevent myocardial I/R injury. 2 Anaesthetized sheep were studied in four groups (n=5-6): control, ischaemic preconditioning (IPC), vehicle and DiOHF (before reperfusion, 5 mg kg(-1), i.v.). The left anterior descending coronary artery was occluded distal to the second diagonal branch for 1 h followed by 2 h of reperfusion. Infarct size, myocardial function, NADPH-activated superoxide generation and biochemical markers of injury were measured. 3 DiOHF (10(-8)-10(-4) m) incubated in vitro with post-I/R myocardium from the vehicle group suppressed superoxide production dose-dependently. 4 DiOHF administered in vivo also significantly reduced superoxide generation in vitro. DiOHF and IPC markedly reduced infarct size, which was 73+/-2% of the area at risk in vehicle, 50+/-4% in DiOHF, 75+/-5% in control and 44+/-4% in IPC. Post-I/R segment shortening within the ischaemic zone was greater in DiOHF (2.3+/-0.7%; P<0.01) and IPC (1.7+/-0.5%; P<0.01) than those in corresponding controls (-1.7+/-0.4; -2.1+/-0.4%). 5 DiOHF and IPC improved coronary blood flow to the ischaemic area and preserved higher levels of nitric oxide metabolites in the venous outflow from the ischaemic zone. 6 DiOHF attenuated superoxide production in post-I/R myocardium, and significantly reduced infarct size and injury following I/R in anaesthetized sheep. The extent of protection by DiOHF is comparable to that afforded by IPC. Thus, DiOHF has clinical potential for improving recovery from acute myocardial infarction and other ischaemic syndromes.

Figure 1

Diagram of experimental protocol: ↑ indicates the time points for blood sampling; ↓ indicates the time points for lidocaine infusion. DiOHF or vehicle was infused intravenously for 20 min commencing 30 min after the onset of ischaemia and continuing until 10 min before reperfusion.

Figure 2

Effect of DiOHF in vitro (a) and in vivo (b) on superoxide generation in postischaemic myocardium. In Figure 2a, ‘Control' represents myocardial tissues preincubated with the solution containing 1% dimethyl sulphoxide, without DiOHF but with the other reagents included (see details of incubation in Methods). The number of myocardial samples is n=6, ^#P<0.01 versus Control. In Figure 2b, n=6 in vehicle, n=5 in DiOHF. ^*P<0.01 versus vehicle (normal zone); ^#P<0.01 versus vehicle (ischaemic zone). All data are expressed as mean±s.e.m.

Figure 3

Effect of I/R on regional myocardial contractility (a) and LAD blood flow (b) in the ischaemic zone among groups at different time points. The solid bar indicates the period of ischaemia and the open bar represents reperfusion. Results are illustrated as mean±s.e.m., n=5 in control, n=6 in vehicle, IPC and DiOHF groups. ^*P<0.01 versus control; ^#P<0.01 versus vehicle.

Figure 4

Effect of DiOHF and IPC on infarct size. LV indicates the volume of left ventricle. Area at risk (AR) is expressed as the percentage of LV and infarct size (IS) is expressed as a percentage of AR. Results are presented as mean±s.e.m. ^*P<0.01 versus control; ^#P<0.01 versus vehicle, n=5 in control, n=6 in vehicle, IPC and DiOHF groups.

Figure 5

Creatine kinase and lactate dehydrogenase levels before ischaemia and after reperfusion. Baseline readings were made after 10–15 min stabilization following surgery and reperfusion readings were obtained 2 h after reperfusion. Data are expressed as mean±s.e.m. ^*P<0.05 versus control; ^#P<0.05 versus vehicle, n=5 in control, n=6 in vehicle, IPC and DiOHF groups.

Figure 6

Plasma levels of nitrite and nitrate (NO_x) before and during ischaemia and after reperfusion in arterial and coronary venous samples and the calculated venous−arterial (V−A) difference. The solid bar indicates the period of ischaemia and the open bar represents reperfusion. All values are mean±s.e.m. ^*P<0.01 versus control; ^#P<0.01 versus vehicle, n=5 in control, n=6 in vehicle, IPC and DiOHF groups.