Nicorandil, a hybrid potassium channel opener and nitrate compound (1), has been shown to have a cardioprotective effect in several experimental animal models of myocardial ischaemia (2). It is well established that nicorandil can act as a potassium channel opener in guinea-pig (3-5) and dog (6-8) myocardium by using electrophysiologic techniques. It has also been reported recently that the myocardial infarct-limiting effect of nicorandil, which is independent of peripheral vasodilatation, is clearly abolished by glibenclamide, an adenosine triphosphate (ATP)-sensitive potassium channel (KATP) blocker in anaesthetised dogs (9). These observations suggest that the cardioprotective effect afforded by nicorandil is likely to be caused by its activity as a potassium channel opener, at least in dogs. However, in rabbit myocardium, there are contradictory reports on the effect of nicorandil. Recently it was reported that nicorandil does not increase outward potassium currents in the isolated ventricular cell from rabbits (10), although other investigators have shown that nicorandil can abbreviate cardiac action potential duration and increase outward currents in isolated rabbit ventricular myocytes (11). In addition, it is not proven whether a nonhypotensive dose of nicorandil has an antiinfarct effect in the rabbit myocardium. Therefore it was interesting to ascertain the effect of nicorandil in our in vivo model of myocardial infarction (12) and to clarify the mechanisms underlying its effect.
The cardioprotective effects of nicorandil have been demonstrated not only in experimental animals but also in human myocardium. For example, during coronary angioplasty, nicorandil significantly reduced the magnitude of ST-segment elevation by balloon inflation (13). We reported that preoperative nicorandil treatment conferred protection against hypoxia/reoxygenation injury in isolated human atrium, by using contractile recovery as an end point for assessing the viability of the muscle (14). Ischaemic preconditioning with a brief period of hypoxia also has been shown to be protective against hypoxia/reoxygenation injury in this human atrial preparation (14). However, we found that the hypoxic preconditioning protocol paradoxically abolished the protection afforded by nicorandil in this preparation (14). As mentioned, however, the end point of cardioprotective efficacy in that study was myocardial contractility, not infarct size. Infarct size is a more precise measure of myocardial response to ischaemia. Therefore it was of value to see whether ischaemic preconditioning could modify any cardioprotective effects of nicorandil by using infarct size as an end point.
The aim of our study was threefold. First, we assessed the infarct size-limiting effect of nicorandil in a well-established rabbit infarct model (12). Second, we investigated whether ischaemic preconditioning could modify any cardioprotective effects of nicorandil by using infarct size as the principal end point. Finally we investigated the underlying mechanism responsible for the effects observed. For this purpose, we compared the cardioprotective effect of nicorandil with that of nitroglycerin, and we also examined the effect of 5-hydroxydecanoate, a KATP blocker (15), on the protective effect of nicorandil.
Male New Zealand White rabbits weighing 2.3-3.2 kg were used throughout. The care and use of animals in this work were in accordance with U.K. Home Office guidelines of the Animals (Scientific Procedures) Act 1986.
Rabbits were anaesthetised with a combination of 40 mg/kg pentobarbitone sodium (i.v.) and 0.15 ml/kg Hypnorm (i.m.). Electrodes were attached to the shaved area on each limb for recording of the surface ECG. After the trachea was cannulated via a midline cervical incision under local anaesthetic (2% lignocaine HCl), animals were ventilated with room air supplemented with O2, and tidal volume was adjusted as necessary throughout the procedure to maintain arterial pH between 7.3 and 7.5, PCO2 at <5.0 kPa and pO2 at >20 kPa. The rate of the ventilation was fixed at 56 cycles/min. The right jugular vein was cannulated with polyethylene tubing for intravenous infusion of nicorandil or saline. The right common carotid artery was cannulated with a short rigid polyethylene cannula attached to a pressure transducer (P23XL; Gould, Valley View, OH, U.S.A.) for continuous recording of arterial blood pressure and intermittent arterial blood gas measurements (AVL993; AVL Medical Instruments UK Ltd, Staffs, U.K.). Rectal temperature was monitored periodically and maintained at 38.5 ± 0.5°C with a heating pad. After a median sternotomy and a pericardiotomy were performed, an anterolateral left coronary artery branch was identified, and a 3-0 silk suture (Mersilk type 546; Ethicon, Edinburgh, U.K.) was passed underneath the vessel at a point approximately halfway between the left atrioventricular groove and the apex. The ends of the suture were threaded through a 1.5-cm polypropylene tube to form a snare. The artery was occluded by pulling the ends of the suture taut and clamping the snare onto the epicardial surface. Snaring of the artery caused epicardial cyanosis and regional hypokinesis within 20-30 s and was usually accompanied by ST-segment elevation in the ECG within 1 min. Reperfusion was instituted by releasing the snare. Successful reperfusion was confirmed by conspicuous blushing of the previous ischaemic myocardium and gradual resolution of the ECG changes. In all animals, the coronary artery was occluded for 30 min, followed by 120-min reperfusion by manipulating the ligature and snare.
At the end of 120-min reperfusion, 1,000 IU heparin sodium was administered before the heart was excised and Langendorff-perfused with saline solution to remove blood. The ligature was tightened again, and zinc-cadmium sulphide microspheres were infused through the aorta to delineate the myocardium at risk under ultraviolet light. After freezing, the heart was sliced transversely from apex to base in 2-mm sections. The slices were defrosted, blotted, and incubated at 37°C with 1% wt/vol triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4) for 10-20 min and fixed in 4% vol/vol formaldehyde solution clearly to distinguish between stained viable tissue and unstained necrotic tissue. The volumes of the infarcted tissue and the tissue at risk were determined by a computerised planimetric technique (Summa Sketch II; Summa Graphics, Seymore, CT, U.S.A.), and the ratio of infarct-to-risk was determined.
The experimental protocols are summarised in Fig. 1. In all animals, index ischaemia was a 30-min coronary occlusion followed by 120-min reperfusion. Animals were divided into the following eight groups. The control group (group 1, CONT) consisted of animals receiving intravenous 0.9% NaCl (1 ml bolus + 0.1 ml/min) 30 min before index ischaemia and continued to the time of reperfusion. In group 2 (NCR), nicorandil was commenced 30 min before coronary occlusion (100 μg/kg bolus + 10 μg/kg/min). Hearts of the group 3 animals were preconditioned with one episode of 5-min coronary occlusion and 10-min reperfusion before the index ischaemia with saline infusion as group 1 (IP). Animals in group 4 were also ischaemically preconditioned under nicorandil infusion as group 2 (NCR + IP). In group 5, nicorandil (100 μg/kg bolus + 10 μg/kg/min, i.v.) was given 5 min before reperfusion and continued throughout the reperfusion period (NCR, R). In group 6, nitroglycerin (10 μg/kg bolus + 1 μg/kg/min, i.v.) was given instead of saline as in group 1 (NTG). In groups 7 (5HD) and 8 (5HD + NCR), 5-hydroxydecanoate (5 mg/kg) was given intravenously as a bolus 5 min before saline and nicorandil infusion, respectively.
We obtained nicorandil as a gift from Chugai Pharmaceutical Co. (Tokyo, Japan), nitroglycerin injection (5 mg/ml) from Faulding Pharmaceuticals Plc (Warwick, U.K.), 5-hydroxydecanoate (sodium salt) from Research Biochemicals International (Natick, MA, U.S.A.), zinc-cadmium sulphide fluorescent microspheres (1-10 μm) from Duke Scientific (Palo Alto, CA, U.S.A.), and TTC from Sigma Chemical (St. Louis, MO, U.S.A.). All other chemicals were of analytical reagent quality. Nicorandil and 5-hydroxydecanoate were dissolved in saline. Nitroglycerin was diluted with saline to desired concentration.
The data are presented throughout as mean ± SEM. The significance of differences in mean values was evaluated by a oneway analysis of variance (ANOVA). When treatment constituted a significant source of variance, Fisher's least significant difference test was used post hoc for predetermined individual group comparisons. The null hypothesis was rejected at p < 0.05.
Mortality and exclusions
A total of 66 rabbits was used for this study. Two animals were lost because of sustained ventricular fibrillation during the 30-min ischaemia (one in group 1, one in group 4). Four hearts (one each in groups 2, 5, 6, and 7) were excluded because of an excessively small or large risk volume <0.4 or >1.6 cc, respectively, which were prospectively determined exclusion criteria. Three experiments were also excluded because of failure of perfusion with zinc-cadmium sulphide microspheres (two in group 1 and one in group 4). The final numbers of animals were nine in group 1, eight in groups 2, 3, 4, and 6, six in group 5, and five in groups 7 and 8.
Myocardial infarct size
Risk volume after coronary ligation was not significantly different between intervention groups at ∼1.0-1.2 cc. Percentages of infarct size in the risk zone (I/R) are shown in Fig. 2. Thirty-minute coronary occlusion and 120-min reperfusion resulted in 39.2 ± 4.3% myocardial infarction within area at risk in the saline-treated control animals (group 1). A nonhypotensive dose of nicorandil (100 μg/kg + 10 μg/kg/min, i.v.), given before and during ischaemia, significantly reduced infarct size to 24.9 ± 2.9% of risk area (group 2, p < 0.01 vs. group 1). Ischaemic preconditioning induced by a single 5-min ischaemia and 10-min reperfusion before 30-min ischaemia also significantly protected ischaemic myocardium from infarction (group 3, I/R = 13.4 ± 4.3%; p < 0.01 vs. group 1). The I/R in the ischaemic preconditioned hearts was significantly smaller than that in nicorandil-treated hearts (p < 0.05). The combination of ischaemic preconditioning with nicorandil showed an intermediate protective efficacy between nicorandil alone and ischaemic preconditioning alone group (group 4, I/R = 18.1 ± 4.2%; p < 0.01 vs. group 1). Nicorandil had no effect on infarct size (I/R = 43.5 ± 3.4%) when it was infused during the reperfusion period (group 5). Nitroglycerin given before and during ischaemia produced a slight, but not statistically significant, infarct-limiting effect (group 6, I/R = 28.9 ± 2.9%; p < 0.05 vs. group 1). The protective effect of nicorandil was abolished by 5-hydroxydecanoate (group 8, I/R = 37.7 ± 5.8%; p < 0.05 vs. group 2), although 5-hydroxydecanoate by itself had no effect on infarct size (group 7, I/R = 38.8 ± 3.6%). Figure 3 shows the relation between risk-zone size and infarct size for each animal. In this figure, all data were divided into two groups, nonprotected (groups 1, 5, 6, 7, and 8) and protected (groups 2, 3, and 4) groups. These plots indicate that infarct size is dependent on the risk-zone size and that distribution of the plots is clearly different between the protected and nonprotected groups, indicating that the reduction in infarct size in the protected group was independent of risk-zone size.
Haemodynamic responses and differences in arterial blood gases
Table 1 describes the changes in heart rate and mean arterial pressure that occurred during the experiments. There were no differences in these haemodynamic parameters between groups throughout the experimental period. Significant differences were not seen in rate-pressure product among any groups (data not shown). Rectal temperature and arterial blood pH were well maintained in the physiological range, and there were no significant differences between groups throughout the experiments. In all groups, arterial pO2 and pCO2 levels were maintained at 20-60 and 3.5-4.5 kPa, respectively.
Infarct-limiting effect of nicorandil in rabbits
Our experimental results demonstrate that an intravenously infused, nonhypotensive dose of nicorandil given before and during ischaemia (early treatment) can reduce myocardial infarction produced by 30-min coronary occlusion followed by 120-min reperfusion in anaesthetised rabbits. This infarct-limiting effect of nicorandil was independent of risk-zone size or systemic haemodynamics, which indicated that the effect of nicorandil was likely to be the result of a direct effect on myocardium. Mizumura et al. (9,16) showed an infarct-limiting effect of early treatment with nicorandil in dogs by using the same dose as we used. They also demonstrated that nicorandil produced a reduction of infarct size when given 10 min before reperfusion and continued throughout the reperfusion period (late treatment; 16). In contrast to the results from these dog experiments, however, late treatment with nicorandil had no protective effect on myocardial infarction in our rabbit model of myocardial infarction. The possible mechanism underlying the protective effect of late treatment with nicorandil in dogs was speculated to be an inhibition of neutrophil activation during reperfusion (16). Neutrophil activation might not be an important factor in the development of myocardial infarction in rabbit ischaemia-reperfused myocardium.
Possible mechanism underlying the protective effect of nicorandil
As nicorandil has both KATP opening and nitrate-like effects, we thought it appropriate to assess the mechanism of cardioprotection afforded by early treatment with this agent by examining whether nitroglycerin, a typical nitrate compound, on its own demonstrated any protective effects compared with nicorandil. If nitroglycerin had no effect on infarction, the protective effect of nicorandil should be caused by its potassium channel-opening activity and not its nitrate-like effect. Nitroglycerin may have demonstrated a weak cardioprotective effect in our model. However, the difference in infarct size between nitroglycerin- and vehicle-treated groups did not reach statistical significance. Interestingly, in pigs, an intracoronary infusion of nitroglycerin exhibited a borderline effect on infarct size (again not statistically significant), although an equihypotensive dose of nicorandil did show a significant infarct-limiting effect (17). In dogs, on the other hand, a nonhypotensive dose of intravenous nitroglycerin significantly reduced infarct size (9,16). These discrepant results might be explained by the difference in the development of collateral circulation, which is very poor in the heart from rabbits and pigs, in contrast with dogs. Although the dose of nitroglycerin we used was reported previously to be a nonhypotensive dose in dogs (9,16), this dose of nitroglycerin caused slight but nonsignificant decreases in blood pressure during 30-min infusion in our experimental conditions (Table 1). Therefore, even if we used a higher dose of nitroglycerin, this could produce more marked hypotension and tachycardia, which would make obscure whether any protective effects of nitroglycerin resulted from its any direct actions on the myocytes (18) or were caused by reduction of preload or afterload or both. In any case, we could not predict the mechanism of nicorandil action from the result of the nitroglycerin study.
We also investigated the effect of 5-hydroxydecanoate, a KATP blocker (15), on its ability to abrogate any protection afforded by nicorandil. Indeed the cardioprotective effect of nicorandil was clearly abolished by 5-hydroxydecanoate. From this result, we conclude that the infarct-limiting effect of nicorandil is most likely mediated by opening of KATP on the myocardial cells. However, we cannot completely rule out the possibility that the contribution of the nitrate-like action of nicorandil may be involved in the overall observed protection, because nitroglycerin appeared to have some marginal protective effects in our experimental model. In dogs, the infarct-limiting effect of nicorandil is abolished by glibenclamide, another KATP blocker, but not by methylene blue, a guanylate cyclase inhibitor (9), which indicates that the effect of nicorandil is a result of its activity as a potassium channel opener but not as a nitrate.
Interaction of ischaemic preconditioning and nicorandil
An important aim of this experiment was to investigate the interaction of cardioprotective effects of nicorandil and ischaemic preconditioning. As mentioned, we had observed in a previous study that the cardioprotective effect of nicorandil against hypoxia/reoxygenation by using an isolated human atrial muscle preparation was abolished by combination with hypoxic preconditioning (14). In this study, we investigated whether this paradoxic result could be reproduced in the in vivo rabbit by using infarct size as the end point. However, we failed to obtain any evidence that ischaemic preconditioning had any suppressive effects on the reduction of infarct size conferred by nicorandil.
The reason for this discrepancy with the result of Carr and Yellon (14) remains uncertain, but it may lie with different experimental conditions between the two studies. First, the end point is different (infarct size in ventricular muscle vs. functional recovery of atrial muscle). Second, species difference (rabbit vs. human muscle) may be relevant. Third, this experiment was conducted in whole animals, whereas one was in the isolated tissue. Fourth, hearts were preconditioned by ischaemia in this experiment, whereas it was done by hypoxia in the previous experiment. Finally, we should pay attention to the difference in route of drug administration between two studies. In this study, we infused nicorandil intravenously. The predictable plasma concentration of nicorandil during intravenous infusion at the dose used is well within the therapeutic window obtained in human (19,20). On the other hand, in our previous experiment (14), nicorandil was orally administered to the patients preoperatively.
The mechanism underlying the limitation of infarct size by ischaemic preconditioning in anaesthetised rabbits has not been clarified completely. However, some experimental evidence indicates the role of KATP in the mechanism of cardioprotection (21). Therefore we suggest from our results that nicorandil and ischaemic preconditioning share the same mechanism (KATP opening) for infarct limitation in our experimental conditions, which is supported by our result that the combination of nicorandil with ischaemic preconditioning produced no further infarct limitation in comparison with ischaemic preconditioning alone.
It has been reported that human myocardium can be preconditioned in situ with single or some repetitive brief ischaemic episodes (22,23). Furthermore, patients with unstable angina also usually have repeated ischaemic episodes and may have myocardium in a preconditioned state (24,25). In this study, nicorandil had infarct-limiting effects in the ischaemic preconditioned hearts as well as in naive hearts. This suggests that the cardioprotective effects of nicorandil are not modified by intermittent ischaemia, as may happen in patients with unstable angina.
Acknowledgment: G. F. Baxter was supported by the Wellcome Trust. We thank the Hatter Foundation for continued support.
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