Asian Cardiovasc Thorac Ann 2007;15:204-209
© 2007 Asia Publishing EXchange Ltd
Nicorandil Attenuates Reperfusion Injury after Long Cardioplegic Arrest
Kyoumi Takarabe, MD,
Yukio Okazaki, MD,
Shinya Higuchi, MD,
Junichi Murayama, MD,
Masafumi Natsuaki, MD,
Tsuyoshi Itoh, MD
Department of Thoracic and Cardiovascular Surgery, Saga Medical School, Saga, Japan
For reprint information contact: Yukio Okazaki, MD Tel: 81 95 234 2345 Fax: 81 95 234 2061 Email: okazaki{at}post.saga-med.ac.jp, Department of Thoracic and Cardiovascular Surgery, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
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ABSTRACT
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The cardioprotective efficacy of nicorandil in cardiac surgery was determined using a surgically relevant 4-hr cardioplegic arrest model. Each isolated rabbit heart was parabiotically blood-perfused using a modified Langendorff column. The magnitude of left ventricular developed pressure and rate of change of developed pressure over time were measured before (baseline) and after ischemia. Nicorandil was administered either pre-ischemia, post-ischemia, pre/post-ischemia, or continuously (before, during, and after ischemia). The endothelium of the coronary artery was observed by scanning electron microscopy. Serum myeloperoxidase activities were also measured. Although pretreatment with nicorandil did not affect recovery of developed pressure, administration of nicorandil after ischemia, or before and after ischemia, enhanced the recovery of developed pressure. Serum myeloperoxidase activity was decreased in the pre/post-ischemia and continuous groups. Endothelial reperfusion injury decreased in all nicorandil-treated groups. Administration of nicorandil attenuated ischemia-reperfusion injury of the myocardium and coronary endothelium while ameliorating leukocyte activation. In the event of unexpected prolonged cardioplegic arrest, administration of nicorandil, even just after declamping, may improve cardiac function. However, pre-ischemia administration alone was not helpful in the heart subjected to prolonged cardioplegic arrest.
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INTRODUCTION
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Nicorandil, N-(2-hydroxyethyl) nicotinamide nitrate, which possesses the hybrid effects of an adenosine triphosphate-sensitive potassium K(ATP) channel opener and a nitrovasodilator, is successfully used for the treatment of angina pectoris. The Impact of Nicorandil in Angina (IONA) study showed that treatment with nicorandil significantly reduced cardiovascular events in patients with stable angina pectoris.1 Notably, a preconditioning effect during percutaneous coronary angioplasty has been reported.2,3 In addition, some studies on the K(ATP) channel opener showed a cardioprotective effect that was beneficial to recovery of contractility, with reduction of infarct size and inhibition of neutrophil activation, in ischemia-reperfusion models.4–10 During cardiac surgery, cardiopulmonary bypass (CPB) causes an inflammatory response that includes activation of leukocytes, which may increase myocardial and endothelial reperfusion injury. Reperfusion injury plays an important role in postoperative myocardial dysfunction. Despite refinements in cardiac protection, the damage to the heart is thought to be potentially considerable, particularly after prolonged cardioplegic arrest with inflammatory responses. This study simulates a prolonged period of cardioplegic arrest with reperfusion of activated blood associated with the inflammatory response to extracorporeal circulation (ECC) to investigate whether nicorandil attenuates reperfusion injury and to determine the most effective timing of its administration in the field of cardiac surgery. Serum myeloperoxidase (MPO) activity was measured in support animals as an indicator of leukocyte activation caused by ECC.
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MATERIALS AND METHODS
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All animals involved in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996. All procedures were approved by the Animal Research Committee of the Saga Medical School. Adult male Japanese white rabbits weighing 2.9 to 4.0 kg were used as support animals. They were anesthetized with intramuscular ketamine (50 mg · kg–1), followed by intravenous pentobarbital sodium (5 to 7 mg · kg–1) and pancuronium bromide (0.08 mg · kg–1). A tracheostomy was performed for mechanical ventilation (model SN-480-5; Shinano Co, Tokyo, Japan) with 100% oxygen throughout the experiment. Anesthesia was maintained with intramuscular ketamine (25 mg · kg–1) every 30 min. After heparinization (500 U · kg–1), the right femoral artery was cannulated (18G; Terumo, Tokyo, Japan) for arterial blood pressure monitoring with a pressure transducer (model RM-6000; Nihon Kohden, Tokyo, Japan). Cannulation to establish ECC was made at the right carotid artery (18G; Terumo, Tokyo, Japan) and the left jugular vein (16G; Terumo, Tokyo, Japan). Adult male Japanese white rabbits weighing 3.0 to 3.6 kg were used as heart and blood donors. They were anesthetized and ventilated in the same manner as the support rabbits. After heparinization (500 U · kg–1), 45 mL of blood was collected from each heart donor animal after the same volume of lactated Ringer’s solution was infused. A rapid cardiectomy was performed through a median sternotomy.
A modified Langendorff column was primed with 45 mL blood from the heart donor rabbit and heparinized (2000 U per column) lactated Ringer’s solution, as previously described.11 Blood was drained actively from the carotid artery of the support animal using a pump (model MP-3; Rikakikai Co. Ltd, Tokyo, Japan) and fed into the modified Langendorff apparatus. The venous flow from the Langendorff column was returned to the jugular vein of the support animal using the same type of pump. The height of the column was set to give a pressure of 80 cm H2O. The systolic blood pressure of the support animal was maintained above 70 mm Hg, and the central venous pressure was maintained between 0 and 2 cm H2O throughout the experiment. The perfusion temperature was kept at 37°C. After cardiectomy, the aorta was cannulated and coronary perfusion was started immediately through the Langendorff column. The remaining blood from the heart donor animal was fed into the chambers of the extracorporeal circuit. A small incision was made at the apex of the left ventricle for venting. After a left atriotomy, a fluid-filled latex balloon was inserted into the left ventricle through the mitral valve and secured in place with a pursestring suture around the mitral annulus. The balloon was connected to a pressure transducer (model RM-6000; Nihon Kohden) for measurement of the left ventricular pressure-volume relationship. The zero-pressure reference was set at the level of the aortic valve. The heart was paced (model EDP 30/s; Biotronik, Inc., Berlin, Germany) at a constant rate throughout the experiment. The coronary blood flow rate was measured directly by timed collection of blood effluent from the coronary sinus. After a 30-min equilibration period, left ventricular developed pressure (LVDP), rate of positive pressure development (+dp/dt), and rate of negative pressure development (–dp/dt) were measured at a left ventricular end-diastolic pressure (LVEDP) of 10 mm Hg, as baseline data. If the baseline LVDP at an LVEDP of 10 mm Hg was less than 60 mm Hg, the heart was excluded from the study. After acquisition of baseline data, the fluid in the latex balloon was adjusted to obtain an LVEDP of 0 mm Hg.
To achieve cardioplegic arrest, 50 mL of St. Thomas’ Hospital solution (composition: NaCl 110.0 mmol · L–1, NaHCO3 10.0 mmol · L–1, KCl 16.0 mmol · L–1, MgCl2 16.0 mmol · L–1, and CaCl2 1.2 mmol · L–1) at 4°C was infused from a height of 80 cm through a separate column, based on surgical procedure. The effluent from the coronary sinus was collected and discarded. After cardiac arrest, the hearts were immersed in saline solution at 20°C for 4 hr. Heart arrest was maintained with additional cardioplegic solution (25 mL) every 30 min during the 4 hr of surgically relevant global ischemia. Blood was recirculated continuously between the support rabbit and the circuit of the Langendorff apparatus at a flow rate of 10 mL · min–1 to simulate the stimulation of leukocytes by ECC. After 4 hr of cardioplegic arrest, heparin (2000 U per column) was added, and the heart was reperfused with blood for 60 min. Left ventricular developed pressure, +dp/dt, and –dp/dt at an LVEDP of 10 mm Hg, as well as coronary flow, were measured 60 min after starting reperfusion. Blood gas analysis was repeated to ensure the stability of the support animals throughout the experiment. At the end of the experiment, St. Thomas’ Hospital solution was perfused into the isolated heart. The tissue was fixed by perfusing 2.5% glutaraldehyde in 0.l M cacodylate buffer with 3% sucrose, at a perfusion pressure of 80 cm H2O.
The dose of nicorandil was set at 2–3 times the clinical dose to enhance its effect. Nicorandil (200 µg · kg–1 per column + 5 µg · kg–1 · min–1) was administered to support animals in groups of 8 as follows: from the start of extracorporeal circulation until the onset of global ischemia (pre-ischemia group); from 10 min before reperfusion until the end of reperfusion (post-ischemia group); from the start of extracorporeal circulation until the onset of global ischemia and again from 10 min before reperfusion until the end of reperfusion (pre/post-ischemia group); continuously from the start of ECC until the end of reperfusion (continuous group). In the control group, nicorandil was not administered.
Blood samples were collected from the carotid artery of the support animal at 3 time points: before ECC, before the ischemic period, and after 1 hr of reperfusion. The blood samples were immediately centrifuged (3,000 rpm for 10 min), and serum samples were frozen (–80°C) until MPO measurement could be performed. The reaction mixture consisted of a serum sample, 20 mM tetramethylbenzidine, 0.3 mM H2O2, 153.8 mM sodium phosphate buffer (pH 5.4), N,N-dimethylformamide, and phosphate-buffered saline in a total volume of 225 µL. The mixture was incubated for 10 min at room temperature. The reaction was terminated by the addition of H2SO4. Myeloperoxidase product was measured in a spectrophotometer at a wavelength of 450 nm. One control sample was diluted by factors of 2–3 to 2–6 to obtain the standard curve. The MPO activity of each sample was measured using the standard curve from the control sample. The increased levels following global ischemia and reperfusion were calculated by comparison with the value before ECC.
For scanning electron microscopy, the heart was stored in the same fixative until it was studied. After the heart was rinsed with 0.l M cacodylate buffer, it was dehydrated through an ethanol series, and freeze dried. The tissue specimen was coated with gold (IB-3 ion coater; Eiko Ltd, Mito, Japan) and observed with a scanning electron microscope (JSM-5200LV; JEOL Ltd., Tokyo, Japan). Endothelial delamination or deposited blood cells indicated injured endothelium, and the surface area of the lesion so defined was measured (Mac Scope; Mitani Co. Ltd., Fukui, Japan).
Results are expressed as the mean ± standard deviation. Statistical analyses were performed by analysis of variance (ANOVA), followed by the Fisher’s parametric least significant difference test, as appropriate. Differences were considered significant at a p value of less than 0.05.
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RESULTS
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The condition of the support animals throughout the ECC was quite stable in all groups, without significant changes in blood pressure or arterial blood gas analysis data. There were no significant differences among groups. The donor heart baseline data for LVDP, +dp/dt, and –dp/dt, at an LVEDP of 10 mm Hg are shown in Table 1
; again, there were no significant differences among the groups. The percentage recovery of LVDP compared with pre-ischemic baseline data is shown in Figure 1. In the control group, the recovery of LVDP was 50.9% ± 8.1% at 60 min after reperfusion. Recovery of LVDP was enhanced in the nicorandil-treated groups, except in the pre-ischemia group. The differences between the control group and the pre-ischemia, post-ischemia, pre/post-ischemia, and continuous groups had p values of 0.6, 0.0091, 0.0008, and 0.0312, respectively. The recovery of +dp/dt compared with pre-ischemic baseline data is shown in Figure 2. In the control group, the recovery of +dp/dt was 52.9% ± 9.8% at 60 min after reperfusion. Recovery of +dp/dt was enhanced in the nicorandil-treated groups, except in the pre-ischemia group. The differences between the control group and the pre-ischemia, post-ischemia, pre/post-ischemia, and continuous groups had p values of 0.7370, 0.0146, 0.0023, and 0.0464, respectively. The recovery of –dp/dt compared with the pre-ischemic baseline data is shown in Figure 3. In the control group, the recovery of –dp/dt was 44.9% ± 10.4% at 60 min after reperfusion. Recovery of –dp/dt was enhanced in the nicorandil-treated groups, except in the pre-ischemia group. The differences between the control group and the pre-ischemia, post-ischemia, pre/post-ischemia, and continuous groups had p values of 0.3772, 0.0244, 0.0020, and 0.0195, respectively.
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Figure 1. Percentage recovery of left ventricular developed pressure (LVDP). *p < 0.05 vs control group.
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The increase of serum MPO activity compared with the pre-ischemic baseline data is shown in Figure 4. In the control group, MPO activity was increased to 307% ± 132% of the baseline level at 60 min after reperfusion, when the total ECC time was approximately 330 min (30 min perfusion before ischemia, 4 hr cardioplegic arrest, and 60 min reperfusion). The increase of MPO activity was ameliorated in the pre/post ischemia nicorandil-treated group, as well as in the continuously nicorandil-treated group. The differences between the control group and the pre-ischemia, post-ischemia, pre/post-ischemia, and continuous groups had p values of 0.8462, 0.0911, 0.0169, and 0.0011, respectively.
When the coronary endothelium was examined using scanning electron microscopy, many endothelial cells were detached; collagen fibers were exposed and had adhered blood cells, particularly in the control group, as reported previously.11,12 The area of such injury was 62.4% ± 16.9% of the observed endothelium in the control group. The injured area of coronary endothelium was decreased in the nicorandil-treated groups ( p < 0.0001 for control vs nicorandil-treated groups; Figure 5).
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Figure 5. Percentage of injured area of the coronary endothelium on scanning electron microscopy, determined by detached endothelium and adhered blood cells. *p < 0.05 vs control group.
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DISCUSSION
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In spite of improved cardioprotective techniques in cardiac surgery, post-cardiotomy cardiogenic shock has been experienced, particularly after prolonged cardioplegic arrest. After a long period of cardioplegic arrest, even with adequate myocardial protection by appropriate administration of cardioplegia, activated leukocytes due to inflammatory responses to prolonged CPB injure the myocardium and coronary endothelium during reperfusion of the heart more severely than after shorter durations of cardioplegic arrest and CPB. In this animal model, myocardial and coronary endothelial damage was clearly demonstrated in the control group with 4 hr of cardioplegic arrest, as reported before.11,12 The contractility of the isolated heart remained significantly decreased, even with 60 min reperfusion after ischemia. If this had been in a clinical setting, circulatory assist might have been needed. This experimental model might partly simulate the damaged heart in post-cardiotomy cardiogenic shock with reperfusion injury associated with activated leukocytes caused by ECC, with increased MPO activity. This study revealed that administration of nicorandil, even after ischemia, attenuated reperfusion injury with improved myocardial contractility, limited coronary endothelial damage, and decreased MPO activity.
Leukocyte activation is reported to be a source of reperfusion injury after ECC.12,13 We measured serum MPO activity in support animals as an indicator of leukocyte activation caused by CPB. Although we performed partial ECC simulating CPB, MPO activity was increased to 307% ± 132% of the baseline level in the pre-ECC period in the control group. The increased MPO activity was significantly suppressed by nicorandil administration in the pre/post-ischemia and continuously treated groups, in which a greater total amount of nicorandil was administered. Previous studies have shown that the K(ATP) channel openers bimakalim, diazoxide, and nicorandil reduce creatinine kinase activity or neutrophil infiltration into the ischemic myocardium following a prolonged period of coronary artery occlusion and reperfusion.4–7,14–16 Pieper and Gross17 reported that nicorandil has a neutrophil-modulating property that is not reversed by a K(ATP) channel blocker. In our experiments, nicorandil reduced leukocyte activity in the serum of the support animals. From these studies, including the present study, it is apparent that nicorandil has potential efficacy in attenuating leukocyte activation.
Nicorandil also attenuates reperfusion injury of the coronary endothelium. Our previous study demonstrated that this type of endothelial reperfusion injury was successfully improved by leukocyte-depleted reperfusion.12 This indicated that better preserved endothelium had little consequence from leukocyte rolling in the nicorandil-treated group. It suggested that nicorandil suppresses the expression of adhesion molecules, and there is a possibility that nicorandil controls transcriptional factor for adhesion molecules.
Contractility was also improved when nicorandil was administered just after ischemia. This means that administration of nicorandil, even just after declamping in the case of unexpected prolonged cardioplegic arrest, may improve cardiac function. If a long duration of cardioplegic arrest is predicted, ischemia-reperfusion injury can be effectively attenuated by administration of nicorandil in both the pre- and post-ischemia periods. However, administration only pre-ischemia did not benefit the prolonged cardioplegic arrested heart. The reason why the recovery of LVDP in the continuous group was less than in the pre/post-ischemia group was thought to be due to a hypotensive dose of nicorandil in the continuous group. More investigation of the dosage will be required.
There are some reports of nicorandil having a preconditioning effect in rabbits when given before and during (but not after) reperfusion.8,9 In the present study, the contractility was better preserved in the post-ischemia group than in the controls, as was suppression of MPO activity with less damage to the coronary endothelium. In contrast, in our preliminary study, we administered nicorandil only during the pre-ischemic period and this failed to ameliorate the functional recovery of isolated hearts. The half-life of the first rapid phase of elimination of nicorandil is approximately 45 min;18 our models required 4 hr of ischemia. Our results confirm the effectiveness of nicorandil infusion during reperfusion, and support the idea that nicorandil may have other cardioprotective mechanisms aside from preconditioning. This means that administration of nicorandil to the patient even just after unexpected prolonged cardioplegic arrest may be effective to ameliorate ischemia-reperfusion injury of the heart in the clinical setting.
Our experimental model had some differences to those of other reports. We attempted to reveal the efficacy of nicorandil administration in the setting of cardiac surgery with prolonged cardioplegic arrest and CPB. We used nicorandil in anticipation not only of its preconditioning effect but also of a cardioprotective or antiinflammatory effect. The other K(ATP) channel openers, bimakalim and diazoxide, have been reported to have a myocardial preconditioning effect and to reduce both the infarcted area and transmural MPO activity, although various modes of usage were tried.4–7 Moreover, nicorandil also has a nitrovasodilative property. This complex of dual effects makes elucidation of the mechanisms of nicorandil more difficult. Although the details of the mechanisms were unknown, administration of nicorandil in the pre-ischemia and reperfusion periods was mandatory for ameliorating reperfusion injury after prolonged cardioplegic arrest.
It was concluded that nicorandil attenuated reperfusion injury of the myocardium and coronary endothelium after prolonged cardioplegic arrest, and depressed MPO activity. In the case of unexpected prolonged cardioplegic arrest, administration of nicorandil even just after declamping may improve cardiac function. An antiinflammatory effect of nicorandil, as well as the K(ATP) channel opening and nitrovasodilating effects, may play a significant role in the attenuation of reperfusion injury after prolonged cardioplegic arrest in the setting of cardiac surgery.
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ACKNOWLEDGMENTS
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The authors wish to express their profound gratitude to Dr. Hayashi for his guidance on the measurement of MPO activity, Mr. Toshimi Tabata and Mr. Shinichi Nakahara for their technical assistance, and Mr. Steven Sager for editing the manuscript.
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REFERENCES
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ABSTRACT
INTRODUCTION
