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Nicorandil reduces myocardial no-reflow by protection of endothelial function via the activation of KATP channel
Clinica Chimica Acta 374 (2006) 100 – 105 www.elsevier.com/locate/clinchim
Nicorandil reduces myocardial no-reflow by protection of endothelial function via the activation of KATP channel Jing-lin Zhao, Yue-jin Yang ⁎, Ji-lin Chen, Lian-ming Kang, Yuan Wu, Run-lin Gao Department of Cardiology, Cardiovascular Institute and Fu-Wai Heart Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Bei Li Shi Road 167, West City District, Beijing, 100037, China Received 23 April 2006; received in revised form 16 May 2006; accepted 29 May 2006 Available online 3 June 2006
Abstract Introduction: It has been found that nicorandil can attenuate myocardial no-reflow. However, the exact cause of this beneficial effect has remained unclear. We investigated whether the beneficial effect of nicorandil on myocardial no-reflow could be partly due to its protection against endothelial dysfunction. Methods: Ligation area and area of no-reflow were determined echocardiographically and pathologically in sixty-two animals randomized into 7 study groups: 9 controls, 9 nicorandil-treated, 8 glibenclamide (KATP channel blocker)-treated, 10 NG-monomethyl-L-arginine (L-NMMA, nonselective nitric oxide synthase antagonist)-treated, 10 nicorandil and glibenclamide-treated, 8 nicorandil and L-NMMA-treated and 8 shamoperated. The acute myocardial infarction and reperfusion model was created with one 3-h occlusion of the left anterior descending coronary artery followed by 2 h of reperfusion. Constitutive nitric oxide synthase (cNOS) activity and inducible nitric oxide synthase (iNOS) activity were also quantified. Results: Compared with the control group, nicorandil significantly improved ventricular function, increased coronary blood flow volume (P < 0.01), decreased area of no-reflow and reduced necrosis area. Nicorandil also increased the cNOS activity and decreased iNOS activity (P < 0.05). L-NMMA and glibenclamide abrogated the effects of nicorandil on ventricular function, coronary blood flow volume, area of noreflow, necrosis area and cNOS activity, but not iNOS activity. Conclusions: The beneficial effect of nicorandil on myocardial no-reflow could be due to its protection of endothelial function via the activation of KATP channel. © 2006 Elsevier B.V. All rights reserved. Keywords: Nicorandil; Acute myocardial infarction; No-reflow
1. Introduction The main goal of reperfusion therapy for acute myocardial infarction (AMI) is to restore both epicardial and microvascular blood flow to the ischemic myocardium. Primary percutaneous coronary intervention (PCI), the preferred treatment for AMI, can achieve normal epicardial coronary flow. However, studies have shown that despite complete restoration of epicardial vessel blood flow, myocardial tissue perfusion evaluated with myocardial contrast echocardiography (MCE) remains incomplete, known as no-reflow phenomenon [1,2], which accounts for 37% [3] of the patients with a first anterior AMI after ⁎ Corresponding author. E-mail address: [email protected] (Y. Yang). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.05.039
receiving coronary reflow. No-reflow has been associated with severe myocardial injury, progressive left ventricular remodeling, congestive heart failure, and poor prognosis [4–6]. Therefore, myocardial tissue perfusion is now accepted as a target of reperfusion therapy for AMI [7]. Although the beneficial effect of nicorandil (a nicotinamide derivative) administrated intravenously before [8] or after ischemia [9,10] on myocardial no-reflow is documented, the exact cause of this beneficial effect has remained unclear. There is evidence that endothelial dysfunction, characterized by a decreased synthesis of endotheliumderived nitric oxide (NO) [11–13], contributes to tissuelevel perfusion [14]. We hypothesized that the beneficial effect of nicorandil on myocardial no-reflow could be partly due to by its protection against endothelial dysfunction. In
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this study, we used a mini-swine model of AMI and reperfusion developed in our laboratory to assess the effects of nicorandil given after ischemia on myocardial no-reflow, constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activity. In addition, to see whether the effect of nicorandil on myocardial no-reflow is also attributable to the activation of KATP channel, the effect of KATP channel blocker glibenclamide was also assessed in the same animal models. 2. Methods The animals and protocol used in the study were approved by the Institutional Animal Care and Use Committee. The miniswines (30 ± 3 kg) were sedated with 10 mg/kg of azaperone intramuscularly, anesthetized with 10 mg/kg of thiopental intravenously. The swines were intubated and ventilated with a respirator (Siemens elema sv 900). A middle thoracotomy was performed, and the heart was suspended in a pericardial cradle. The middle and distal portion of left anterior descending coronary artery (LAD) was dissected free from surrounding tissue, and was encircled by a suture. The 2 ends of the suture were threaded through a length of plastic tubing, forming a snare, which could be tightened to achieve coronary artery occlusion. The right femoral artery and vein were cannulated for hemodynamic monitoring and contrast agent injection respectively. An ultrasonic flow probe was placed proximal to the site of occlusion. 2.1. Experimental protocol Sixty-two animals were randomized into 7 study groups: 9 controls, 9 nicorandil-treated, 8 glibenclamide (KATP channel blocker [15])-treated, 10 N G -monomethyl-L-arginine (LNMMA, nonselective nitric oxide synthase antagonist [16])treated, 10 nicorandil and glibenclamide-treated, 8 nicorandil and L-NMMA-treated and 8 sham-operated. In nicorandiltreated animals, the dose of nicorandil employed was 1.5-fold higher than that in the previous clinical study [10] based on body surface area [17]. Nicorandil (donated by Libang Pharma Co., Ltd., Xi-an) was thus given as a 120 μg/kg i.v. bolus, followed by an infusion administered at a rate of 4 μg/kg/min starting from 30 min before reperfusion to the end of protocol. In the animals treated with glibenclamide, glibenclamide (0.3 mg/kg) was administered i.v. bolus 30 min before coronary occlusion. In the animals treated with L-NMMA, LNMMA (15 mg/kg) was injected 30 min before and 2.5 h after coronary occlusion and 30 min after reperfusion. In the animals treated with combination of glibenclamide or LNMMA with nicorandil, 0.3 mg/kg glibenclamide or 15 mg/kg L-NMMA was also given, combined with nicorandil administered from 30 min before reperfusion to the end of protocol. Control animals received the same amount of saline intravenously. The former six groups were subjected to 3 h of coronary occlusion followed by 2 h of reperfusion. In the sham-operated animals, LAD was only encircled by a suture, but not occluded.
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2.2. Hemodynamics Fluid-filled multilumen balloon flotation (Swan-Ganz) and 7/8F pigtail catheters were inserted percutaneously under fluoroscopic guidance through the femoral vein and artery for cardiac output (CO) and left ventricular pressure measurements respectively. Cardiac output (CO) was measured with a flowdirected, thermodilution method with Edward's cardiac output computer. Three consecutive readings for CO were used for the final computation. The variability of thermodilution measurements in our laboratory was ± 3%. Hemodynamic data measurements were repeated at baseline, at 3 h of LAD occlusion and at 2 h of reperfusion. Coronary blood flow volume (CBV) was digitally measured by the ultrasonic flow probe connected to a flowmeter (Nikon Kohden Corp.) at baseline, immediately after release of occlusion (3 h) and at 2 h of reperfusion. 2.3. MCE evaluation Echocardiography was performed with HP 5500 machine (Philips Ultrasound). The transducer was fixed in position to obtain the same short-axis images of the left ventricle at the midpapillary muscle level. A warm-water bath acted as an acoustic interface between the heart and the transducer. A bolus of 0.05 ml/kg of sonovue (Bracco Inc, Geneva, Switzerland) was injected intravenously as a slow bolus during 30 s followed by 5-ml saline flush. For each MCE, enddiastolic images were acquired at a pulsing interval of 4 cardiac cycles during contrast injection to allow complete beam replenishment and demarcation between perfused and nonperfused tissue. The myocardial ligation area (LA) and the area of no-reflow (ANR) were identified as the region of unopacified myocardium by MCE at 3 h of LAD occlusion and at 2 h of reperfusion respectively. Ligation area, area of no-reflow and left ventricular wall area (LVWA) were traced and measured. Ligation area was expressed as a percentage of LVWA, whereas area of no-reflow was expressed as a percentage of ligation area. 2.4. Histopathological evaluation After completion of the experimental protocol, area of noreflow was delineated by intra-atrial injection of 1 ml/kg of the fluorescent dye thioflavin S (Sigma Chem. Co). The LAD was then reoccluded, and Evans blue dye was injected into the left atrium to determine ligation area. The swine was then euthanized and the heart explanted. After explantation, 5–6 LV slices of the heart were cut parallel to the atrioventricular groove. Under ultraviolet light in a dark room, the areas not perfused by thioflavin S were identified. Ligation area was defined as the region unstained by Evans blue, while area of noreflow was defined as the non-fluorescent area within ligation area. Six samples (each of 2 mm3) were immediately taken from transmural myocardium in the normal, reflow or no-reflow region of two slices, washed thoroughly with saline and snapfrozen in liquid nitrogen for the measurement of NOS activity.
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Fig. 1. The variation of area of no-reflow (%) and necrosis area (%). ⁎P < 0.05, †P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB, MCE, LA, ANR and NA represent nicorandil, glibenclamide, myocardial contrast echocardiography, ligation area, area of no-reflow and necrosis area respectively. Nicorandil reduced area of no-reflow and necrosis area. L-NMMA and glibenclamide abrogated the effects of nicorandil on area of no-reflow and necrosis area.
The other slices were incubated in a 1% solution of triphenyltetrazolium chloride (TTC) for 15 min at 37 °C. Regions that failed to demonstrate red staining were considered to represent necrosis area (NA). The outlines of the LVWA, ligation area, area of no-reflow and necrosis area were calculated. Ligation area was expressed as percentage of the LVWA; area of no-reflow and necrosis area as percentage of ligation area. 2.5. NOS activity assay Constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activities were determined using the [ 3 H] L -arginine–[ 3 H] L -citrulline conversion [18,19]. Briefly, the tissue lysates were sonicated with lysis buffer for 5 s twice on ice and centrifuged at 12,000×g for 5 min at 4 °C. The supernatant was collected and incubated with the buffer under one of three conditions: (a) calcium and calmodulin (containing cNOS and iNOS activity), (b) EDTA/ EGTA (containing iNOS activity), and (c) EDTA/EGTA plus nitro-L-arginine methylester (1 mmol/l; to account for nonspecific radioactivity and non-NOS-related conversion of [3H]L-arginine). These 3 incubation conditions allowed us to differentiate between calcium-dependent (cNOS) and calcium-independent (iNOS) NOS activity. All data were standardized to the protein level in the homogenate, with the Table 1 The variation of CBV (ml/min) Group
Baseline
3 h occlusion
2 h reperfusion
Sham Control NIC L-NMMA GLB NIC ± GLB NIC ± L-NMMA
29.5 ± 3.87 31.15 ± 4.14 30.12 ± 4.15 32.01 ± 5.11 31.57 ± 3.98 31.15 ± 4.14 31.15 ± 4.14
30.76 ± 4.42 15.75 ± 3.05⁎ 22.79 ± 3.1⁎,† 15.23 ± 4.12⁎ 14.37 ± 4.52⁎ 15.33 ± 3.64⁎ 14.89 ± 3.67⁎
30.21 ± 4.02 14.26 ± 4.15⁎ 22.17 ± 4.19⁎,† 15.71 ± 3.84⁎ 14.11 ± 3.51⁎ 15.06 ± 3.26⁎ 15.14 ± 5.31⁎
⁎P < 0.01 vs. baseline; †P < 0.05 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. CBV, NIC and GLB represent coronary blood flow volume, nicorandil and glibenclamide respectively.
protein concentration determined by a microplate modification of the Bradford [20] assay and expressed as U/mg protein. Table 2 The variation of hemodynamic data Group
Baseline
3 h occlusion
2 h reperfusion
Sham (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
115 ± 5 3.9 ± 2.0 2.67 ± 0.12
116 ± 4 3.8 ± 1.1 2.54 ± 0.09
116 ± 2 3.8 ± 1.9 2.65 ± 0.15
Control (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
116 ± 4 4.0 ± 1.5 2.58 ± 0.36
100 ± 4‡ 7.1 ± 2.0‡ 1.36 ± 0.29‡
109 ± 2†,§ 6.0 ± 1.3† 1.79 ± 0.25†
NIC (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
116 ± 6 3.9 ± 1.5 2.54 ± 0.47
99 ± 6† 6.9 ± 1.9‡ 1.37 ± 0.26‡
113 ± 2⁎,§ 4.3 ± 1.7⁎,§ 2.28 ± 0.25⁎,§
L-NMMA (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
114 ± 5 3.8 ± 0.9 2.61 ± 0.24
97 ± 4‡ 7.0 ± 1.6‡ 1.31 ± 0.17‡
107 ± 3†,§ 5.8 ± 2.1† 1.83 ± 0.27†
GLB (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
114 ± 3 4.0 ± 2.1 2.56 ± 0.16
98 ± 3‡ 6.8 ± 1.1‡ 1.36 ± 0.14‡
107 ± 2†,§ 5.9 ± 1.6† 1.74 ± 0.24†
NIC + L-NMMA (n = 8) LVSP (mm Hg) 114 ± 5 LVEDP (mm Hg) 3.9 ± 1.1 CO (l/min) 2.58 ± 0.36
99 ± 4‡ 7.2 ± 1.3‡ 1.41 ± 0.29‡
108 ± 3†,§ 6.1 ± 1.0† 1.79 ± 0.15†
NIC + GLB (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
100 ± 3‡ 7.0 ± 1.3‡ 1.39 ± 0.23‡
108 ± 3†,§ 5.9 ± 1.1† 1.84 ± 0.28†
114 ± 3 4.0 ± 1.7 2.6 ± 0.35
⁎P < 0.05 vs. control. †P < 0.05, ‡P < 0.01 vs. baseline. §P < 0.05 vs. 3 h ischemia. Data are expressed as the mean value ± S.E.M. NIC, GLB, LVSP, LVEDP and CO represent nicorandil, glibenclamide, left ventricular systolic pressure, left ventricular end-diastolic pressure and cardiac output respectively.
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Fig. 2. The variation of myocardial tissue cNOS activity (expressed as U/mg protein). ⁎P < 0.01 vs. normal region; †P < 0.01 vs. reflow region; ‡P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB and cNOS represent nicorandil, glibenclamide and constitutive nitric oxide synthase. Nicorandil increased cNOS activity. L-NMMA and glibenclamide abrogated the effect of nicorandil on cNOS activity.
2.6. Statistical methods Data are expressed as mean ± S.E.M. Comparisons of data among all stages were performed with repeated-measures ANOVA followed by Student–Newman–Keuls test for multiple comparison. Comparisons of ligation area, area of no-reflow and necrosis area among groups were done with one-way ANOVA followed by Dunnett test for multiple comparison with control. A value of P < 0.05 (2-sided) was considered statistically significant.
reflow was also similar (78.5% and 82.3% by the two methods, respectively), with final necrosis area reaching 99% of ligation area. Compared with the control group, area of noreflow in nicorandil group was significantly decreased to 51.1–53.4% (P < 0.01) with final necrosis area significantly decreased to 87.6% of ligation area (P < 0.05). There was no significant difference in area of no-reflow and necrosis area among L -NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on area of no-reflow and necrosis area (Fig. 1).
3. Results 3.2. Coronary blood flow volume Six mini-swines (2 receiving L-NMMA, 2 receiving nicorandil and glibenclamide, 1 receiving nicorandil and 1 control) died of ventricular fibrillation during the ischemia period and they were excluded. Therefore, 8 animals were evaluated in each group. 3.1. No-reflow and infarct size There was no significant difference in ligation area on MCE and pathological evaluation between 5 treated and control groups (P > 0.05). In the control group, the area of no-
In the control group, CBV was significantly declined at immediately after release of 3-h occlusion and at 2 h of reperfusion (all P < 0.01). In the nicorandil group, CBV was also significantly declined at the 2 time points above compared with the baseline (P < 0.01), but was significantly higher than that in the control group (P < 0.05). There was no significant difference in CBV among L-NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effect of nicorandil on CBV (Table 1).
Fig. 3. The variation of myocardial tissue iNOS activity (expressed as U/mg protein). ⁎P < 0.01 vs. normal region; †P < 0.01 vs. reflow region; ‡P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB and iNOS represent nicorandil, glibenclamide and inducible nitric oxide synthase. Nicorandil decreased iNOS activity. L-NMMA and glibenclamide did not abrogate the effect of nicorandil on iNOS activity.
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3.3. Hemodynamics In control group, left ventricular systolic pressure (LVSP), and cardiac output (CO) significantly declined (P < 0.05– 0.01), while left ventricular end-diastolic pressure (LVEDP) significantly increased at 2 h of reperfusion (P < 0.01). Compared with those in the control group, CO and LVEDP significantly recovered at 2 h of reperfusion in nicorandil group (all P < 0.05). There was no significant difference in hemodynamic data among L-NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on hemodynamic data (Table 2). 3.4. The effects of nicorandil on cNOS activity and iNOS activity in the myocardium In the control and nicorandil groups, cNOS activity in the reflow and no-reflow myocardium was significantly lower than that in normal myocardium (P < 0.01), while the iNOS activity was significantly lower than that in normal myocardium (P < 0.01). In nicorandil group, cNOS activity or iNOS activity was significantly higher or lower than that in the control group (P < 0.01). In the L-NMMA group, cNOS activity and iNOS activity were significantly lower than in the control group (P < 0.05). There was no significant difference in cNOS and iNOS activity between glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on cNOS activity, but not iNOS activity (Figs. 2 and 3). 4. Discussion Nicorandil has a potassium channel-opening effect that leads to the activation of KATP channels and induces nitric oxide in the same way as nitrates, resulting in increased intracellular cyclic GMP [21] . These effects of nicorandil are known to dilate large pericardial vessels in the same action as nitrates including nitroglycerin, and also to increase the coronary blood flow by dilating small coronary resistance vessels [21]. Currently, it has been found to attenuate no-reflow. However, the exact cause of this beneficial effect remains unclear. The present study demonstrated that nicorandil decreased the area of no-reflow and improved CBV. Our data directly established that nicorandil exerts a favorable effect on microvascular perfusion after restoration of flow in epicardial vessels, which is in agreement with the reports of Ito et al. [9] and Ono et al. [10]. The proposed mechanism of the no-reflow phenomenon is multifactorial. Animal and postmortem histologic studies have demonstrated varying degrees of small-vessel vasospasm, endothelial gap and bleb formation, neutrophil plugging of capillaries as well as microvascular compression from myocytes, interstitial edema, and hemorrhage after recanalization [22,23]. The mechanism by which nicorandil administrated before reperfusion is beneficial in the reduction of the no-reflow is thought to be involved in KATP channels opening and microvascular vessels dilating.
Our study provides another possible mechanism for the beneficial effect of nicorandil on myocardial no-reflow. Endothelial dysfunction can be characterized by a decreased synthesis of endothelium-derived nitric oxide (NO). NO synthase (NOS) isoforms are the enzymes responsible for NO generation. NOS isoforms are divided into two categories according to calmodulin and calcium: cNOS (including neuronal NOS (type 1) and endothelial NOS (type 3)) and iNOS (type 2). The cNOS and iNOS were mainly produced by the vascular endothelium and macrophage respectively [24]. The evidence which data provided showed that NO synthesized through cNOS has cardiac protection role, while high concentrations of NO which synthesized through iNOS rapidly interacts with oxygen to yield the potent oxidant peroxynitrite (ONOO−) and subsequently induces and exacerbates myocardial ischemia reperfusion injury [25]. The present study demonstrated that nicorandil increased constitutive NOS activity and decreased inducible NOS activity, and L-NMMA (nonselective nitric oxide synthase antagonist) abrogated the effects of nicorandil on cNOS activity, but not iNOS activity, which is in agreement with the report of Shigeo et al. [26], implying the beneficial effect of nicorandil may be also partly due to its protection of endothelial function. They further thought that the effect of nicorandil on cNOS activity was mediated by increasing intracellular calcium via the KATP channel, because the KATP channel inhibitor glibenclamide inhibited it. Our study also showed that the necrosis area comprised 99% of the ligation area in a mini-swine model of AMI (3 h) and reperfusion, while nicorandil decreased necrosis area to 87.6%, which is similar with the reports of Lamping et al. [27] and Tsuchida et al. [28]. The mechanism of nicorandil in reducing infarct size is not well defined and may be explained by (1) preventing myocardial no-reflow, (2) activating the KATP channels and (3) inhibitory effect on reactive oxygen species formation. The data from this study also showed that nicorandil improved ventricular function, which is in agreement with the report of Auchampach et al. [29]. The beneficial effect of nicorandil on ventricular function was due not only to decreased myocardial necrosis and attenuation of myocardial stunning but also to preservation of microvascular integrity and improved myocardial tissue perfusion during AMI and reperfusion. 5. Study limitations This study has several limitations. Because we assessed infarct size at 2 h of reperfusion, the ultimate infarct size may be larger. However, since all animals were evaluated at the same point, the comparative result cannot be affected. Results were observed in a short-term experimental setting, and no long-term data are available. In conclusion, the present study demonstrated that nicorandil attenuated myocardial no-reflow, associated with increased cNOS activity and decreased iNOS activity. The present study also showed that L-NMMA and glibenclamide abrogated the effects of nicorandil on cNOS activity. These results suggested that the beneficial effect of
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nicorandil on myocardial no-reflow was due to improvement of endothelial function via the activation of KATP channel. Acknowledgement This study was supported, in part, by a grant-in-aid (30572439) from the National Natural Science Foundation of China. References [1] Ito H, Okamura A, Iwakura K, et al. Myocardial perfusion patterns related to thrombolysis in myocardial infarction perfusion grades after coronary angioplasty in patients with acute anterior wall myocardial infarction. Circulation 1996;93:1993–9. [2] van't Hof AW, Liem A, de Boer MJ, et al. Clinical value of 12-lead electrocardiogram after successful reperfusion therapy for acute myocardial infarction. Zwolle Myocardial infarction Study Group. Lancet 1997;350:615–9. [3] Ito H, Maruyama A, Iwakura K, et al. Clinical implications of the ‘noreflow’ phenomenon: a predictor of complications and left ventricular remodeling in perfused anterior wall myocardial infarction. Circulation 1996;93:223–8. [4] Wu KC, Zerhouni EA, Judd RM, et al. Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation 1998;97:765–72. [5] Swinburn JM, Lahiri A, Senior R. Intravenous myocardial contrast echocardiography predicts recovery of dysynergic myocardium early after acute myocardial infarction. J Am Coll Cardiol 2001;38:19–25. [6] Reffelmann T, Kloner RA. The “no-reflow” phenomenon: basic science and clinical correlates. Heart 2002;87:162–8. [7] Gersh BJ. Optimal management of acute myocardial infarction at the down of the next millennium. Am Heart J 1999;138:188–202. [8] Zhao JL, Yang YJ, You SJ, et al. Beneficial effects of nicorandil on myocardial no-reflow state in a mini-swine model of acute myocardial infarction and reperfusion. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 2005;17:421–5. [9] Ito H, Taniyama Y, Iwakura K, et al. Intravenous nicorandil can preserve microvascular integrity and myocardial viability in patients with reperfused anterior wall myocardial infarction. J Am Coll Cardiol 1999;33:654–60. [10] Ono H, Osanai T, Ishizaka H, et al. Nicorandil improves cardiac function and clinical outcome in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention: role of inhibitory effect on reactive oxygen species formation. Am Heart J 2004;148:E15. [11] Linke A, Recchia F, Zhang X, et al. Acute and chronic endothelial dysfunction: implications for the development of heart failure. Heart Fail Rev 2003;8:87–97. [12] Kawashima S, Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol 2004;24: 998–1005.
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Nicorandil reduces myocardial no-reflow by protection of endothelial function via the activation of KATP channel Jing-lin Zhao, Yue-jin Yang ⁎, Ji-lin Chen, Lian-ming Kang, Yuan Wu, Run-lin Gao Department of Cardiology, Cardiovascular Institute and Fu-Wai Heart Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Bei Li Shi Road 167, West City District, Beijing, 100037, China Received 23 April 2006; received in revised form 16 May 2006; accepted 29 May 2006 Available online 3 June 2006
Abstract Introduction: It has been found that nicorandil can attenuate myocardial no-reflow. However, the exact cause of this beneficial effect has remained unclear. We investigated whether the beneficial effect of nicorandil on myocardial no-reflow could be partly due to its protection against endothelial dysfunction. Methods: Ligation area and area of no-reflow were determined echocardiographically and pathologically in sixty-two animals randomized into 7 study groups: 9 controls, 9 nicorandil-treated, 8 glibenclamide (KATP channel blocker)-treated, 10 NG-monomethyl-L-arginine (L-NMMA, nonselective nitric oxide synthase antagonist)-treated, 10 nicorandil and glibenclamide-treated, 8 nicorandil and L-NMMA-treated and 8 shamoperated. The acute myocardial infarction and reperfusion model was created with one 3-h occlusion of the left anterior descending coronary artery followed by 2 h of reperfusion. Constitutive nitric oxide synthase (cNOS) activity and inducible nitric oxide synthase (iNOS) activity were also quantified. Results: Compared with the control group, nicorandil significantly improved ventricular function, increased coronary blood flow volume (P < 0.01), decreased area of no-reflow and reduced necrosis area. Nicorandil also increased the cNOS activity and decreased iNOS activity (P < 0.05). L-NMMA and glibenclamide abrogated the effects of nicorandil on ventricular function, coronary blood flow volume, area of noreflow, necrosis area and cNOS activity, but not iNOS activity. Conclusions: The beneficial effect of nicorandil on myocardial no-reflow could be due to its protection of endothelial function via the activation of KATP channel. © 2006 Elsevier B.V. All rights reserved. Keywords: Nicorandil; Acute myocardial infarction; No-reflow
1. Introduction The main goal of reperfusion therapy for acute myocardial infarction (AMI) is to restore both epicardial and microvascular blood flow to the ischemic myocardium. Primary percutaneous coronary intervention (PCI), the preferred treatment for AMI, can achieve normal epicardial coronary flow. However, studies have shown that despite complete restoration of epicardial vessel blood flow, myocardial tissue perfusion evaluated with myocardial contrast echocardiography (MCE) remains incomplete, known as no-reflow phenomenon [1,2], which accounts for 37% [3] of the patients with a first anterior AMI after ⁎ Corresponding author. E-mail address: [email protected] (Y. Yang). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.05.039
receiving coronary reflow. No-reflow has been associated with severe myocardial injury, progressive left ventricular remodeling, congestive heart failure, and poor prognosis [4–6]. Therefore, myocardial tissue perfusion is now accepted as a target of reperfusion therapy for AMI [7]. Although the beneficial effect of nicorandil (a nicotinamide derivative) administrated intravenously before [8] or after ischemia [9,10] on myocardial no-reflow is documented, the exact cause of this beneficial effect has remained unclear. There is evidence that endothelial dysfunction, characterized by a decreased synthesis of endotheliumderived nitric oxide (NO) [11–13], contributes to tissuelevel perfusion [14]. We hypothesized that the beneficial effect of nicorandil on myocardial no-reflow could be partly due to by its protection against endothelial dysfunction. In
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this study, we used a mini-swine model of AMI and reperfusion developed in our laboratory to assess the effects of nicorandil given after ischemia on myocardial no-reflow, constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activity. In addition, to see whether the effect of nicorandil on myocardial no-reflow is also attributable to the activation of KATP channel, the effect of KATP channel blocker glibenclamide was also assessed in the same animal models. 2. Methods The animals and protocol used in the study were approved by the Institutional Animal Care and Use Committee. The miniswines (30 ± 3 kg) were sedated with 10 mg/kg of azaperone intramuscularly, anesthetized with 10 mg/kg of thiopental intravenously. The swines were intubated and ventilated with a respirator (Siemens elema sv 900). A middle thoracotomy was performed, and the heart was suspended in a pericardial cradle. The middle and distal portion of left anterior descending coronary artery (LAD) was dissected free from surrounding tissue, and was encircled by a suture. The 2 ends of the suture were threaded through a length of plastic tubing, forming a snare, which could be tightened to achieve coronary artery occlusion. The right femoral artery and vein were cannulated for hemodynamic monitoring and contrast agent injection respectively. An ultrasonic flow probe was placed proximal to the site of occlusion. 2.1. Experimental protocol Sixty-two animals were randomized into 7 study groups: 9 controls, 9 nicorandil-treated, 8 glibenclamide (KATP channel blocker [15])-treated, 10 N G -monomethyl-L-arginine (LNMMA, nonselective nitric oxide synthase antagonist [16])treated, 10 nicorandil and glibenclamide-treated, 8 nicorandil and L-NMMA-treated and 8 sham-operated. In nicorandiltreated animals, the dose of nicorandil employed was 1.5-fold higher than that in the previous clinical study [10] based on body surface area [17]. Nicorandil (donated by Libang Pharma Co., Ltd., Xi-an) was thus given as a 120 μg/kg i.v. bolus, followed by an infusion administered at a rate of 4 μg/kg/min starting from 30 min before reperfusion to the end of protocol. In the animals treated with glibenclamide, glibenclamide (0.3 mg/kg) was administered i.v. bolus 30 min before coronary occlusion. In the animals treated with L-NMMA, LNMMA (15 mg/kg) was injected 30 min before and 2.5 h after coronary occlusion and 30 min after reperfusion. In the animals treated with combination of glibenclamide or LNMMA with nicorandil, 0.3 mg/kg glibenclamide or 15 mg/kg L-NMMA was also given, combined with nicorandil administered from 30 min before reperfusion to the end of protocol. Control animals received the same amount of saline intravenously. The former six groups were subjected to 3 h of coronary occlusion followed by 2 h of reperfusion. In the sham-operated animals, LAD was only encircled by a suture, but not occluded.
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2.2. Hemodynamics Fluid-filled multilumen balloon flotation (Swan-Ganz) and 7/8F pigtail catheters were inserted percutaneously under fluoroscopic guidance through the femoral vein and artery for cardiac output (CO) and left ventricular pressure measurements respectively. Cardiac output (CO) was measured with a flowdirected, thermodilution method with Edward's cardiac output computer. Three consecutive readings for CO were used for the final computation. The variability of thermodilution measurements in our laboratory was ± 3%. Hemodynamic data measurements were repeated at baseline, at 3 h of LAD occlusion and at 2 h of reperfusion. Coronary blood flow volume (CBV) was digitally measured by the ultrasonic flow probe connected to a flowmeter (Nikon Kohden Corp.) at baseline, immediately after release of occlusion (3 h) and at 2 h of reperfusion. 2.3. MCE evaluation Echocardiography was performed with HP 5500 machine (Philips Ultrasound). The transducer was fixed in position to obtain the same short-axis images of the left ventricle at the midpapillary muscle level. A warm-water bath acted as an acoustic interface between the heart and the transducer. A bolus of 0.05 ml/kg of sonovue (Bracco Inc, Geneva, Switzerland) was injected intravenously as a slow bolus during 30 s followed by 5-ml saline flush. For each MCE, enddiastolic images were acquired at a pulsing interval of 4 cardiac cycles during contrast injection to allow complete beam replenishment and demarcation between perfused and nonperfused tissue. The myocardial ligation area (LA) and the area of no-reflow (ANR) were identified as the region of unopacified myocardium by MCE at 3 h of LAD occlusion and at 2 h of reperfusion respectively. Ligation area, area of no-reflow and left ventricular wall area (LVWA) were traced and measured. Ligation area was expressed as a percentage of LVWA, whereas area of no-reflow was expressed as a percentage of ligation area. 2.4. Histopathological evaluation After completion of the experimental protocol, area of noreflow was delineated by intra-atrial injection of 1 ml/kg of the fluorescent dye thioflavin S (Sigma Chem. Co). The LAD was then reoccluded, and Evans blue dye was injected into the left atrium to determine ligation area. The swine was then euthanized and the heart explanted. After explantation, 5–6 LV slices of the heart were cut parallel to the atrioventricular groove. Under ultraviolet light in a dark room, the areas not perfused by thioflavin S were identified. Ligation area was defined as the region unstained by Evans blue, while area of noreflow was defined as the non-fluorescent area within ligation area. Six samples (each of 2 mm3) were immediately taken from transmural myocardium in the normal, reflow or no-reflow region of two slices, washed thoroughly with saline and snapfrozen in liquid nitrogen for the measurement of NOS activity.
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Fig. 1. The variation of area of no-reflow (%) and necrosis area (%). ⁎P < 0.05, †P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB, MCE, LA, ANR and NA represent nicorandil, glibenclamide, myocardial contrast echocardiography, ligation area, area of no-reflow and necrosis area respectively. Nicorandil reduced area of no-reflow and necrosis area. L-NMMA and glibenclamide abrogated the effects of nicorandil on area of no-reflow and necrosis area.
The other slices were incubated in a 1% solution of triphenyltetrazolium chloride (TTC) for 15 min at 37 °C. Regions that failed to demonstrate red staining were considered to represent necrosis area (NA). The outlines of the LVWA, ligation area, area of no-reflow and necrosis area were calculated. Ligation area was expressed as percentage of the LVWA; area of no-reflow and necrosis area as percentage of ligation area. 2.5. NOS activity assay Constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activities were determined using the [ 3 H] L -arginine–[ 3 H] L -citrulline conversion [18,19]. Briefly, the tissue lysates were sonicated with lysis buffer for 5 s twice on ice and centrifuged at 12,000×g for 5 min at 4 °C. The supernatant was collected and incubated with the buffer under one of three conditions: (a) calcium and calmodulin (containing cNOS and iNOS activity), (b) EDTA/ EGTA (containing iNOS activity), and (c) EDTA/EGTA plus nitro-L-arginine methylester (1 mmol/l; to account for nonspecific radioactivity and non-NOS-related conversion of [3H]L-arginine). These 3 incubation conditions allowed us to differentiate between calcium-dependent (cNOS) and calcium-independent (iNOS) NOS activity. All data were standardized to the protein level in the homogenate, with the Table 1 The variation of CBV (ml/min) Group
Baseline
3 h occlusion
2 h reperfusion
Sham Control NIC L-NMMA GLB NIC ± GLB NIC ± L-NMMA
29.5 ± 3.87 31.15 ± 4.14 30.12 ± 4.15 32.01 ± 5.11 31.57 ± 3.98 31.15 ± 4.14 31.15 ± 4.14
30.76 ± 4.42 15.75 ± 3.05⁎ 22.79 ± 3.1⁎,† 15.23 ± 4.12⁎ 14.37 ± 4.52⁎ 15.33 ± 3.64⁎ 14.89 ± 3.67⁎
30.21 ± 4.02 14.26 ± 4.15⁎ 22.17 ± 4.19⁎,† 15.71 ± 3.84⁎ 14.11 ± 3.51⁎ 15.06 ± 3.26⁎ 15.14 ± 5.31⁎
⁎P < 0.01 vs. baseline; †P < 0.05 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. CBV, NIC and GLB represent coronary blood flow volume, nicorandil and glibenclamide respectively.
protein concentration determined by a microplate modification of the Bradford [20] assay and expressed as U/mg protein. Table 2 The variation of hemodynamic data Group
Baseline
3 h occlusion
2 h reperfusion
Sham (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
115 ± 5 3.9 ± 2.0 2.67 ± 0.12
116 ± 4 3.8 ± 1.1 2.54 ± 0.09
116 ± 2 3.8 ± 1.9 2.65 ± 0.15
Control (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
116 ± 4 4.0 ± 1.5 2.58 ± 0.36
100 ± 4‡ 7.1 ± 2.0‡ 1.36 ± 0.29‡
109 ± 2†,§ 6.0 ± 1.3† 1.79 ± 0.25†
NIC (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
116 ± 6 3.9 ± 1.5 2.54 ± 0.47
99 ± 6† 6.9 ± 1.9‡ 1.37 ± 0.26‡
113 ± 2⁎,§ 4.3 ± 1.7⁎,§ 2.28 ± 0.25⁎,§
L-NMMA (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
114 ± 5 3.8 ± 0.9 2.61 ± 0.24
97 ± 4‡ 7.0 ± 1.6‡ 1.31 ± 0.17‡
107 ± 3†,§ 5.8 ± 2.1† 1.83 ± 0.27†
GLB (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
114 ± 3 4.0 ± 2.1 2.56 ± 0.16
98 ± 3‡ 6.8 ± 1.1‡ 1.36 ± 0.14‡
107 ± 2†,§ 5.9 ± 1.6† 1.74 ± 0.24†
NIC + L-NMMA (n = 8) LVSP (mm Hg) 114 ± 5 LVEDP (mm Hg) 3.9 ± 1.1 CO (l/min) 2.58 ± 0.36
99 ± 4‡ 7.2 ± 1.3‡ 1.41 ± 0.29‡
108 ± 3†,§ 6.1 ± 1.0† 1.79 ± 0.15†
NIC + GLB (n = 8) LVSP (mm Hg) LVEDP (mm Hg) CO (l/min)
100 ± 3‡ 7.0 ± 1.3‡ 1.39 ± 0.23‡
108 ± 3†,§ 5.9 ± 1.1† 1.84 ± 0.28†
114 ± 3 4.0 ± 1.7 2.6 ± 0.35
⁎P < 0.05 vs. control. †P < 0.05, ‡P < 0.01 vs. baseline. §P < 0.05 vs. 3 h ischemia. Data are expressed as the mean value ± S.E.M. NIC, GLB, LVSP, LVEDP and CO represent nicorandil, glibenclamide, left ventricular systolic pressure, left ventricular end-diastolic pressure and cardiac output respectively.
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Fig. 2. The variation of myocardial tissue cNOS activity (expressed as U/mg protein). ⁎P < 0.01 vs. normal region; †P < 0.01 vs. reflow region; ‡P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB and cNOS represent nicorandil, glibenclamide and constitutive nitric oxide synthase. Nicorandil increased cNOS activity. L-NMMA and glibenclamide abrogated the effect of nicorandil on cNOS activity.
2.6. Statistical methods Data are expressed as mean ± S.E.M. Comparisons of data among all stages were performed with repeated-measures ANOVA followed by Student–Newman–Keuls test for multiple comparison. Comparisons of ligation area, area of no-reflow and necrosis area among groups were done with one-way ANOVA followed by Dunnett test for multiple comparison with control. A value of P < 0.05 (2-sided) was considered statistically significant.
reflow was also similar (78.5% and 82.3% by the two methods, respectively), with final necrosis area reaching 99% of ligation area. Compared with the control group, area of noreflow in nicorandil group was significantly decreased to 51.1–53.4% (P < 0.01) with final necrosis area significantly decreased to 87.6% of ligation area (P < 0.05). There was no significant difference in area of no-reflow and necrosis area among L -NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on area of no-reflow and necrosis area (Fig. 1).
3. Results 3.2. Coronary blood flow volume Six mini-swines (2 receiving L-NMMA, 2 receiving nicorandil and glibenclamide, 1 receiving nicorandil and 1 control) died of ventricular fibrillation during the ischemia period and they were excluded. Therefore, 8 animals were evaluated in each group. 3.1. No-reflow and infarct size There was no significant difference in ligation area on MCE and pathological evaluation between 5 treated and control groups (P > 0.05). In the control group, the area of no-
In the control group, CBV was significantly declined at immediately after release of 3-h occlusion and at 2 h of reperfusion (all P < 0.01). In the nicorandil group, CBV was also significantly declined at the 2 time points above compared with the baseline (P < 0.01), but was significantly higher than that in the control group (P < 0.05). There was no significant difference in CBV among L-NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effect of nicorandil on CBV (Table 1).
Fig. 3. The variation of myocardial tissue iNOS activity (expressed as U/mg protein). ⁎P < 0.01 vs. normal region; †P < 0.01 vs. reflow region; ‡P < 0.01 vs. control group. Data are expressed as the mean value ± S.E.M.; n = 8 per group. NIC, GLB and iNOS represent nicorandil, glibenclamide and inducible nitric oxide synthase. Nicorandil decreased iNOS activity. L-NMMA and glibenclamide did not abrogate the effect of nicorandil on iNOS activity.
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3.3. Hemodynamics In control group, left ventricular systolic pressure (LVSP), and cardiac output (CO) significantly declined (P < 0.05– 0.01), while left ventricular end-diastolic pressure (LVEDP) significantly increased at 2 h of reperfusion (P < 0.01). Compared with those in the control group, CO and LVEDP significantly recovered at 2 h of reperfusion in nicorandil group (all P < 0.05). There was no significant difference in hemodynamic data among L-NMMA, glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on hemodynamic data (Table 2). 3.4. The effects of nicorandil on cNOS activity and iNOS activity in the myocardium In the control and nicorandil groups, cNOS activity in the reflow and no-reflow myocardium was significantly lower than that in normal myocardium (P < 0.01), while the iNOS activity was significantly lower than that in normal myocardium (P < 0.01). In nicorandil group, cNOS activity or iNOS activity was significantly higher or lower than that in the control group (P < 0.01). In the L-NMMA group, cNOS activity and iNOS activity were significantly lower than in the control group (P < 0.05). There was no significant difference in cNOS and iNOS activity between glibenclamide and control groups (P > 0.05). In contrast, L-NMMA and glibenclamide abrogated the effects of nicorandil on cNOS activity, but not iNOS activity (Figs. 2 and 3). 4. Discussion Nicorandil has a potassium channel-opening effect that leads to the activation of KATP channels and induces nitric oxide in the same way as nitrates, resulting in increased intracellular cyclic GMP [21] . These effects of nicorandil are known to dilate large pericardial vessels in the same action as nitrates including nitroglycerin, and also to increase the coronary blood flow by dilating small coronary resistance vessels [21]. Currently, it has been found to attenuate no-reflow. However, the exact cause of this beneficial effect remains unclear. The present study demonstrated that nicorandil decreased the area of no-reflow and improved CBV. Our data directly established that nicorandil exerts a favorable effect on microvascular perfusion after restoration of flow in epicardial vessels, which is in agreement with the reports of Ito et al. [9] and Ono et al. [10]. The proposed mechanism of the no-reflow phenomenon is multifactorial. Animal and postmortem histologic studies have demonstrated varying degrees of small-vessel vasospasm, endothelial gap and bleb formation, neutrophil plugging of capillaries as well as microvascular compression from myocytes, interstitial edema, and hemorrhage after recanalization [22,23]. The mechanism by which nicorandil administrated before reperfusion is beneficial in the reduction of the no-reflow is thought to be involved in KATP channels opening and microvascular vessels dilating.
Our study provides another possible mechanism for the beneficial effect of nicorandil on myocardial no-reflow. Endothelial dysfunction can be characterized by a decreased synthesis of endothelium-derived nitric oxide (NO). NO synthase (NOS) isoforms are the enzymes responsible for NO generation. NOS isoforms are divided into two categories according to calmodulin and calcium: cNOS (including neuronal NOS (type 1) and endothelial NOS (type 3)) and iNOS (type 2). The cNOS and iNOS were mainly produced by the vascular endothelium and macrophage respectively [24]. The evidence which data provided showed that NO synthesized through cNOS has cardiac protection role, while high concentrations of NO which synthesized through iNOS rapidly interacts with oxygen to yield the potent oxidant peroxynitrite (ONOO−) and subsequently induces and exacerbates myocardial ischemia reperfusion injury [25]. The present study demonstrated that nicorandil increased constitutive NOS activity and decreased inducible NOS activity, and L-NMMA (nonselective nitric oxide synthase antagonist) abrogated the effects of nicorandil on cNOS activity, but not iNOS activity, which is in agreement with the report of Shigeo et al. [26], implying the beneficial effect of nicorandil may be also partly due to its protection of endothelial function. They further thought that the effect of nicorandil on cNOS activity was mediated by increasing intracellular calcium via the KATP channel, because the KATP channel inhibitor glibenclamide inhibited it. Our study also showed that the necrosis area comprised 99% of the ligation area in a mini-swine model of AMI (3 h) and reperfusion, while nicorandil decreased necrosis area to 87.6%, which is similar with the reports of Lamping et al. [27] and Tsuchida et al. [28]. The mechanism of nicorandil in reducing infarct size is not well defined and may be explained by (1) preventing myocardial no-reflow, (2) activating the KATP channels and (3) inhibitory effect on reactive oxygen species formation. The data from this study also showed that nicorandil improved ventricular function, which is in agreement with the report of Auchampach et al. [29]. The beneficial effect of nicorandil on ventricular function was due not only to decreased myocardial necrosis and attenuation of myocardial stunning but also to preservation of microvascular integrity and improved myocardial tissue perfusion during AMI and reperfusion. 5. Study limitations This study has several limitations. Because we assessed infarct size at 2 h of reperfusion, the ultimate infarct size may be larger. However, since all animals were evaluated at the same point, the comparative result cannot be affected. Results were observed in a short-term experimental setting, and no long-term data are available. In conclusion, the present study demonstrated that nicorandil attenuated myocardial no-reflow, associated with increased cNOS activity and decreased iNOS activity. The present study also showed that L-NMMA and glibenclamide abrogated the effects of nicorandil on cNOS activity. These results suggested that the beneficial effect of
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