Ann Thorac Surg 2000;70:1601-1606
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Cardioprotective effects of FK409, a nitric oxide donor, after isolated rat heart preservation for 16 hours
Jie-Min Zhang, MDa,
Kazumasa Orihashi, MDa,
Taijiro Sueda, MDa,
Yuichiro Matsuura, MDa
a First Department of Surgery, Hiroshima University School of Medicine, Hiroshima, Japan
Address reprint requests to Dr Zhang, First Department of Surgery, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8551, Japan
e-mail: jiemin{at}mcai.med.hiroshima-u.ac.jp
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Abstract
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Background. We examined the cardioprotective effects of FK409, a nitric oxide donor, after isolated rat heart preservation.
Methods. FK409 was administered to the hearts in pretreatment (FK409-1 group), during ischemia (FK409-2), or during reperfusion (FK409-3). The combined nitrate and nitrite level, coronary flow, cardiac function, coronary vasodilatory response, creatine kinase (CK), and myocardial water content were evaluated after the hearts had been preserved in University of Wisconsin solution at 0°C for 16 hours.
Results. The release of nitrate and nitrite increased in reperfusion between the 20-to-40-second measurement and the measurement at 40 minutes, and the recovery of cardiac function was significantly improved in the FK409 groups. The coronary vasodilatory response to acetylcholine chloride was enhanced in the FK409-1 and FK409-2 groups. CK release decreased in FK409 groups after 15 minutes in reperfusion.
Conclusions. This study suggests that FK409 has the best protective effect on cardiac function and coronary endothelial function when it is administered in the ischemic period, a less protective effect when administered during pretreatment, and the least protective effect when FK409 is given during reperfusion after heart preservation for 16 hours.
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Introduction
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Impaired cardiac function remains a major problem after prolonged preservation of donor hearts. Recent studies suggest that reduced production of nitric oxide (NO), endothelial damage, or both might play a critical role in ischemia-reperfusion injury [1, 2]. Reperfusion injury can be responsible for the diminished release of endogenous NO because of dysfunction of the reperfused endothelium [2]. Therefore, one key to extending the safe ischemic time of a cardiac allograft is to improve metabolism of NO in the coronary endothelium during ischemia and reperfusion [3]. On the other hand, the crucial effect of NO as a potential modulator of a variety of physiologic processes has received much attention in recent years [2, 3]. A number of studies have demonstrated that exogenous administration of L-arginine or NO donor reverses endothelial dysfunction, improves coronary flow, and enhances cardiac performance in postischemic reperfusion [1, 3, 4].
FK409—(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide—is a NO donor. It possesses both potent vasorelaxant and antiplatelet activities because of the NO that is generated spontaneously from the compound [5, 6]. To date, the role of FK409 as a NO donor in either causing or ameliorating organ damage during prolonged hypothermic heart preservation has not been extensively studied. This study was designed to examine (1) whether FK409 enhances the recovery of myocardial and endothelial function after hypothermic preservation for 16 hours; and (2) whether the best timing for administering FK409 to obtain a cardioprotective effect is during pretreatment, in the ischemic period, or in the reperfusion period.
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Material and methods
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Isolated rat heart preservation
Thirty-two male Wistar rats weighing 290 to 370 g were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and mechanically ventilated by means of a tracheostomy. In each rat, the heart was exposed by median sternotomy. The rat received systemic heparin (1000 U/kg), and then the heart was arrested with an intravenous injection of 1.5 to 3.5 mL of 4°C Young solution (containing 74 mmol/L potassium citrate and 220 mmol/L magnesium sulfate). The heart was excised, immediately immersed in 4°C Krebs-Henseleit buffer (KHB) solution, and quickly perfused from the aorta with 60 mL/kg of oxygenated University of Wisconsin solution at 4°C, using less than 38 mm Hg pressure continuously. The heart was submersed in 40 mL of University of Wisconsin solution and surrounded with crushed ice in a large refrigerated room at 0°C for 16 hours.
Experimental groups
Nonstorage group
Eight hearts that had not been preserved were mounted on the Langendorff apparatus (UPH-W2, Unique Medical Co, Ltd, Tokyo, Japan); the hearts were perfused in the Langendorff model (L mode) for 34 minutes and then in the working model (W mode) for 15 minutes to assess the cardiac function under normal aerobic conditions (Fig 1).
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Fig 1. The protocol shows that the hearts were administered FK409 either before storage, in ischemia, or during reperfusion. Each heart was reperfused using both the Langendorff model and the working model. Hemodynamic indices were recorded, and changes of coronary flow (CF), combined nitrite and nitrate level (NO), and creatine kinase (CK), as well as increased percentage of CF, were measured after heart preservation at 0°C for 16 hours. The ventricular specimens were obtained after the experiment was finished. (ACh = acetylcholine chloride.)
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Cold-storage groups: control group
Six hearts were simply preserved for 16 hours.
Cold-storage groups: drug treatment groups
For the FK409-1 group, six hearts were administered intravenous FK409 (10 µmol/kg) [7] for 60 minutes before being excised. In the FK409-2 group (n = 6), FK409 (50 µmol/L) was added into oxygenated, cold-storage University of Wisconsin solution. The six hearts of the FK409-3 group were administered FK409 (5 µmol/L) [8] during the whole reperfusion period after the hearts had been stored for 16 hours.
Each heart was mounted on a Langendorff apparatus by the aorta and perfused with KHB solution at a constant pressure of 60 mm Hg for 15 minutes in L mode. KHB solution was filtered (40-µm filter), equilibrated with 95% O2 and 5% CO2, and maintained at 37°C. This circuit was not recycled. Meanwhile, KHB perfusate was monitored by blood gas analysis apparatus. The measured processes of coronary flow (CF), combined nitrate and nitrite levels (to measure NO), creatine kinase (CK), and increased percentage of CF are given later in the article. Following cannulation of the left atrium through the auricle, the Langendorff apparatus was switched to W mode, with a left atrium perfusion pressure of 10 mm Hg and an afterload of 60 mm Hg for measurements of hemodynamic indices. These included aortic flow, CF, systolic pressure, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and rate of left ventricular pressure rise or fall (±dp/dt). Aortic flow was recorded with an electromagnetic flowmeter. Left ventricular minute work (LVW), calculated as cardiac output x (systolic pressure - left atrial preload), served as an index of mechanical function. The LVDP, LVEDP, and ±dp/dt (in mm Hg/s) were measured by puncturing the left ventricular apex with a pressure transducer (AP-641G Type and EQ-601G Type, Nihon Koden Inc, Hiroshima, Japan). The hearts were paced in an atrial mode at 200 beats/min.
Nitrate and nitrite levels, coronary flow, and creatine kinase
Coronary effluents were collected after 20 to 40 seconds, 5 minutes, 15 minutes, 25 minutes, and 40 minutes in reperfusion. Meanwhile, CF (in mL/min) was also measured. Combined nitrate and nitrite levels (to measure NO) of the coronary effluent were determined with NO2/NO3 Assay Kit-F (Fluorometric, Dojindo Co, Tokyo, Japan) by reacting each sample with Griss reagent. Absorption was measured at 
ex 365 nm/em 450 nm [9]. CK level in the coronary effluent was measured after 15 and 30 minutes in reperfusion by DRI-CHEM 5500 (Fujifile Inc, Tokyo, Japan). The activity of CK was expressed as IU/L [10].
Coronary vasodilatory response to acetylcholine chloride
The vascular dilatory function of endothelial cells was assessed by the increased percentage of CF in response to acetylcholine chloride (ACh) at 10-8 mol/L. The basal CF was measured for 1 minute after the hearts had been perfused with the KHB solution on L mode for 15 minutes. Then the hearts were perfused with KHB solution containing 10-8 mol/L of ACh for 3 minutes. The content of CF was measured at 1, 2, and 3 minutes after the infusion of ACh. An increased percentage of CF after administration of ACh was calculated and expressed per minute using the following formula: increase of CF (%) = [CF (with ACh) - basal CF]/basal CF x 100.
Myocardial water content
A ventricular specimen was weighed after the experiment was finished and then reweighed after the specimen had been dried at 80°C for 24 hours. Myocardial water content was calculated using the following formula: myocardial water content (%) = (wet weight - dry weight)/wet weight x 100.
Sources of drugs
FK409 was provided by Fujisawa Pharmaceutical Company, Limited (Osaka, Japan); the University of Wisconsin solution was from Du Pont Pharmaceuticals Co, Inc (Wilmington, DE) imported by Fujisawa Pharmaceutical Co; and the ACh was from Daiichi Pharmaceutical, Incorporated (Tokyo, Japan).
All animals received humane care in compliance with the "Principals of Laboratory Animal Care" formulated by the National Society for Medical Research and with the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences (NIH publication 85-23, revised 1985). This experiment was also reviewed by the Committee of the Ethics on Animal Experiments in the Faculty of Medicine, Hiroshima University, and was performed under the Guidelines for Animal Experiments in the Faculty of Medicine, Hiroshima University, and the law (no. 105) and notification (no. 6) of the Japanese government.
Statistical analysis
Results for each group are expressed as the mean ± standard deviation. The values among three or more groups were compared with the Fisher’s Protected Least Significant Difference test. A series of data was compared among more than three groups by means of two-way repeated measures analysis of variance by using statistic software StatView 5.0 (SAS Institute Inc, Cary, NC). Statistical significance was accepted at p values less than 0.05.
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Results
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Recovery of cardiac function
Indices of cardiac function measured in W mode are summarized in Tables 1 and 2. Recovery of aortic flow, CF, cardiac output (CO), systolic pressure, and LVW in FK409 groups (except for CF of FK409-3) were all significantly better than corresponding values in the control group. Indices of LVDP, LVEDP, and ±dp/dt max in FK409 groups were significantly enhanced compared with the control group. Among the cold-storage groups, LVW, systolic pressure, LVDP, and ±dp/dt max recovered best in the FK409-2 group.
Changes of coronary flow
The CF in L mode was significantly higher in the FK409-1 and FK409-2 groups than in the control group (Fig 2 ; p = 0.0327 and p = 0.0249, respectively). However, there was no significant difference between the FK409-3 group and the control group.
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Fig 2. Effects of FK409 on coronary flow (CF). CF (in mL/min) was measured in Langendorff reperfusion. Data are shown as the mean ± standard deviation. (* p = 0.0327 for FK409-1 group versus control group;  p = 0.0249 for FK409-2 group versus control group by means of two-way repeated measures analysis of variance.)
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Changes of combined nitrate and nitrite level
Nitrate and nitrite changes in coronary effluent in reperfusion are shown in Figure 3. The combined nitrate and nitrite content in the FK409 groups was significantly higher than in the control group (p < 0.0001, p < 0.0001, and p < 0.0001, for FK409-1, FK409-2, and FK409-3 groups, respectively). Because the FK409-3 group included 5 µmol/L of FK409, an NO donor, in KHB solution, the nitrate and nitrite content was highest in this group, compared with all cold-storage groups.
Coronary vasodilatory response to acetylcholine chloride
Administration of a vasodilatory agent resulted in a reproducible increase in CF without causing any significant changes in the systolic pressure. The vasodilatory response to ACh is shown in Figure 4. The increased percentage of CF in the FK409-1 and FK409-2 groups was significantly higher than in the control group (p = 0.0089 and p = 0.0009, respectively). However, the FK409-3 group did not show a significant increase of CF compared with the control group (p = 0.2823).
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Fig 4. Coronary vasodilation in response to 10-8 mol/L of acetylcholine chloride (ACh). Data are shown as the mean ± standard deviation. The basal coronary flow (CF) was measured for 1 minute after the hearts had been perfused with the KHB solution in Langendorff model for 15 minutes. Then the hearts were perfused with KHB solution containing 10-8 mol/L of ACh for 3 minutes. The content of CF was measured at 1, 2, and 3 minutes after the infusion of ACh. An increased percentage of CF after perfusion with ACh was calculated and expressed per minute. (* p = 0.0089 for FK409-1 group versus control group; p = 0.0009 for FK409-2 group versus control group by means of two-way repeated measures analysis of variance.)
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Changes of creatine kinase level
The CK level in the coronary effluent in the FK409 groups after 15 minutes in reperfusion was significantly lower than that in the control group (p < 0.05 in FK409-1 group or p < 0.001 in FK409-2 and FK409-3 groups). In addition, the CK level in the FK409-2 group was significantly lower than that of the FK409-1 group at 15 minutes (p < 0.05). After 30 minutes in reperfusion, the CK level in the FK409-2 group was significantly lower than that in the control group (Fig 5).
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Fig 5. Changes of creatine kinase (CK) in coronary effluent. Data are shown as the mean ± standard deviation. Levels of CK (IU/L) in the coronary effluent were measured at 15 minutes and 30 minutes during reperfusion in the cold-storage groups. (* p < 0.05 and ** p < 0.01 for the FK409 groups versus control group; p < 0.05 for the FK409-2 group versus the FK409-1 group.)
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Myocardial water content
Myocardial water content in the nonstorage group was significantly lower than that in the control group (p = 0.0351). However, there was no significant difference of myocardial water content among cold-storage groups (Table 3).
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Comment
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The present study has shown that exogenous administration of the NO donor FK409 elicited: (1) an improved recovery of cardiac function (in terms of aortic flow, cardiac output, systolic pressure, LVW, LVEDP, and ±dp/dt max) and CF; (2) less myocardial damage (as measured by CK levels); and (3) protection of vascular endothelial function (coronary vasodilatory response and NO release) in postischemic reperfusion after heart preservation for 16 hours. The best effect was achieved when FK409 was given during the ischemic period. Ischemia followed by reperfusion is known to impair endothelial function [11]. A decrease in NO production is now recognized as an important feature of ischemia-reperfusion injury [3], because of the vital cardioprotective role of endogenous NO [12]. Reduction in basal NO release is known to adversely influence cardiac mechanical function [3]. Endothelial dysfunction is manifested mainly by a reduced vasodilatory response to endothelium-dependent agents [3, 13]. In our study, the coronary vasodilatory response evaluated by increased percentage of CF was significantly higher and the NO content in the coronary effluent increased in the FK409-1 and FK409-2 groups. These results indicate that FK409 may have a protective effect on the coronary vascular endothelium.
Recent studies have shown that decreased endothelial damage can be induced by incorporation of a source of NO in the storage solution during reperfusion [14]. Although prolonged preservation in University of Wisconsin solution causes a profound depression in cardiac function [14], as evidenced by the poor recovery of CF, cardiac output, LVW, LVEDP, and ±dp/dt max in the control group, depression of the cardiac function was reduced in the FK409 groups in our study.
Although this study could not elucidate the mechanism of the beneficial effect provided by FK409, there are several possible such mechanisms. During hypothermic storage, NO released from FK409 may directly regulate the level of guanylate cyclase enzyme, facilitating cyclic guanosine monophosphate formation and maintaining a dilated vascular bed [12, 14]. Another role of NO is to inhibit xanthine oxidase, the proximal superoxide-producing enzyme responsible for oxidant production and endothelial cell damage during organ or tissue reperfusion [15]. Hassoun and colleagues reported that endothelial xanthine oxidase has been shown to be inactivated by exogenous NO [15]. The fact that the best results appeared in the FK409-2 group may be explained by these protective mechanisms.
Elevation of cyclic guanosine monophosphate content has been shown to favorably influence myocardial energy substrate metabolism by reducing the rate of myocardial glycolysis [16]. On myocardial metabolism, CK content in the coronary effluent in the FK409 groups significantly decreased, indicating that NO may improve myocardial metabolism in ischemia reperfusion.
Another possible mechanism of cardioprotection is that the presence of NO throughout the hypothermic storage period and early in the reperfusion period might prevent adhesion and activation of neutrophils, with the resultant localized production of cytokines, and oxidants that can be harmful to the endothelium [17]. However, the study of Katori and colleagues showed that intercellular adhesion molecule-1 expression in donor endothelial cells was not different between rat hearts pretreated with FK409 and those in the control group [7]. Because our model did not contain blood-borne cellular elements, the contribution of neutrophils and platelets to ischemia-reperfusion injury could not be examined. This is a limitation of this study.
The cardioprotective effect was less when FK409 was administered during reperfusion. After heart preservation for 16 hours, the myocardium and coronary vascular endothelium might be seriously impaired, and exogenous administration of NO could be insufficient to improve the myocardial and endothelial function in reperfusion, in spite of a high concentration of FK409.
In conclusion, FK409 had a protective effect on cardiac function and on coronary endothelial function in isolated rat hearts preserved for 16 hours. This protective effect was strongest when FK409 was administered in ischemia, less when administered as a pretreatment, and least when given during reperfusion. Future studies are still needed to determine the mechanism of these effects of FK409, to evaluate the effect in comparison with other NO donors, and to examine the influence of blood-borne elements.
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Acknowledgments
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The authors thank Mr Kazunori Iwase, Ms Sachiko Hidaka, and Ms Taiko Yoneya for their excellent technical assistance on experimental preparation and measurement of NO in this experiment.
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Footnotes
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Fujisawa Pharmaceutical Company, Limited, provided the FK409, and provided funding for this study.
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Accepted for publication April 24, 2000.
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Abstract
Introduction
p < 0.0001 for FK409-3 group versus other groups by means of two-way repeated measures analysis of variance.)