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Cardioprotective effect induced by brief exposure to nitric oxide before myocardial ischemia–reperfusion in vivo
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 7 (2002) 210–216 www.academicpress.com
Cardioprotective effect induced by brief exposure to nitric oxide before myocardial ischemia–reperfusion in vivo Andrey V. Gourine,* Aliaksandr A. Bulhak, Adrian T. Gonon, John Pernow, and Per-Ove Sj€ oquist Department of Cardiology, Karolinska Hospital, Stockholm, Sweden Received 21 February 2002
Abstract Administration of nitric oxide (NO) donors during ischemia and reperfusion protects from myocardial injury. However, whether administration of an NO donor during a brief period prior to ischemia protects the myocardium and the endothelium against ischemia–reperfusion injury in vivo is unknown. To study this possibility anesthetized pigs were subjected to 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. In initial dose-finding experiments, vehicle or three different doses of the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP; 0.1; 0.5; 2:5 lmol) were infused into the LAD for 3 min starting 13 min during ischemia. Only the 0:5 lmol dose of SNAP reduced infarct size (from 85 3% of the area at risk in the vehicle group to 63 3% in the SNAP-treated group; p < 0:01). There were no significant differences in hemodynamics in the vehicle and SNAP groups during ischemia–reperfusion. Endothelium-dependent dilatation of coronary microvasculature induced by substance P was larger in the SNAP group than in the vehicle group. Myeloperoxidase activity was lower in the ischemic/reperfused myocardial area of pigs given SNAP (4:97 0:61 U/g) than in vehicle-treated pigs (8:45 0:25 U/g; p < 0:05). It is concluded that intracoronary administration of the NO donor SNAP for a brief period before ischemia reduces infarct size, attenuates neutrophil accumulation, and improves endothelial function. These results suggest that NO exerts a classic preconditioning-like protection against ischemia–reperfusion injury in vivo in a narrow concentration range. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: S-nitroso-N-acetyl-D ,L -penicillamin; Coronary circulation; Endothelial function; Myeloperoxidase; Ischemia–reperfusion; Nitric oxide; Neutrophils
Nitric oxide (NO)1 is produced by several cell types within the heart, including vascular endothelial cells [1] and cardiac myocytes [2,3] from L -arginine by the enzyme NO synthase (NOS). Recently, much attention has been focused on the role of NO during myocardial ischemia–reperfusion. Several studies have shown that L -arginine or NO donors given during ischemia and reperfusion evoked cardioprotective effects by limiting infarct size, maintaining endothelial function, and improving myocardial contractility [4–8]. These effects of
*
Corresponding author. Fax:+46-8-31-1044. E-mail address: [email protected] (A.V. Gourine). 1 Abbreviations used: NO, nitric oxide; NOS, NO synthase; PC, preconditioning; SNAP, S-nitroso-N-acetyl-D ,L -penicillamin; MAP, mean arterial pressure; HR, heart rate; LAD, left anterior descending coronary artery; MPO, myeloperoxidase; RPP, rate-pressure product.
NO during ischemia–reperfusion have been attributed to several factors including decrease in vascular tone [9], decrease in myocardial oxygen consumption [10], inhibition of platelet aggregation and prevention of platelet and neutrophil adherence to the vascular endothelium [11,12] and scavenging of superoxide [1]. Previous studies have also demonstrated that endogenous NO can serve as a trigger and a mediator of late preconditioning (PC) against cardiac dysfunction and infarction [13,14]. Recent studies on myocytes in culture and the isolated rat heart indicate that the NO donor S-nitroso-N-acetylD ,L -penicillamin (SNAP), which spontaneously releases NO, induces early protective effects against ischemia– reoxygenation injury by preserving cell viability and improving myocardial contractility [15–17]. However, whether NO exerts early (classic) PC-like protection against myocardial and endothelial injury under in vivo
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 1 1 4 - 3
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conditions is not known. The hypothesis that we tested in the present study was that intracoronary administration of the NO donor SNAP during a brief period before the onset of ischemia protects the myocardium against subsequent ischemia–reperfusion injury in anesthetized pigs.
Methods Animal preparation The study was approved by the regional ethical committee for laboratory animal experiments. Pigs of either sex (n ¼ 22) were premedicated with ketamine hydrochloride (20 mg/kg i.m.) and atropine sulfate (0.1 mg/kg i.m.). Anesthesia was induced by injection of sodium pentobarbital (20 mg/kg. i.v.) and maintained by a continuous infusion (2–4 mg/kg/h i.v.). The animals were intubated and mechanically ventilated with air and oxygen. Respiratory rate and tidal volume were adjusted to keep arterial blood pH, pO2 , and pCO2 within the physiological range. Rectal temperature was kept at 38.5– 39.0 °C by means of a heated operating table. A 7 French catheter was positioned in the superior caval vein through the internal jugular vein for drug and fluid administration. Another 7 French catheter was positioned in the descending aorta via the left femoral artery for sampling of blood and for measurement of mean arterial pressure (MAP) via a Statham P23Db transducer. Heart rate (HR) was determined from the arterial pressure curve. All variables were continuously recorded on a Grass polygraph (Model 7). The heart was exposed via a sternotomy. A ligature was placed around the left anterior descending coronary artery (LAD) at a position from which the distal third of the artery would be occluded by tightening the ligature. An ultrasonic flow probe (Transonic Systems, New York, NY) was placed around the artery just proximal to the snare for measurement of blood flow. The flow probe was connected to a Transonic 208 blood flow meter. A needle (0.4 mm OD) connected to a catheter was introduced into the LAD just distal to the snare. Experimental protocol The protocol was designed to perform experiments in two steps. In the first set of experiments, including only a minimum number of experimental animals, a dosefinding of the infarct-limiting effect of SNAP was performed. When an effective dose was found, additional experiments were performed in the second step of the protocol by increasing the number of animals in the effective SNAP dose group and in the vehicle group to enable proper statistical analyses. In the first part of the study, 15 pigs were randomly assigned to four different groups. These animals were subjected to 45 min of coronary artery ligation followed
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by 4 h of reperfusion. One group was given vehicle (saline, n ¼ 4) and three groups were given different doses of SNAP (0:1 lmol, n ¼ 4; 0:5 lmol, n ¼ 4; 2:5 lmol; n ¼ 3) into the LAD for 3 min starting 13 min before the onset of ischemia. In these initial experiments the final infarct size was evaluated (see below). The 0:5 lmol dose of SNAP was the only dose that showed a clear tendency to have a cardioprotective effect. This dose was therefore chosen in the subsequent part of the study (see below). Seven additional animals were randomly assigned to the vehicle (n ¼ 3) and the 0:5 lmol SNAP ðn ¼ 4Þ groups and were subjected to LAD ligation and reperfusion as described above. Endothelium-dependent responses of coronary microvasculature were evaluated at the end of reperfusion by intracoronary infusion of substance P (0.02 and 0:2 lg=min at a rate of 2 ml/min) immediately before the animals were sacrificed. Determination of infarct size At the end of the experiment LAD was reoccluded and 2% Evans blue was injected into the left atrium to outline the ischemic myocardium, after which the pigs were killed by injection of a high dose of potassium chloride into the left atrium. The heart was rapidly extirpated. The atria and the right ventricle were removed. The left ventricle was cut into 0.8- to 1-cm-thick slices perpendicular to the heart base–apex axis. The slices were then incubated in 0.8% triphenyltetrazolium chloride at 37 °C, which stained the viable myocardium red, to measure the extent of myocardial necrosis [18]. The extent of myocardial necrosis and the area at risk were determined by planimetry by a person who was blinded from the treatment group. Determination of myocardial myeloperoxidase activity Myocardial samples from the nonischemic and the ischemic/reperfused areas of the left ventricle were frozen in liquid nitrogen and stored frozen at )80 °C. Myeloperoxidase (MPO) activity was analyzed according to a method described previously in detail [19]. Briefly, the frozen myocardial samples were thawed and diluted with 10 volumes 0.5% hexadecyltrimethyl ammonium bromide buffer (Sigma, St Louis, MO) in 50 mM phosphate buffer. They were homogenized with a Ultra-Turrax homogenizer (Labassco, Sweden). The homogenates were centrifuged at 5000g for 30 min at 2 °C. Thereafter, 50 ll of the supernatant was mixed with 50 ll of O-dianisidine hydrochloride (1.67 mg/ml; Sigma) and 0.05% H2 O2 in phosphate buffer. The absorbance was read in a spectrophotometer (Victor 2, Wallac, Sweden) at 460 nm at 30 and 90 s. The tissue MPO activity is expressed in units/g cardiac tissue. One unit of MPO is defined as that quantity of enzyme hydrolyzing 1 mmol peroxide/min at 25 °C.
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Chemicals and statistical analysis Ketamine hydrochloride was purchased from Parke– Davis (USA), sodium pentobarbital was from Apotekebolaget (Sweden), atropine sulfate and sodium heparin were from Lovens (Denmark), and SNAP and substance P were from Sigma. SNAP and substance P were dissolved in saline. All values are presented as mean SE. Differences between the vehicle and the 0:5 lmol SNAP groups were calculated with the nonparametric Mann–Whitney u test. Differences within groups were calculated with nonparametric Kruskal–Wallis analysis of variance for paired observations, followed by Dunn’s test. A p < 0:05 was considered statistically significant. No statistical evaluation was performed following the preliminary dose-finding experiments due to the limited number of animals included in that part.
Fig. 1. Increase in LAD blood flow evoked by intracoronary infusion of the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP) before ischemia. The animals were given SNAP (0.1, 0.5, or 2:5 lmol) into the LAD for 3 min starting 13 min before the onset of ischemia. Data are presented as the means SE.
Results Mortality and exclusions from the study Of 22 initially included pigs 12 developed ventricular fibrillation. Of these pigs 8 were successfully defibrillated by DC shocks (10–20 J) to sinus rhythm and were included in the final analysis of the study. The remaining 4 pigs (1 in the vehicle group, 2 in the 0:5 lmol SNAP group, and 1 in the 2:5 lmol SNAP group) were excluded from the study. Dose-finding experiments Infusion of 0:1 lmol SNAP caused a small increase (10%) in coronary blood flow, whereas 0.5 and
2:5 lmol SNAP increased blood flow by 38 and 47%, respectively (Fig. 1). The effect of SNAP on LAD blood flow was short lasting and the flow had returned to preinfusion levels within 7–8 min after cessation of the infusion. Thus, there was no remaining hemodynamic effect of the SNAP infusion at the start of the ischemic period (Table 1). The infarct size in the three animals of the vehicle group in the dose-finding experiments was 88 5%. The infarct size of the animals given 0.1, 0.5, and 2:5 lmol of SNAP was 89 5, 59 5, and 82 5%, respectively. Based on this finding, a clear trend toward a decrease in infarct size only by 0:5 lmol of SNAP, we selected this dose for the subsequent part of the study.
Table 1 Hemodynamic data before and after SNAP administration Group
Variable
Preinfusion
Preischemia
Vehicle
MAP HR RPP LAD flow MAP HR RPP LAD flow MAP HR RPP LAD flow MAP HR RPP LAD flow
111 9 134 5 18333 1530 16 2 110 12 139 15 18107 1348 13 1 111 12 117 19 16833 3357 18 3 101 5 141 19 16962 2412 22 2
110 9 132 4 17807 1027 16 2 109 11 140 14 18080 1040 13 1 112 14 121 25 17155 4499 18 3 98 9 141 17 17787 3597 22 3
SNAP ð0:1 lmolÞ
SNAP (0:5 lmol)
SNAP (2:5 lmol)
Values are presented as the means SE. MAP, mean arterial pressure (mm Hg); HR, heart rate (beats/min); RPP, rate-pressure product (beats/ min mm Hg); LAD flow; left anterior descending coronary artery flow (ml/min).
90 4 146 5 15673 865 32 6 91 6 128 11 14399 1550 41 7 92 7 125 13 14522 1922
99 4 133 8 16167 1360
SNAP n ¼ 6
103 6 128 7 16132 1036 14 1 102 8 120 13 15295 1798 18 2 104 6 129 7 16521 1226 15 2 103 6 117 11 15083 1829 18 2 MAP HR RPP LAD flow MAP HR RPP LAD flow Vehicle n ¼ 6
Values are presented as the means SE. Significant differences from preischemic values are shown; *p < 0:05. MAP, mean arterial pressure (mm Hg); HR, heart rate (beats/min); RPP, ratepressure product (beats/min mm Hg). LAD flow, left anterior descending coronary artery flow (ml/min).
85 2 164 11 17690 1214 13 4 84 7 130 10 13827 1382 14 2 90 3 153 9 17338 1284 25 5 85 4 132 12 14313 1596 22 5
91 4 149 12 16317 1498 25 7 86 4 125 12 13463 1585 30 5
60 45
Preischemia Preinfusion Variable Group
Table 2 Hemodynamic data before drug infusion, before ischemia, at the end of ischemia, and during reperfusion
120 Reperfusion, min Ischemia, min
180
240
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Hemodynamics MAP, HR, rate-pressure product (RPP), and LAD blood flow, before SNAP administration, before ischemia, at the end of ischemia, and during reperfusion in the groups which received vehicle and 0:5 lmol SNAP are presented in Table 2. There were no significant differences in hemodynamics before drug administration and during ischemia in the different groups. MAP decreased significantly during reperfusion in both groups and there were no significant differences in MAP in the groups. HR increased in both groups at the end of reperfusion but the changes were statistically significant only in the vehicle-treated group. RPP did not differ between the groups during ischemia and reperfusion. LAD blood flow increased approximately twofold at the onset of reperfusion in both groups and there were no significant differences between the groups. At the end of reperfusion LAD blood flow had returned to preischemic levels. Infarct size Fig. 2 depicts the infarct size expressed as a percentage of the area at risk in both experimental groups. The infarct size was 85 3% of the area at risk in the vehicle group. The infarct size was reduced to 63 3% in the group given 0:5 lmol SNAP (p < 0:01 vs vehicle). The area at risk in relation to the left ventricle in the vehicle group (16 2%) was not significantly different from that in the SNAP-treated group (20 2%).
Fig. 2. Infarct size expressed as percentage of the area at risk after 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle ðn ¼ 6Þ or the NO donor S-nitroso-N-acetyl-D ,L penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 before the onset of ischemia. Data are presented as the means SE. Significant differences from the vehicle group are shown; **p < 0:01.
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MPO activity There was no difference in MPO activity in the nonischemic myocardium in the vehicle and the 0:5 lmol SNAP group (Fig. 4). The MPO activity was significantly higher in the ischemic/reperfused than in the nonischemic myocardium in both groups. However, the MPO activity was almost two times higher in the ischemic/reperfused myocardium of the vehicle group than in the SNAP group (Fig. 4). Fig. 3. Increase in LAD blood evoked by intracoronary infusion of substance P (0.02 and 0:2 lg= min) at the end of reperfusion to pigs subjected to 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle ðn ¼ 6Þ or the NO donor S-nitrosoN-acetyl-D ,L -penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 min before ischemia. Data are presented as the means SE. Significant differences from the vehicle group are shown; *p < 0:05.
Endothelial function Endothelium-dependent responses of coronary microvasculature were evaluated by intracoronary administration of substance P (0.02 and 0:2 lg= min) at the end of reperfusion. There were no differences in preinfusion flow in the groups (Table 2; 240 min reperfusion). Both doses of substance P increased LAD blood flow significantly more in the group given SNAP than in the group given vehicle (Fig. 3).
Fig. 4. Myeloperoxidase activity in nonischemic myocardium and myocardium subjected to 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle (n ¼ 6) or the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 min before the onset of ischemia. Data are presented as the means SE. Significant differences between the vehicle group are shown; *p < 0:05.
Discussion The aim of this study was to investigate whether an intracoronary infusion of the NO donor SNAP during a brief period before ischemia protects the myocardium from a subsequent episode of ischemia–reperfusion. The main findings are that administration of SNAP resulted in reduction in infarct size, attenuated neutrophil accumulation in the ischemic/reperfused myocardium, and enhanced endothelial function in the coronary microvasculature. The protective effect of SNAP was present within a narrow dose range. It has previously been demonstrated that administration of L -arginine or NO donors decreases myocardial injury when given during the ischemic and/or reperfusion periods [4–8]. In addition, L -arginine may exert an antiarrhythmic effect when administered before ischemia [20]. The present study is the first to show that a brief (3min) exposure to SNAP ending 10 min before the start of ischemia results in reduction in infarct size and improved endothelial function during a subsequent period of ischemia–reperfusion in vivo. The hemodynamic effects of SNAP were very short lasting and were not present at the start of ischemia. This finding indicates that exogenous NO is capable of evoking PC-like cardioprotection by reduction in infarct size and improving endothelial function. This concept has not been documented before. Several different mechanisms of action have been proposed to explain the cardioprotection afforded by augmentation of NO levels in ischemia–reperfusion models. These mechanisms include preservation of endothelial function [6,9], reduction of neutrophil adhesion to the vascular endothelium [7], and inactivation of superoxide anion [1]. Furthermore, since NO has been suggested to be involved in the regulation of calcium homeostasis [21] and myocardial contractile function [22], it is reasonable to assume that NO donors can influence calcium influx during ischemia–reperfusion. Rakhit et al. [16,17] demonstrated that the NO donor SNAP and short-term episodes of hypoxia preserved myocardial cell viability during long-term ischemia via activation of the eNOS isoform through a cGMP-dependent mechanism inhibiting calcium influx. This cardioprotective effect appeared in a very narrow dose
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range. In addition, Hotta et al. [3] demonstrated that myocyte mitochondria are protected by SNAP via a mechanism related to the inhibition of calcium influx. Our present results are consistent with these observations and suggest that NO can induce a classic PC-like cardioprotective effect in a pig model of regional ischemia–reperfusion under in vivo conditions. In addition, recent data indicate that certain NO donors, which spontaneously release NO, can mediate their effects via inhibition of mitochondrial respiration. NO modulates mitochondrial function through reversible and irreversible interactions with respiratory chain complexes [23–25]. Physiological concentrations of NO inhibit cytochrome oxidase (complex IV) in a reversible manner [23]. The reversible interaction may play an important role in the physiological regulation of mitochondrial respiration by reducing oxygen consumption without causing myocardial high-energy phosphate depletion. This NO-mediated effect may be beneficial during ischemia. However, exposure to a high concentration of NO can irreversibly inhibit complex I by S-nitrosation of critical thiols in the enzyme complex [25]. Furthermore, a high concentration of NO promotes removal of oxygen from the extracellular medium which was dependent on the time of exposure to NO and on the NO concentration [26]. These effects of NO maybe deleterious during ischemia–reperfusion. The infarct-limiting effect of SNAP in the present study exists in a narrow dose range. Thus, the dosefinding experiments revealed that the protective action of SNAP was absent when the dose was reduced fivefold or increased fivefold. This narrow dose range is in accordance with the observations on isolated myocytes in which 1 mM SNAP protected from hypoxia–reoxygenation, whereas 2 mM SNAP appeared to be cytotoxic [17]. It is possible that high-concentration SNAP irreversibly inhibits mitochondrial respiration as discussed above. Another possibility is that NO reacts with superoxide anion to form the potent oxidant peroxynitrite, which is toxic to cardiac myocytes. A high concentration of SNAP may result in large amounts of peroxynitrite. In the present study SNAP was demonstrated to be short acting and its vascular effect was absent at the start of ischemia. Therefore, it seems unlikely that NO released from SNAP before the onset of ischemia resulted in any significant levels of peroxynitrite since no or little superoxide is present in the preischemic period. The endothelial function of coronary microvasculature was evaluated at the end of reperfusion by local administration of substance P, which is known to evoke endothelium-dependent dilatation in pigs [27]. The increase in LAD blood flow in response to both doses of substance P were significantly larger in the SNAP-treated group than in the vehicle group. These results indicate that endothelium-dependent vasodilatation to substance P measured at the end of reperfusion was
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better maintained in the SNAP group during ischemia– reperfusion. We did not evaluate endothelium-independent vasodilator responses in these experiments. Several previous studies have demonstrated that only endothelium-dependent, not endothelium-independent, coronary dilator responses of coronary microvasculature are attenuated by ischemia–reperfusion [9,28,29]. Myocardial MPO activity was significantly lower in the SNAP-treated group than in the vehicle group, which indicates that SNAP reduced neutrophil infiltration into the area at risk. Neutrophils are known to contribute to the development of ischemia–reperfusion injury [30]. It is therefore reasonable to suggest that administration of SNAP resulted in maintained endothelial function which prevented neutrophil adhesion to the vascular endothelium and subsequent infiltration of neutrophils into the myocardium [11,12]. However, the exact mechanisms by which SNAP improved endothelial function and reduced MPO activity is beyond the aim of the present investigation and should be explored in future studies. In conclusion, a brief period of intracoronary administration of the NO donor SNAP before ischemia results in reduced infarct size, attenuated neutrophil infiltration, and improved endothelial function. These results suggest that NO liberated from SNAP exerts an early PC-like effect against ischemia–reperfusion injury in a narrow concentration range under in vivo conditions. Acknowledgments This study was supported by grants from the Swedish Medical Research Council (10857), the Swedish Heart and Lung Foundation, and the Karolinska Institute. Marita Wallin is gratefully acknowledged for technical assistance. References [1] R.M. Palmer, D.S. Ashton, S. Moncada, Vascular endothelial cells synthesize nitric oxide from L -arginine, Nature 333 (1988) 664–666. [2] K.Y. Xu, D.L. Huso, T.M. Dawson, D.S. Bredt, L.C. Becker, Nitric oxide synthase in cardiac sarcoplasmic reticulum, Proc. Natl. Acad. Sci. USA 96 (1999) 657–662. [3] Y. Hotta, H. Otsuka-Murakami, M. Fujita, J. Nakagawa, M. Yajima, W. Liu, N. Ishikawa, N. Kawai, T. Masumizu, M. Kohno, Protective role of nitric oxide synthase against ischemia– reperfusion injury in guinea pig myocardial mitochondria, Eur. J. Pharmacol. 380 (1999) 37–48. [4] M.R. Siegfried, J. Erhardt, T. Rider, X.L. Ma, A.M. Lefer, Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion, J. Pharmacol. Exp. Ther. 260 (1992) 668–675. [5] A.S. Weyrich, X.L. Ma, A.M. Lefer, The role of L -arginine in ameliorating reperfusion injury after myocardial ischemia in the cat, Circulation 86 (1993) 279–288.
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Biology and Chemistry
Nitric Oxide 7 (2002) 210–216 www.academicpress.com
Cardioprotective effect induced by brief exposure to nitric oxide before myocardial ischemia–reperfusion in vivo Andrey V. Gourine,* Aliaksandr A. Bulhak, Adrian T. Gonon, John Pernow, and Per-Ove Sj€ oquist Department of Cardiology, Karolinska Hospital, Stockholm, Sweden Received 21 February 2002
Abstract Administration of nitric oxide (NO) donors during ischemia and reperfusion protects from myocardial injury. However, whether administration of an NO donor during a brief period prior to ischemia protects the myocardium and the endothelium against ischemia–reperfusion injury in vivo is unknown. To study this possibility anesthetized pigs were subjected to 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. In initial dose-finding experiments, vehicle or three different doses of the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP; 0.1; 0.5; 2:5 lmol) were infused into the LAD for 3 min starting 13 min during ischemia. Only the 0:5 lmol dose of SNAP reduced infarct size (from 85 3% of the area at risk in the vehicle group to 63 3% in the SNAP-treated group; p < 0:01). There were no significant differences in hemodynamics in the vehicle and SNAP groups during ischemia–reperfusion. Endothelium-dependent dilatation of coronary microvasculature induced by substance P was larger in the SNAP group than in the vehicle group. Myeloperoxidase activity was lower in the ischemic/reperfused myocardial area of pigs given SNAP (4:97 0:61 U/g) than in vehicle-treated pigs (8:45 0:25 U/g; p < 0:05). It is concluded that intracoronary administration of the NO donor SNAP for a brief period before ischemia reduces infarct size, attenuates neutrophil accumulation, and improves endothelial function. These results suggest that NO exerts a classic preconditioning-like protection against ischemia–reperfusion injury in vivo in a narrow concentration range. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: S-nitroso-N-acetyl-D ,L -penicillamin; Coronary circulation; Endothelial function; Myeloperoxidase; Ischemia–reperfusion; Nitric oxide; Neutrophils
Nitric oxide (NO)1 is produced by several cell types within the heart, including vascular endothelial cells [1] and cardiac myocytes [2,3] from L -arginine by the enzyme NO synthase (NOS). Recently, much attention has been focused on the role of NO during myocardial ischemia–reperfusion. Several studies have shown that L -arginine or NO donors given during ischemia and reperfusion evoked cardioprotective effects by limiting infarct size, maintaining endothelial function, and improving myocardial contractility [4–8]. These effects of
*
Corresponding author. Fax:+46-8-31-1044. E-mail address: [email protected] (A.V. Gourine). 1 Abbreviations used: NO, nitric oxide; NOS, NO synthase; PC, preconditioning; SNAP, S-nitroso-N-acetyl-D ,L -penicillamin; MAP, mean arterial pressure; HR, heart rate; LAD, left anterior descending coronary artery; MPO, myeloperoxidase; RPP, rate-pressure product.
NO during ischemia–reperfusion have been attributed to several factors including decrease in vascular tone [9], decrease in myocardial oxygen consumption [10], inhibition of platelet aggregation and prevention of platelet and neutrophil adherence to the vascular endothelium [11,12] and scavenging of superoxide [1]. Previous studies have also demonstrated that endogenous NO can serve as a trigger and a mediator of late preconditioning (PC) against cardiac dysfunction and infarction [13,14]. Recent studies on myocytes in culture and the isolated rat heart indicate that the NO donor S-nitroso-N-acetylD ,L -penicillamin (SNAP), which spontaneously releases NO, induces early protective effects against ischemia– reoxygenation injury by preserving cell viability and improving myocardial contractility [15–17]. However, whether NO exerts early (classic) PC-like protection against myocardial and endothelial injury under in vivo
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 1 1 4 - 3
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conditions is not known. The hypothesis that we tested in the present study was that intracoronary administration of the NO donor SNAP during a brief period before the onset of ischemia protects the myocardium against subsequent ischemia–reperfusion injury in anesthetized pigs.
Methods Animal preparation The study was approved by the regional ethical committee for laboratory animal experiments. Pigs of either sex (n ¼ 22) were premedicated with ketamine hydrochloride (20 mg/kg i.m.) and atropine sulfate (0.1 mg/kg i.m.). Anesthesia was induced by injection of sodium pentobarbital (20 mg/kg. i.v.) and maintained by a continuous infusion (2–4 mg/kg/h i.v.). The animals were intubated and mechanically ventilated with air and oxygen. Respiratory rate and tidal volume were adjusted to keep arterial blood pH, pO2 , and pCO2 within the physiological range. Rectal temperature was kept at 38.5– 39.0 °C by means of a heated operating table. A 7 French catheter was positioned in the superior caval vein through the internal jugular vein for drug and fluid administration. Another 7 French catheter was positioned in the descending aorta via the left femoral artery for sampling of blood and for measurement of mean arterial pressure (MAP) via a Statham P23Db transducer. Heart rate (HR) was determined from the arterial pressure curve. All variables were continuously recorded on a Grass polygraph (Model 7). The heart was exposed via a sternotomy. A ligature was placed around the left anterior descending coronary artery (LAD) at a position from which the distal third of the artery would be occluded by tightening the ligature. An ultrasonic flow probe (Transonic Systems, New York, NY) was placed around the artery just proximal to the snare for measurement of blood flow. The flow probe was connected to a Transonic 208 blood flow meter. A needle (0.4 mm OD) connected to a catheter was introduced into the LAD just distal to the snare. Experimental protocol The protocol was designed to perform experiments in two steps. In the first set of experiments, including only a minimum number of experimental animals, a dosefinding of the infarct-limiting effect of SNAP was performed. When an effective dose was found, additional experiments were performed in the second step of the protocol by increasing the number of animals in the effective SNAP dose group and in the vehicle group to enable proper statistical analyses. In the first part of the study, 15 pigs were randomly assigned to four different groups. These animals were subjected to 45 min of coronary artery ligation followed
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by 4 h of reperfusion. One group was given vehicle (saline, n ¼ 4) and three groups were given different doses of SNAP (0:1 lmol, n ¼ 4; 0:5 lmol, n ¼ 4; 2:5 lmol; n ¼ 3) into the LAD for 3 min starting 13 min before the onset of ischemia. In these initial experiments the final infarct size was evaluated (see below). The 0:5 lmol dose of SNAP was the only dose that showed a clear tendency to have a cardioprotective effect. This dose was therefore chosen in the subsequent part of the study (see below). Seven additional animals were randomly assigned to the vehicle (n ¼ 3) and the 0:5 lmol SNAP ðn ¼ 4Þ groups and were subjected to LAD ligation and reperfusion as described above. Endothelium-dependent responses of coronary microvasculature were evaluated at the end of reperfusion by intracoronary infusion of substance P (0.02 and 0:2 lg=min at a rate of 2 ml/min) immediately before the animals were sacrificed. Determination of infarct size At the end of the experiment LAD was reoccluded and 2% Evans blue was injected into the left atrium to outline the ischemic myocardium, after which the pigs were killed by injection of a high dose of potassium chloride into the left atrium. The heart was rapidly extirpated. The atria and the right ventricle were removed. The left ventricle was cut into 0.8- to 1-cm-thick slices perpendicular to the heart base–apex axis. The slices were then incubated in 0.8% triphenyltetrazolium chloride at 37 °C, which stained the viable myocardium red, to measure the extent of myocardial necrosis [18]. The extent of myocardial necrosis and the area at risk were determined by planimetry by a person who was blinded from the treatment group. Determination of myocardial myeloperoxidase activity Myocardial samples from the nonischemic and the ischemic/reperfused areas of the left ventricle were frozen in liquid nitrogen and stored frozen at )80 °C. Myeloperoxidase (MPO) activity was analyzed according to a method described previously in detail [19]. Briefly, the frozen myocardial samples were thawed and diluted with 10 volumes 0.5% hexadecyltrimethyl ammonium bromide buffer (Sigma, St Louis, MO) in 50 mM phosphate buffer. They were homogenized with a Ultra-Turrax homogenizer (Labassco, Sweden). The homogenates were centrifuged at 5000g for 30 min at 2 °C. Thereafter, 50 ll of the supernatant was mixed with 50 ll of O-dianisidine hydrochloride (1.67 mg/ml; Sigma) and 0.05% H2 O2 in phosphate buffer. The absorbance was read in a spectrophotometer (Victor 2, Wallac, Sweden) at 460 nm at 30 and 90 s. The tissue MPO activity is expressed in units/g cardiac tissue. One unit of MPO is defined as that quantity of enzyme hydrolyzing 1 mmol peroxide/min at 25 °C.
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Chemicals and statistical analysis Ketamine hydrochloride was purchased from Parke– Davis (USA), sodium pentobarbital was from Apotekebolaget (Sweden), atropine sulfate and sodium heparin were from Lovens (Denmark), and SNAP and substance P were from Sigma. SNAP and substance P were dissolved in saline. All values are presented as mean SE. Differences between the vehicle and the 0:5 lmol SNAP groups were calculated with the nonparametric Mann–Whitney u test. Differences within groups were calculated with nonparametric Kruskal–Wallis analysis of variance for paired observations, followed by Dunn’s test. A p < 0:05 was considered statistically significant. No statistical evaluation was performed following the preliminary dose-finding experiments due to the limited number of animals included in that part.
Fig. 1. Increase in LAD blood flow evoked by intracoronary infusion of the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP) before ischemia. The animals were given SNAP (0.1, 0.5, or 2:5 lmol) into the LAD for 3 min starting 13 min before the onset of ischemia. Data are presented as the means SE.
Results Mortality and exclusions from the study Of 22 initially included pigs 12 developed ventricular fibrillation. Of these pigs 8 were successfully defibrillated by DC shocks (10–20 J) to sinus rhythm and were included in the final analysis of the study. The remaining 4 pigs (1 in the vehicle group, 2 in the 0:5 lmol SNAP group, and 1 in the 2:5 lmol SNAP group) were excluded from the study. Dose-finding experiments Infusion of 0:1 lmol SNAP caused a small increase (10%) in coronary blood flow, whereas 0.5 and
2:5 lmol SNAP increased blood flow by 38 and 47%, respectively (Fig. 1). The effect of SNAP on LAD blood flow was short lasting and the flow had returned to preinfusion levels within 7–8 min after cessation of the infusion. Thus, there was no remaining hemodynamic effect of the SNAP infusion at the start of the ischemic period (Table 1). The infarct size in the three animals of the vehicle group in the dose-finding experiments was 88 5%. The infarct size of the animals given 0.1, 0.5, and 2:5 lmol of SNAP was 89 5, 59 5, and 82 5%, respectively. Based on this finding, a clear trend toward a decrease in infarct size only by 0:5 lmol of SNAP, we selected this dose for the subsequent part of the study.
Table 1 Hemodynamic data before and after SNAP administration Group
Variable
Preinfusion
Preischemia
Vehicle
MAP HR RPP LAD flow MAP HR RPP LAD flow MAP HR RPP LAD flow MAP HR RPP LAD flow
111 9 134 5 18333 1530 16 2 110 12 139 15 18107 1348 13 1 111 12 117 19 16833 3357 18 3 101 5 141 19 16962 2412 22 2
110 9 132 4 17807 1027 16 2 109 11 140 14 18080 1040 13 1 112 14 121 25 17155 4499 18 3 98 9 141 17 17787 3597 22 3
SNAP ð0:1 lmolÞ
SNAP (0:5 lmol)
SNAP (2:5 lmol)
Values are presented as the means SE. MAP, mean arterial pressure (mm Hg); HR, heart rate (beats/min); RPP, rate-pressure product (beats/ min mm Hg); LAD flow; left anterior descending coronary artery flow (ml/min).
90 4 146 5 15673 865 32 6 91 6 128 11 14399 1550 41 7 92 7 125 13 14522 1922
99 4 133 8 16167 1360
SNAP n ¼ 6
103 6 128 7 16132 1036 14 1 102 8 120 13 15295 1798 18 2 104 6 129 7 16521 1226 15 2 103 6 117 11 15083 1829 18 2 MAP HR RPP LAD flow MAP HR RPP LAD flow Vehicle n ¼ 6
Values are presented as the means SE. Significant differences from preischemic values are shown; *p < 0:05. MAP, mean arterial pressure (mm Hg); HR, heart rate (beats/min); RPP, ratepressure product (beats/min mm Hg). LAD flow, left anterior descending coronary artery flow (ml/min).
85 2 164 11 17690 1214 13 4 84 7 130 10 13827 1382 14 2 90 3 153 9 17338 1284 25 5 85 4 132 12 14313 1596 22 5
91 4 149 12 16317 1498 25 7 86 4 125 12 13463 1585 30 5
60 45
Preischemia Preinfusion Variable Group
Table 2 Hemodynamic data before drug infusion, before ischemia, at the end of ischemia, and during reperfusion
120 Reperfusion, min Ischemia, min
180
240
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Hemodynamics MAP, HR, rate-pressure product (RPP), and LAD blood flow, before SNAP administration, before ischemia, at the end of ischemia, and during reperfusion in the groups which received vehicle and 0:5 lmol SNAP are presented in Table 2. There were no significant differences in hemodynamics before drug administration and during ischemia in the different groups. MAP decreased significantly during reperfusion in both groups and there were no significant differences in MAP in the groups. HR increased in both groups at the end of reperfusion but the changes were statistically significant only in the vehicle-treated group. RPP did not differ between the groups during ischemia and reperfusion. LAD blood flow increased approximately twofold at the onset of reperfusion in both groups and there were no significant differences between the groups. At the end of reperfusion LAD blood flow had returned to preischemic levels. Infarct size Fig. 2 depicts the infarct size expressed as a percentage of the area at risk in both experimental groups. The infarct size was 85 3% of the area at risk in the vehicle group. The infarct size was reduced to 63 3% in the group given 0:5 lmol SNAP (p < 0:01 vs vehicle). The area at risk in relation to the left ventricle in the vehicle group (16 2%) was not significantly different from that in the SNAP-treated group (20 2%).
Fig. 2. Infarct size expressed as percentage of the area at risk after 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle ðn ¼ 6Þ or the NO donor S-nitroso-N-acetyl-D ,L penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 before the onset of ischemia. Data are presented as the means SE. Significant differences from the vehicle group are shown; **p < 0:01.
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MPO activity There was no difference in MPO activity in the nonischemic myocardium in the vehicle and the 0:5 lmol SNAP group (Fig. 4). The MPO activity was significantly higher in the ischemic/reperfused than in the nonischemic myocardium in both groups. However, the MPO activity was almost two times higher in the ischemic/reperfused myocardium of the vehicle group than in the SNAP group (Fig. 4). Fig. 3. Increase in LAD blood evoked by intracoronary infusion of substance P (0.02 and 0:2 lg= min) at the end of reperfusion to pigs subjected to 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle ðn ¼ 6Þ or the NO donor S-nitrosoN-acetyl-D ,L -penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 min before ischemia. Data are presented as the means SE. Significant differences from the vehicle group are shown; *p < 0:05.
Endothelial function Endothelium-dependent responses of coronary microvasculature were evaluated by intracoronary administration of substance P (0.02 and 0:2 lg= min) at the end of reperfusion. There were no differences in preinfusion flow in the groups (Table 2; 240 min reperfusion). Both doses of substance P increased LAD blood flow significantly more in the group given SNAP than in the group given vehicle (Fig. 3).
Fig. 4. Myeloperoxidase activity in nonischemic myocardium and myocardium subjected to 45 min of ischemia followed by 4 h of reperfusion. The animals were given either vehicle (n ¼ 6) or the NO donor S-nitroso-N-acetyl-D ,L -penicillamin (SNAP, n ¼ 6) into the LAD for 3 min starting 13 min before the onset of ischemia. Data are presented as the means SE. Significant differences between the vehicle group are shown; *p < 0:05.
Discussion The aim of this study was to investigate whether an intracoronary infusion of the NO donor SNAP during a brief period before ischemia protects the myocardium from a subsequent episode of ischemia–reperfusion. The main findings are that administration of SNAP resulted in reduction in infarct size, attenuated neutrophil accumulation in the ischemic/reperfused myocardium, and enhanced endothelial function in the coronary microvasculature. The protective effect of SNAP was present within a narrow dose range. It has previously been demonstrated that administration of L -arginine or NO donors decreases myocardial injury when given during the ischemic and/or reperfusion periods [4–8]. In addition, L -arginine may exert an antiarrhythmic effect when administered before ischemia [20]. The present study is the first to show that a brief (3min) exposure to SNAP ending 10 min before the start of ischemia results in reduction in infarct size and improved endothelial function during a subsequent period of ischemia–reperfusion in vivo. The hemodynamic effects of SNAP were very short lasting and were not present at the start of ischemia. This finding indicates that exogenous NO is capable of evoking PC-like cardioprotection by reduction in infarct size and improving endothelial function. This concept has not been documented before. Several different mechanisms of action have been proposed to explain the cardioprotection afforded by augmentation of NO levels in ischemia–reperfusion models. These mechanisms include preservation of endothelial function [6,9], reduction of neutrophil adhesion to the vascular endothelium [7], and inactivation of superoxide anion [1]. Furthermore, since NO has been suggested to be involved in the regulation of calcium homeostasis [21] and myocardial contractile function [22], it is reasonable to assume that NO donors can influence calcium influx during ischemia–reperfusion. Rakhit et al. [16,17] demonstrated that the NO donor SNAP and short-term episodes of hypoxia preserved myocardial cell viability during long-term ischemia via activation of the eNOS isoform through a cGMP-dependent mechanism inhibiting calcium influx. This cardioprotective effect appeared in a very narrow dose
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range. In addition, Hotta et al. [3] demonstrated that myocyte mitochondria are protected by SNAP via a mechanism related to the inhibition of calcium influx. Our present results are consistent with these observations and suggest that NO can induce a classic PC-like cardioprotective effect in a pig model of regional ischemia–reperfusion under in vivo conditions. In addition, recent data indicate that certain NO donors, which spontaneously release NO, can mediate their effects via inhibition of mitochondrial respiration. NO modulates mitochondrial function through reversible and irreversible interactions with respiratory chain complexes [23–25]. Physiological concentrations of NO inhibit cytochrome oxidase (complex IV) in a reversible manner [23]. The reversible interaction may play an important role in the physiological regulation of mitochondrial respiration by reducing oxygen consumption without causing myocardial high-energy phosphate depletion. This NO-mediated effect may be beneficial during ischemia. However, exposure to a high concentration of NO can irreversibly inhibit complex I by S-nitrosation of critical thiols in the enzyme complex [25]. Furthermore, a high concentration of NO promotes removal of oxygen from the extracellular medium which was dependent on the time of exposure to NO and on the NO concentration [26]. These effects of NO maybe deleterious during ischemia–reperfusion. The infarct-limiting effect of SNAP in the present study exists in a narrow dose range. Thus, the dosefinding experiments revealed that the protective action of SNAP was absent when the dose was reduced fivefold or increased fivefold. This narrow dose range is in accordance with the observations on isolated myocytes in which 1 mM SNAP protected from hypoxia–reoxygenation, whereas 2 mM SNAP appeared to be cytotoxic [17]. It is possible that high-concentration SNAP irreversibly inhibits mitochondrial respiration as discussed above. Another possibility is that NO reacts with superoxide anion to form the potent oxidant peroxynitrite, which is toxic to cardiac myocytes. A high concentration of SNAP may result in large amounts of peroxynitrite. In the present study SNAP was demonstrated to be short acting and its vascular effect was absent at the start of ischemia. Therefore, it seems unlikely that NO released from SNAP before the onset of ischemia resulted in any significant levels of peroxynitrite since no or little superoxide is present in the preischemic period. The endothelial function of coronary microvasculature was evaluated at the end of reperfusion by local administration of substance P, which is known to evoke endothelium-dependent dilatation in pigs [27]. The increase in LAD blood flow in response to both doses of substance P were significantly larger in the SNAP-treated group than in the vehicle group. These results indicate that endothelium-dependent vasodilatation to substance P measured at the end of reperfusion was
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better maintained in the SNAP group during ischemia– reperfusion. We did not evaluate endothelium-independent vasodilator responses in these experiments. Several previous studies have demonstrated that only endothelium-dependent, not endothelium-independent, coronary dilator responses of coronary microvasculature are attenuated by ischemia–reperfusion [9,28,29]. Myocardial MPO activity was significantly lower in the SNAP-treated group than in the vehicle group, which indicates that SNAP reduced neutrophil infiltration into the area at risk. Neutrophils are known to contribute to the development of ischemia–reperfusion injury [30]. It is therefore reasonable to suggest that administration of SNAP resulted in maintained endothelial function which prevented neutrophil adhesion to the vascular endothelium and subsequent infiltration of neutrophils into the myocardium [11,12]. However, the exact mechanisms by which SNAP improved endothelial function and reduced MPO activity is beyond the aim of the present investigation and should be explored in future studies. In conclusion, a brief period of intracoronary administration of the NO donor SNAP before ischemia results in reduced infarct size, attenuated neutrophil infiltration, and improved endothelial function. These results suggest that NO liberated from SNAP exerts an early PC-like effect against ischemia–reperfusion injury in a narrow concentration range under in vivo conditions. Acknowledgments This study was supported by grants from the Swedish Medical Research Council (10857), the Swedish Heart and Lung Foundation, and the Karolinska Institute. Marita Wallin is gratefully acknowledged for technical assistance. References [1] R.M. Palmer, D.S. Ashton, S. Moncada, Vascular endothelial cells synthesize nitric oxide from L -arginine, Nature 333 (1988) 664–666. [2] K.Y. Xu, D.L. Huso, T.M. Dawson, D.S. Bredt, L.C. Becker, Nitric oxide synthase in cardiac sarcoplasmic reticulum, Proc. Natl. Acad. Sci. USA 96 (1999) 657–662. [3] Y. Hotta, H. Otsuka-Murakami, M. Fujita, J. Nakagawa, M. Yajima, W. Liu, N. Ishikawa, N. Kawai, T. Masumizu, M. Kohno, Protective role of nitric oxide synthase against ischemia– reperfusion injury in guinea pig myocardial mitochondria, Eur. J. Pharmacol. 380 (1999) 37–48. [4] M.R. Siegfried, J. Erhardt, T. Rider, X.L. Ma, A.M. Lefer, Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion, J. Pharmacol. Exp. Ther. 260 (1992) 668–675. [5] A.S. Weyrich, X.L. Ma, A.M. Lefer, The role of L -arginine in ameliorating reperfusion injury after myocardial ischemia in the cat, Circulation 86 (1993) 279–288.
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