Interact CardioVasc Thorac Surg 2009;8:629-634. doi:10.1510/icvts.2008.195933
© 2009 European Association of Cardio-Thoracic Surgery
Institutional report - Experimental |
Sivelestat reduces myocardial ischemia and reperfusion injury in rat hearts even when administered after onset of myocardial ischemia
Masaru Kambe*,
Ryuzo Bessho,
Masahiro Fujii,
Masami Ochi and
Kazuo Shimizu
Department of Biological Regulation and Regenerative Surgery, Nippon Medical School Graduate School of Medicine, 1-1-5, Sendagi, Bunkyo, Tokyo, Japan
Received 9 October 2008;
received in revised form 13 February 2009;
accepted 17 February 2009
 This paper was presented at the 19th World Congress of the International Society for Heart Research (2007) in Bologna, Italy.
*Corresponding author. Department of Thoracic and Cardiovascular Surgery, Nippon Medical School Chiba-Hokuso Hospital, 1715, Kamagari, Inba, Chiba 270-1694, Japan. Tel.: +81-476-99-1835; fax: +81-476-99-1921.
E-mail address: shaw{at}nms.ac.jp (M. Kambe).
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Abstract
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Sivelestat, a neutrophil elastase inhibitor, has been shown to attenuate pulmonary injury during ischemia and reperfusion by improving microcirculation and may be effective as a cardioprotective agent. Isolated rat hearts were Langendorff-perfused (constant pressure, 75 mmHg) with oxygenated Krebs–Henseleit bicarbonate buffer (KHB). The optimal sivelestat concentration at 19 µmol/l was revealed because left ventricular developed pressure (LVDP) recovery in 19 µmol/l sivelestat was highest among 0.19, 1.9, 19, 190, and 1900 µmol/l sivelestat (26±10, 33±7, 56±5*, 35±2, and 15±5%, respectively; *P<0.01). In order to examine the optimal administration timing, sivelestat was administered at pre- and post-ischemic phases. LVDP recovery and troponin-T were observed in pre-, post-ischemic sivelestat groups and control. After 60 min-reperfusion, LVDP recoveries were 42±10*, 45±19*, and 14±5%, respectively (*P<0.01 compared to control), and troponin-T values were 4±1, 2±1**, and 8±2, respectively (**P<0.05 compared to control). Acetylcholine-induced increase in coronary flow was also investigated to examine the sivelestat's cardioprotective mechanism. Ischemia–reperfusion (I/R) impaired the acetylcholine-induced increase in coronary flow (maximal changes: sham, 125±11%; I/R, 98±3; P<0.01) and this impairment was attenuated by sivelestat-perfusion at reperfusion (maximal change: 112±7%; P<0.05 vs. I/R). Sivelestat attenuates coronary endothelial ischemia–reperfusion injury and improves myocardial protection even when administered at the reperfusion period. This suggests a role for sivelestat in the preservation of coronary endothelial function enhancing myocardial protection.
Key Words: Ischemia/reperfusion; Myocardial protection; Endothelium
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1. Introduction
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The essential qualification for the salvage of viable myocardium and the conservation of cardiac function after an ischemic event is timely reperfusion [1]. Although reperfusion salvages myocardium that would ultimately die in its absence, restoring blood flow to the myocardium carries the potential to exaggerate the injury of ischemia [2]. This is called a reperfusion injury. A reperfusion injury slightly offsets the optimal recovery of myocardium achieved in cardiac surgery, percutaneous coronary intervention and other recanalization therapies.
Sivelestat, a specific neutrophil elastase inhibitor which is already used clinically as an agent for acute lung injury or acute respiratory distress syndrome [3], is known to simultaneously have the effect of attenuating ischemia and reperfusion injury in the lung [4]. Microscopic remarks have revealed that sivelestat attenuates ischemia and reperfusion injury in pulmonary microcirculation by suppressing endothelial permeability [4], preventing the endothelial and surrounding tissue from swelling (edema) or by preventing neutrophils from sticking to capillary walls [5].
A hypothesis was established that the administration of sivelestat sodium might attenuate ischemia and reperfusion injury in the heart and have a protective effect on myocardium, so the first experiment was conducted to investigate the efficacy of this agent. Consequently, further experiments were conducted to examine the optimal timing for the administration of sivelestat and to investigate the mechanism of action for endothelial preservation.
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2. Material and methods
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2.1. Animals
Adult male Wistar rats (240–300 g body weight) were used (Saitama experiment animals, Saitama, Japan). All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care,’ formulated by the National Society for Medical Research, and the ‘Guide for the Care and Use of Laboratory Animals,’ published by the National Institute of Health (NIH Publication No. 85-23, revised 1996). Additionally, this study was approved by the Animal Ethics Committee of Nippon Medical School. The rats were anesthetized with pentobarbital (50 mg/kg i.p.) and anticoagulated with heparin (1000 IU/kg i.v.).
2.2. Heart isolation and perfusion
The hearts were quickly excised and immersed in cold (4 °C) Krebs–Henseleit bicarbonate buffer (KHB). The aorta was then cannulated, and the heart was perfused at 37 °C in the Langendorff mode with KHB at constant pressure (75 mmHg), as previously described [6]. The heart was prepared by inserting a saline-filled vinyl balloon into the left ventricle via the left atrium, and the balloon was connected to a pressure transducer to measure left ventricular pressure. The balloon's pressure was then adjusted to a range between 3 and 8 mmHg of the left ventricular end-diastolic pressure (LVEDP). All hearts were equilibrated with 20 min of aerobic perfusion, and baseline readings of left ventricular systolic pressure (LVSP, mmHg), LVEDP (mmHg), heart rate (beats/min), and coronary flow (ml/min) were measured. Left ventricular developed pressure (LVDP) was calculated (LVSP minus LVEDP). At the time of baseline readings, hearts were excluded if the acceptable ranges of LVDP (>100 mmHg), heart rate (>220 beats/min), and coronary flow (8–16 ml/min) were not met.
2.3. Perfusion medium
Composition of KHB was as follows: NaCl, 118.5 mmol/l; NaHCO3, 25.0 mmol/l; KCl, 4.8 mmol/l; MgSO4, 1.2 mmol/l; KH2PO4, 1.18 mmol/l; CaCl2, 1.4 mmol/l; and glucose, 11.0 mmol/l.
Sivelestat was dissolved in KHB to 19 µmol/l (10 µg/ml) sivelestat-dissolved KHB, which has been discovered to be the effective concentration for clinical use to achieve lung protection [7].
Acetylcholine chloride (ACh) was dissolved in distilled water and then diluted in oxygenated KHB to 1 µmol/l -ACh-dissolved KHB, as previously described [8].
2.4. Perfusion protocol
For all perfusion protocols, each heart was subjected to a 20 min equilibration period of aerobic KHB perfusion at 37 °C. After equilibration, the hearts were subjected to one of the three perfusion protocols as shown in Fig. 1a–c.
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Fig. 1. (a) Experimental perfusion protocols of the preliminary study. In this protocol, all hearts were perfused at a constant pressure equivalent to 75 mmHg before and after global ischemia. First, the hearts were assigned to two groups (n=6/group): After a 20-min equilibration period, the hearts were then subjected to 30 min of global ischemia followed by either control (i): 60 min of reperfusion by KHB; or sivelestat (ii): 19 µmol/l (10 µg/ml) of sivelestat sodium-dissolved KHB infused for 10 min at a constant pressure (75 mmHg) prior to 60 min of KHB reperfusion. After 60 min of reperfusion, myocardial function was then measured. Secondly, thirty-six hearts were divided into six groups (n=6/group): control (i): same as above; 1/100 Sivelestat (S) (ii): 0.19 µmol/l; 1/10 S (iii): 1.9 µmol/l; S (iv): administered 19 µmol/l (10 µg/ml) sivelestat dissolved in KHB solution for 10 min prior to reperfusion; 10 S (v): 0.19 mmol/l; 100 S (vi): 1.9 mmol/l, respectively. All protocols were followed by a further 60 min of reperfusion, after which recovery of myocardial function was measured. (b) Experimental perfusion protocol of Study 1. In Study 1, the hearts were assigned to one of three groups (n=6/group) before being subjected to 30 min of global ischemia: control (i): additional 10 min perfusion with KHB and no other treatment throughout the protocol; pre-sivelestat (ii): additional 10 min perfusion with 19 µmol/l sivelestat in perfusate; reperfusion-sivelestat (iii) additional 10 min perfusion only at the beginning of reperfusion with 19 µmol/l sivelestat in perfusate. All protocols were followed by a further 60 min of reperfusion, after which recovery of myocardial function was measured. During the reperfusion period, effluent was collected and the troponin T level was evaluated. (c) Experimental perfusion protocol of Study 2. In Study 2, three groups of hearts (n=6/group) were studied. The hearts were aerobically perfused (20 min); then were subjected to either: sham (i): 30 min aerobic perfusion (non-ischemia); control (ii): 30 min of global (37 °C) ischemia (GI); Sivelestat (iii): sivelestat sodium (19 µmol/l) infused immediately after 30 min GI. The hearts were then reperfused for a further 60 min and coronary flow was measured (as a baseline value). Subsequently, all hearts were aerobically perfused with acetylcholine-dissolved (1 µmol/l) KH for 1 min and the change in coronary flow was recorded for the next 10 min.
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2.4.1. Preliminary study: does sivelestat have an effect on ischemia and reperfusion injury and at what dose does it work best?
After 20 min of an equilibration period, the hearts were subjected to 30 min of global ischemia followed by either (i) control: 60 min reperfusion with KHB; or (ii) sivelestat: 10 min perfusion with 19 µmol/l (10 µg/ml) sivelestat-dissolved KHB prior to 60 min of KHB reperfusion, after which the recovery of myocardial function (LVDP) was measured (Fig. 1a). Then, the hearts of the Sivelestat group were randomly assigned to one of five sivelestat concentration groups (i.e. 0.19, 1.9, 19, 190, and 1900 µmol/l; n=6/group).
2.4.2. Study 1: does the timing of sivelestat administration influence protection?
In Study 1, the sivelestat concentration was fixed at 19 µmol/l. The hearts were assigned to one of three groups (n=6/group) before being subjected to 30 min of global (37 °C) ischemia: (i) control: an additional 10-min perfusion with KHB (no treatment); (ii) pre-sivelestat: an additional 10-min perfusion with 19 µmol/l sivelestat-dissolved KHB; (iii) reperfusion-sivelestat: an additional 10-min perfusion at the beginning of the reperfusion with 19 µmol/l sivelestat-dissolved KHB. All protocols were followed by a further 60 min of reperfusion, after which recovery of myocardial function was measured (Fig. 1b).
To evaluate for myocardial damage, the coronary effluents were collected and troponin T was measured by Electrochemiluminescence immunoassay.
2.4.3. Study 2: does sivelestat influence acetylcholine-induced increase in coronary flow?
After 20 min of equilibration period, hearts (n=6/group) were subjected to either: (i) sham; (ii) control: 30 min global (37 °C) ischemia; (iii) sivelestat: 10 min perfusion at the beginning of reperfusion with 19 µmol/l sivelestat-dissolved KHB. All hearts were reperfused for 60 min and coronary flow was measured, then 1 µmol/l acetylcholine was infused for 1 min and coronary flow changes were recorded for the following 10 min (Fig. 1c). As the perfusion pressure was maintained at a constant, increases in coronary flow were considered to be relaxations and reductions in coronary flow to be constrictions, of the coronary vasculature.
2.5. Expression of results
Post-ischemic recovery of LVDP, heart rate, and coronary flow were expressed as a percentage of the baseline values; LVEDP and troponin T were expressed as an absolute value (LVEDP: mmHg; troponin T: ng/ml).
2.6. Statistics
Statistical analysis was performed using SPSS. All data are expressed as mean±S.D. Comparisons between groups were assessed for significance by ANOVA or repeated measures ANOVA, as appropriate; if significance was established, post-hoc analysis was assessed by the Bonferroni test, which allowed for multiple comparisons. The Student t-test was used to compare the two means.
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3. Results
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3.1. Preliminary study: does sivelestat have an effect on ischemia and reperfusion injury and at what dose does it work best?
The pilot dose–response study established an optimal concentration of sivelestat at 19 µmol/l.
Firstly, the recovery of LVDP in the sivelestat group had better values, and was more rapid than the control group. Recovery percentages of LVDP after 60 min of reperfusion were (i) 16±6% and (ii) 35±5% (P<0.001, Fig. 2a).
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Fig. 2. Recovery of function and the dose–response curve in hearts subjected to the preliminary study. (a) Recovery of LVDP with reperfusion duration (min) in the preliminary study, expressed as a percentage of the pre-ischemic control value. Values are the mean of 6 hearts/group±S.D. LVDP: left ventricular developed pressure. *P<0.01,  P<0.001 vs. another group. Filled squares, control; filled circles, sivelestat-treated hearts. (b) Recovery of LVDP at the end of 60 min reperfusion in the respective concentration of the sivelestat treatment groups, expressed as a percentage of the pre-ischemic control value. Values are the mean of 6 hearts/group±S.D. *P<0.01 vs. other groups. 1/100 S: 0.19 µmol/l sivelestat; 1/10 S: 1.9 µmol/l sivelestat; S: 19 µmol/l sivelestat; 10 S: 190 µmol/l sivelestat; 100 S: 1.9 mmol/l sivelestat, respectively.
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Secondly, the sivelestat dose–response curve for LVDP recovery (Fig. 2b) demonstrated a bell-shaped curve with increased concentrations of sivelestat. (Baseline and final recovery of all parameters are shown in Table 1.)
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Table 1 Baseline value and final percentage of recovery of LVDP, heart rate, coronary flow, and final LVEDP value for the preliminary study
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3.2. Study 1: does the timing of sivelestat administration influence protection?
The mean baseline values of LVDP, coronary flow, heart rate, and LVEDP after the equilibration period are shown in Table 2. There were no significant differences between the groups for any of these values.
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Table 2 Baseline value and percentage of recovery of LVDP, heart rate, coronary flow, and final LVEDP value in Study 1
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Post-ischemic recovery of LVDP (Fig. 3a) for hearts not treated by sivelestat had a low degree of recovery (reaching a plateau value at approximately 10% of baseline), whereas those treated by sivelestat recovered gradually and reached a value around 40%. It is interesting to note that the recovery of LVDP did not differ significantly whether sivelestat was administered prior to, or subsequent to, global ischemia.
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Fig. 3. (a) Recovery of LVDP with reperfusion duration (min) in Study 1, expressed as a percentage of the pre-ischemic control value. Values are the mean of 6 hearts/group±S.D. *P<0.005, P<0.05 vs. the non-treated group. Filled squares, 10 min treatment at the beginning of reperfusion with sivelestat; open squares, 10 min treatment prior to global ischemia with sivelestat; filled circles, no treatment (control). (b) Transition of LVEDP with reperfusion duration (min) in Study 1, expressed as absolute value. Values are the mean of 6 hearts/group±S.D. *P<0.05 vs. treated groups. Filled squares, 10 min treatment at the beginning of reperfusion with sivelestat; open squares, 10 min treatment prior to global ischemia with sivelestat; filled circles, no treatment (control). (c) Troponin T values during the reperfusion period in Study 1. Values are the mean of 6 hearts/group±S.D. *P<0.05 vs. the non-treated group. ‘Pre’, 10 min treatment prior to global ischemia with sivelestat; ‘Reperfusion’, hearts of 10 min treatment at the beginning of reperfusion with sivelestat.
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Elevated post-ischemic LVEDP in the non-treated group had a higher tendency during the reperfusion period and was significantly higher (P<0.05) at the end of reperfusion than the other two groups (Table 2, Fig. 3b). Recovery of heart rate and coronary flow (Table 2) were similar for all groups, however, the higher tendency existed in the sivelestat-treatment groups with regards to coronary flow.
The troponin T of group (iii) was significantly smaller (P<0.05) than non-treated group (Fig. 3c). The value of group (ii) was smaller than that of group (i) but did not have a significant difference.
3.3. Study 2: does sivelestat influence acetylcholine-induced increase in coronary flow?
Ischemia–reperfusion (I/R) impaired the acetylcholine-induced increase in coronary flow (percent pre-acetylcholine-infusion value; maximal changes: (i) sham, 125±11%; (ii) I/R, 98±3; P<0.01) and this impairment was attenuated by sivelestat-perfusion at reperfusion (maximal change: (iii) sivelestat, 112±7%; P<0.05) (Fig. 4).
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Fig. 4. The reaction of coronary flow to ACh in Study 2. Height of the bar indicates the increase or decrease in coronary flow at a constant pressure (75 mmHg) expressed as a percentage compared to the baseline values (just before the administration of ACh). Values are the mean of 6 hearts/group±S.D. *P<0.05 vs. control, **P<0.005 vs. control.
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4. Discussion
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This study has revealed interesting data regarding the role of sivelestat in myocardial ischemia and reperfusion injury, including the finding that administration at the time of reperfusion has comparable effects to pre-ischemic treatment. Sivelestat was also found to have a protective effect against ischemia and reperfusion injury in the coronary endothelium. Furthermore, these protective effects were obtained in the crystalloid perfusion model, in which neutrophils are not involved.
Sivelestat, a novel neutrophil elastase inhibitor, has been clinically applied in the respiratory field [3]. Recent studies have shown that sivelestat reduces and prevents tissue ischemia and reperfusion injury, not just in the lung but in other organs [9, 10]. The hearts in this study, however, were perfused with a buffer solution, so that few neutrophils could have infiltrated them, which prompted the significant question as to why sivelestat ameliorates myocardial ischemia and reperfusion injury in the crystalloid perfusion model that is neutrophil-free.
Initially, this study was designed using the blood perfusion model. Fortuitously, the results of the pilot crystalloid-perfused study presented interesting data regarding the role of sivelestat in myocardial ischemia and reperfusion injury. Previous studies have demonstrated [11, 12] that post-ischemic reperfusion may produce endothelial dysfunction, since it reduces endothelium-dependent coronary vasodilatation, and endothelial dysfunction may reduce myocardial function. A hypothesis that the myocardial protective effect of sivelestat is based on endothelial protection was suggested, therefore coronary endothelial function after ischemia and reperfusion was investigated. Ischemia and reperfusion may impair not only myocardial function but the coronary endothelial-dependent dilative function [12]. The same phenomenon was observed in this study: cardiac acetylcholine-induced relaxation was impaired in the heart exposed to post-ischemic reperfusion and this impairment was attenuated by sivelestat. This study suggests that sivelestat has a protective effect on coronary endothelium, which may eventually bring about a myocardial protective effect against ischemia and reperfusion. This is in agreement with the animal data and suggests that the injury that occurs during reperfusion is largely attributable to ischemia and reperfusion-induced endothelial dysfunction [11, 13].
Although Ueno et al. demonstrated in 2001 that treatment with sivelestat resulted in lower CK-MB, lactate, and inflammatory cytokine levels in a heart transplantation model [14], the present study still contains the interesting finding that the timing of sivelestat treatment does not matter. This is interesting since sivelestat is a neutrophil elastase inhibitor, and neutrophils are of particular importance in the reperfusion phase of ischemia and reperfusion injury. In this regard, it has been demonstrated in in-vivo models [15] that ischemia and reperfusion consist of a dual phase, in which proinflammatory cytokines are released by epithelial cells and macrophages, after which neutrophils and lymphocytes are recruited into the organ and endothelial cells, mainly during reperfusion. Therefore, it would appear that the optimal timing for sivelestat administration would be during reperfusion, to prevent endothelial reperfusion injury under conditions where neutrophils are present. In this study, however, the hearts were perfused with a buffer solution instead of whole blood, so that few neutrophils could have infiltrated the heart. This fact indicates that sivelestat's protective mechanism does not lie in neutrophil elastase inhibition, but in some other effect, perhaps the demonstrated effect on the endothelium. Nevertheless, the preservation of endothelium-dependent vasodilatation does not necessarily mean a mechanism of myocardial protection, it may just be a parallel phenomenon. Further studies are needed to verify sivelestat's true mechanism of action.
Coronary artery disease is a highly complicated condition. The hearts used in this study were isolated from healthy rats. It is unlikely that any protective effect seen would be efficacious in the diseased heart, particularly in diseased endothelium. Moreover, it remains to be determined to what extent these results in rat hearts correspond to human hearts.
This study demonstrated some interesting beneficial effects of sivelestat. This novel pharmacological post-conditioning effect of myocardium and endothelium warrant further study to determine the efficacy of this potentially beneficial alternative to conventional post-conditioning procedures.
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Abstract
1. Introduction
