reperfusion injury in rats

reperfusion injury in rats

Clinical Biochemistry 38 (2005) 943 – 947 Antiarrhythmic effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia/reperfusion injury in r...

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Clinical Biochemistry 38 (2005) 943 – 947

Antiarrhythmic effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia/reperfusion injury in rats Shiang-Suo Huang a, Sheng-Ming Liu a, Su-Man Lin b, Pei-Hu Liao a, Ruey-Hseng Lin a, Yu-Chia Chen c, Chun-Lien Chih b, Shen-Kou Tsai b,* a Department of Pharmacology and Institute of Medicine, College of Medicine, Chung Shan Medical University, Taichung, Taiwan Department of Anesthesiology, College of Medicine, National Yang-Ming University and Buddhist Tzu-Chi University and Hospital, Taipei Veterans General Hospital, 201, Shih-Pai Road, Second, Taipei, Taiwan c Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan

b

Received 3 May 2005; received in revised form 22 June 2005; accepted 6 July 2005 Available online 10 August 2005

Abstract Objectives: The present study was designed to determine the antiarrhythmic effect of caffeic acid phenethyl ester (CAPE), an active component of propolis, which exhibits antioxidant properties, in rats subjected to myocardial ischemia and ischemia – reperfusion (I/R) injury. Design and methods: Rats were subjected to 30 min coronary artery occlusion for evaluating the effect of CAPE on the myocardial ischemia injury. While in the myocardial I/R injury study, the coronary artery was ligated for a 5-min period of ischemia followed by a 30min period of reperfusion. Animals were pretreated with or without CAPE before coronary artery ligation and the severity of myocardial ischemia- and I/R-induced arrhythmias and mortality were compared. Results: Pretreatment of CAPE (0.1 and 1 Ag/kg) not only reduced both the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) but also decreased the mortality during the myocardial ischemia and I/R injury period. Conclusions: Our results suggest that CAPE is a potent antiarrhythmic agent with cardioprotective effects in myocardial ischemia and I/ R injury rats. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Antiarrhythmic agent; Caffeic acid phenethyl ester (CAPE); Heart; Ischemia; Reperfusion; Rat

Introduction There are many studies to report the role of free radicals on ischemia and I/R injury. Because of the potent and unstable characteristics of free radicals, it is easy for free radicals to interact with functional groups in living tissue, resulting in cellular membrane destruction and even cellular death [1,2]. An important consequence of myocardial ischemia or I/R injury is the occurrence of potentially lethal ventricular arrhythmia. Free radical scavenger and antioxidants become important strategies for the treatment of myocardial ischemia or I/R injury and the suppression of ventricular arrhythmia. * Corresponding author. Fax: +11 886 2 28751597. E-mail address: [email protected] (S.-K. Tsai).

Propolis is a natural product collected from honeybees and has been used in traditional Chinese medicine [3]. The biological activities of propolis have been reported to include free radical scavenging activity [4,5], anti-inflammatory effects [6], and antibacterial activity [7]. CAPE (caffeic acid phenethyl ester), one of the main components of propolis extract, exhibits antioxidant activity [8,9], vasorelaxant effects [10], anti-inflammatory effects [11], anticarcinogenic activities [12], and immunomodulatory effects [13]. CAPE also has been shown to prevent I/R injury in kidneys [14] and spinal cord [15]. Recently, CAPE has been shown to reduce I/R-induced myocardial infarction [16]. However, there is no literature that considers CAPE for possible use as a therapeutic drug in treating the acute symptoms of myocardial ischemia or I/R injury-induced arrhythmia. Therefore, in the present study,

0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.07.003

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we evaluated the antiarrhythmic effects of CAPE on myocardial ischemia or I/R injury in anesthetized rats subjected to transient coronary artery occlusion and reperfusion.

Materials and methods

releasing the tension applied to the ligature (operated groups) [19]. Successful ligation of the coronary artery was validated by observation of a decrease in arterial pressure and ECG changes (increase in R wave and ST segment elevation) indicative of ischemia. Sham-operated animals underwent all surgical procedures, except that the silk passing around the left coronary artery was not tied (sham groups).

Animals and chemicals Drug administration The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Sprague – Dawley rats (National Lab. Animal Breeding and Research Center, Taipei, Taiwan) weighing 250 – 300 g were used. These animals were housed in a room with controlled temperature (24 T 1-C) and humidity (55 T 5%) under a 12:12-h light – dark cycle. They were allowed free access to food and water. CAPE was purchased from Sigma Chemical Company (St. Louis, MO, USA).

CAPE (0.01, 0.1, or 1 Ag/kg) or vehicle (dimethyl sulfoxide – 0.9% NaCl, 1:104; v/v) was infused via a jugular vein 15 min before coronary artery occlusion. No effect of vehicle on ischemia- and I/R-induced arrhythmia at such a

Animal preparation Myocardial ischemia was produced by a temporary tightening of the silk ligature around the left main coronary artery as previously described [17,18]. Briefly, male Sprague – Dawley rats were anesthetized with urethane (1.25 g/kg i.p.) and placed on an operating table. The trachea was cannulated for artificial respiration and the jugular vein was cannulated for drug administration. Polyethylene catheters (PE-50) were inserted into the common carotid artery for continuous monitoring of heart rate (HR) and arterial blood pressure (BP) by a Statham P23 XL transducer and displayed on a Gould RS-3400 physiological recorder (Gould, Cleveland, OH, USA). A standard lead-1 electrocardiogram (ECG) was also recorded by attaching silver electrodes to the extremities of animals. After tracheotomy, the animals were ventilated with room air by a respirator for small rodents (Model 131, NEMI, USA) with a stroke volume of 12 mL/kg body weight and at a rate of 60 strokes/min to maintain normal pO2, pCO2, and pH parameters (blood gas analyzer, GEM-5300 I.L. CO, USA). The chest was opened by a left thoracotomy, followed by sectioning the fourth and fifth ribs, approximately 2 mm to the left of the sternum. The heart was quickly expressed out of the thoracic cavity, inverted and a 6/0 silk ligature was placed around the left main coronary artery. The heart was repositioned in the chest and the animal was allowed to recover for 15 min. Animals in which the procedure produced arrhythmia or a sustained decrease in BP to less than 70 mm Hg were not included in the study. A small plastic snare formed from a Portex P-270 cannula was threaded through the ligature and placed in contact with the heart. The coronary artery then was occluded by tightening the ligature and reperfusion was achieved by

Fig. 1. (A) Systolic (closed symbols) and diastolic (open symbols) arterial blood pressure change in vehicle (0.01% DMSO) ( , o), and three doses of CAPE at 0.01 Ag/kg (0, >), 0.1 Ag/kg (r, q), and 1Ag/kg ( , g) during myocardial ischemia in rats. (B) Heart rate change in vehicle (0.01% DMSO) ( ), and three doses of CAPE at 0.01 Ag/kg (0), 0.1 Ag/kg (r), and 1Ag/kg ( ) in rats subjected to myocardial ischemia. Each experimental group contains ten rats. Results are expressed as mean T SEM. The differences between vehicle and three doses of CAPE infused animals were not statistically significant (ANOVA). 0 indicates coronary ligation.

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Table 1 Effect of CAPE on coronary ligation (30 min) induced arrhythmias in anesthetized rats n

Sham Vehicle CAPE 1 Ag/kg

4 4

Operated (coronary ligation) Vehicle 17 CAPE 0.01 Ag/kg 16 CAPE 0.1 Ag/kg 12 CAPE 1 Ag/kg 10

Ventricular tachycardia

Ventricular fibrillation

Mortality

Incidence (%)

Duration (s)

Incidence (%)

Duration (s)

(%)

– –

– –

– –

– –

– –

94 100 75 50*

73.1 55.1 23.4 17.8

71 63 33 20*

209.8 197.7 46.9 31.5

T T T T

35.1 24.6 12.8* 5.5*

T T T T

53.4 55.7 19.2* 23.5*

59 50 25 20

Vehicle is 0.01% DMSO in normal saline; n = number of experiments; values for duration of VT and VF are shown as the mean T SEM. * Statistical difference at the level of P < 0.05 as compared with vehicle.

Statistics

concentration. Animals were randomly allocated to each drug treatment and vehicle group.

Data were expressed as mean T standard error of mean (SEM). The difference of BP, HR, duration of VT and VF between vehicle and drug treatment groups in the arrhythmia study was carried out by using analysis of variance (ANOVA) followed by Bonferroni’s test. The difference in the percentage incidence of VT, VF, and mortality was analyzed with a v 2 test. P < 0.05 was considered to be statistically significant.

Evaluation of arrhythmia For evaluating the effect of CAPE on myocardial ischemia or I/R injury, the coronary artery was occluded for 30 min or 5 min followed by 30 min reperfusion. Before and during the ischemia or I/R period, heart rate (HR), blood pressure (BP), and ECG changes were recorded simultaneously on a personal computer with a wave form data analysis software (AcqKnowledge, Biopac System, Goleta, CA, USA). Ventricular ectopic activity was evaluated according to the diagnostic criteria advocated by the Lambeth Convention [20]. The ECGs were analyzed to determine incidence and duration of ventricular tachyarrhythmias, including ventricular tachycardia (VT) and ventricular fibrillation (VF), in surviving as well as the nonsurviving animals. VF was defined as (1) the development of a chaotic, irregular rapid left ventricular cardiomyogram (LVCMG); (2) the loss of pulsatile left ventricular pressure (LVP); and (3) the loss of grossly observable, regular ventricular contraction. VT was defined as a rapid LVCMG with a regular and stable rhythm and a regular cyclic change in LVP with small amplitude. In rats with irreversible VF, the duration of VF was recorded up to the time when BP fell to <15 mm Hg.

Results Hemodynamic changes during coronary artery occlusion Injection of CAPE via the jugular vein did not change the BP, nor the HR in rats subjected to myocardial ischemia (Fig. 1). No significant difference was seen among control and drug-treated groups. Ischemia- and I/R-induced rhythm disturbances Effects of coronary ligation-elicited arrhythmias in anesthetized rats are shown in Table 1. In the ischemiainduced arrhythmia study, severe ventricular arrhythmias occurred at 6 –7 min and peaked at 8– 12 min after coronary

Table 2 Effect of CAPE on reperfusion (30 min) induced arrhythmias in anesthetized rats n

Sham Vehicle CAPE 1 Ag/kg Operated (I/R) Vehicle CAPE 0.01 Ag/kg CAPE 0.1 Ag/kg CAPE 1 Ag/kg

Ventricular tachycardia

4 4

15 14 8 8

Ventricular fibrillation

Mortality

Incidence (%)

Duration (s)

Incidence (%)

Duration (s)

(%)

– –

– –

– –

– –

– –

100 57* 38* 38*

24.6 15.4 2.4 2.0

73 50 25* 13*

186.4 127.0 41.8 40.6

T T T T

11.2 10.4* 1.6* 1.5*

T T T T

42.4 38.7 39.3* 40.6*

Vehicle is 0.01% DMSO in normal saline; n = number of experiments; values for duration of VT and VF are shown as the mean T SEM. * Statistical difference at the level of P < 0.05 as compared with vehicle.

53 50 13 13

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occlusion. In the vehicle-treated group, 94% (16/17) of rats exhibited VT (73.1 T 35.1 s in duration), and 71% (12/17) of rats exhibited VF (209.8 T 53.4 s). However, for rats treated with CAPE at doses of 0.1 and 1 Ag/kg 15 min prior to coronary occlusion, the duration of VT (23.4 T 12.8 s, 17.8 T 5.5 s) and VF (46.9 T 19.2 s, 31.5 T 23.5 s) significantly decreased, respectively, and the incidence of VT and VF reduced significantly only in rats with 1 Ag/kg CAPE administration. The mortality declined from 59% to 20% in rats treated with 1 Ag/kg CAPE (Table 1). Effects of myocardial I/R-elicited arrhythmias in anesthetized rats are shown in Table 2. In this study, we selected a 5-min period of ischemia followed by a 30-min period of reperfusion in order to produce the maximal effect of the rhythm disturbance [21]. During reperfusion injury, rats treated with all three concentrations of CAPE significantly reduced the incidence (57%, 38%, 38% vs. 100%, P < 0.05) and duration (15.4 T 10.4 s, 2.4 T 1.6 s, 2.0 T 1.5 s vs. 24.6 T 11.2 s, P < 0.05) of VT, but the incidence and duration of VF was significantly reduced only at the doses of 0.1 (25% vs. 73%, 41.8 T 39.3 s vs. 186.4 T 42.4 s) and 1 Ag/kg CAPE (13% vs. 73%, 40.6 T 40.6 s vs. 186.4 T 42.4 s) treatment compared to that of vehicle-treated rats. The mortality rate declined from 53% to 13% in rats treated with 1 Ag/kg CAPE (Table 2).

Discussion Myocardial ischemia and I/R injury will induce ventricular arrhythmia resulting in circulation collapse and end in sudden death [22,23]. The underlying pathophysiological mechanisms that have been suggested include a burst production of reactive oxygen intermediates (superoxide radical, hydrogen peroxide, hydroxyl radical, singlet oxygen) [2], and intracellular calcium overload [24] that occurs during early moments of myocardial ischemia and I/R might be involved. Therefore, effective inhibition of oxygen free radical production or elimination of oxygen free radicals can reduce the ventricular arrhythmia and sudden death caused by ischemia and I/R injury [2,25]. In the present study, we provide evidence that in anesthetized rats the administration of CAPE (0.1 and 1 Ag/kg) prior to coronary artery occlusion reduced the mortality and significantly suppressed both the incidence and duration of VT and VF during myocardial ischemia and I/R injury. These results indicated that CAPE possesses the robust cardioprotective effect against myocardial ischemia and I/R injury. The cardioprotective effect of CAPE may be due to the antioxidant activity in biological systems. During myocardial ischemia and I/R period, the xanthine/xanthine oxidase system and rapid accumulation of neutrophils in the damaged tissue are considered to be the potential source of free radicals. CAPE at a concentration of 10 AM completely blocks the production of reactive oxygen species in human neutrophils and in the xanthine/xanthine oxidase system [9].

The production of oxygen-derived free radicals during myocardial ischemia and the I/R period may be suppressed by the antioxidant activity of CAPE. Recently, Cicala et al. reported the vasorelaxant effect of CAPE is dependent upon the presence of functional endothelium and, at high concentrations, upon an inhibitory effect on Ca2+ movements through the cell membrane and within the vascular smooth muscle cell [10]. The vasodilative effect of CAPE may improve myocardial perfusion during myocardial ischemia. CAPE also increases tissue NO levels to protect the testes from torsion/detorsion injuries [26]. Furthermore, CAPE has been shown to increase eNOS activity to prevent ischemia – reperfusion injury [15,27]. During myocardial ischemia and I/R period, CAPE may suppress superoxide radical production to prevent NO interaction with superoxide anion, which then preserves the benefits of the physiological function of NO including autoregulatory modulation of coronary blood flow [28] and inhibition of platelet aggregation [29]. NO relaxes vessels and could protect myocardium against ischemia and I/R injury through coronary vasodilatation and prevent the blockage of microvascular blood flow in the ischemic region via anti-platelet aggregation. NO precursors like larginine have been reported to have cardioprotective effects in the ischemic myocardium against myocardial arrhythmia and infarction [30]. It is reasonable to speculate that the protective mechanism of CAPE in the coronary artery occlusion and reperfusion rat is partially mediated via NO. In conclusion, our study presents the first evidence that CAPE could effectively protect myocardium against myocardial ischemia- and I/R-induced arrhythmia and mortality. We would speculate that the beneficial cardioprotective effect of CAPE may be due to its antioxidant activity, vasodilating effect, upregulation of NO, or all these mechanisms combined.

Acknowledgments This study was supported by grant NSC 93-2314-B-010047 from the National Science Council and Academic Sinica, and grant VGH 94-340 from Taipei Veterans General Hospital, Taipei, Taiwan.

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