行政院國家科學委員會專題研究計畫 成果報告
3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) 還 原酵素阻斷素, statins 對肺靜脈及心房心肌細胞之電生理
與心律不整作用
計畫類別： 個別型計畫
計畫編號： NSC94-2314-B-038-061-
執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日 執行單位： 臺北醫學大學醫學系
計畫主持人： 陳亦仁 共同主持人： 陳耀昌
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中 華 民 國 95 年 9 月 18 日
3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, statins 對肺靜脈及心房心肌細胞之電生理與心律不整作用
研究計畫中英文摘要 中文摘要
心房顫動乃是臨床上常見之心律不整，且會造成心臟功能不良以及腦中風，
雖然許多抗心律不整藥物已經被用於治療與預防心房顫動，然大多具有相當之副 作用而無法長期使用。最近研究顯示 HMG-GA 還原酵素阻礙素 statins，乃是廣泛 用於治療高血脂症之藥物，被發現可減少心房顫動之產生，然而其電生理機轉以 及預防心房顫動的原因仍不清楚，再則，是否不同的 statins 會有不同的效果也 未明瞭。
肺靜脈已知是引發心房顫動之病灶所在，過去的研究已知肺靜脈含心肌細胞 且有其特有之電生理特性可引發心律不整活性，長時間心房電刺激，以及使用發 炎物質都會增加肺靜脈心肌細胞引發心律不整活性，反之，一氧化氮則被發現可 用抑制肺靜脈心肌心律不整之作用，由於 statins 已知會增加一氧化氮之生理活 性以及明顯之抗發炎作用，這些結果顯示，statins 或可藉者抑制肺靜脈心肌之 心律不整活性而達到其抑制心房顫動的效果，因此本研究旨在探討 statins 對肺 靜脈之心律不整活性之作用。
方法： 傳統電極記錄記錄肺靜脈之動作電位，以及收縮力在接受 Simastatin 0.1 、 1μM 後之變化，以及在 1μM 之 Simastatin 下，使用 L-NAME 100μM 後 的變化。
結果：Simastatin 在 1μM 的濃度下可以於 1 小時後抑制肺靜脈心肌細胞之自 動性從 1.7±0.1Hz 到 1.5±0.1Hz，並在 2 小時後到達平穩的抑制狀態約 1.4±0.1 Hz。這個動作可以被 100μM 的 L-NAME 所抑制而回復肺靜脈之節律。再則 1μM Simastatin 可以稍微延長肺靜脈心肌之動作電位從 88±7 ms 到 93±7 ms。
結論：本實驗顯示 Simastatin 可以抑制肺靜脈引發心律不整活性，且此機 轉與一氧化碳之產生有關。這些結果可能是造成 Statin 減少心房顫動的機轉。
英文摘要
Atrial fibrillation is the most common cardiac arrhythmia seen in clinical practice and induce cardiac dysfunction and stroke. Although several antiarrhythmic drugs have been used in treating and preventing atrial fibrillation, drugs with little adverse effects effectively to prevent atrial fibrillation. Recent studies have shown that use of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, statins reduce the occurrences of atrial fibrillation clinically. However, knowledge about the electrophysiological effects of statins on
cardiomyocytes and the mechanisms of anti- fibrillation were limited and it is not clear whether different statins may have different cardiac effects. Pulmonary veins (PVs) were known to be important sources of ectopic beats with the initiation of paroxysmal atrial fibrillation. Our previous have found that PVs have cardiomyocytes with distinct
electrophysiological characteristics and arrhythmogenic activities.
Long-term rapid atrial pacing and inflammatory cytokine increased PV arrhythmogneic activity. In contrast, nitric oxide was found to decrease PV arrhythmogenic activity with the reduction of atrial fibrillation.
Because statins increases nitric oxide bioavailability and has
anti-infllammation effects, it is possible that the statins may inhibit atrial fibrillation through the decrease of PV arrhythmogenic activity.
Therefore, the purposes of the present study are to investigate the effects of statins on the arrhythmogenic activity of PV cardiomyocytes.
Methods: Conventional microelectrodes were used to record the action potential
(AP) and contractility in isolated rabbit PV tissue specimens before and after
the administration of simvastatin (0.1, 1 µM). L-NAME (100 µM)was administrated in the presence of simvastatin (1 µM).
Results: Simvastatin (1 µM, but not 0.1 µM) decrease the PV firing rates from 1.7±0.1 to 1.5±0.1 Hz at one hour and achieve steady state firing rates of 1.4±0.1 Hz at 2 hour. This effect is reversed after the
administration of L-NAME (100 µM, inhibitor of nitric oxide production).
Moreover, simvastatin (1 µM) mildly prolonged the action potential duration from 88±7 ms to 93±7 ms (n=5).
Conclusion
We demonstrated the simvastatin may decrease the PV arrhythmogenesis through the production of nitric oxide. These results may underlie the anti-arrhythmic potential of statin and result in the decrease of atrial fibrillation.
Key Word: Atrial fibrillation, electrophysiology, HMG-CoA, pulmonary vein,
Introduction
Atrial fibrillation is the most common cardiac arrhythmia seen in clinical practice and induce cardiac dysfunction and stroke [1-2]. Although several antiarrhythmic drugs have been used in treating and preventing atrial fibrillation, drugs with little adverse effects effectively to prevent atrial fibrillation, especially in the presence of heart failure and myocardial ischemia were still limited clinically. Recent studies have shown that use of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, statins reduces the occurrences of atrial
fibrillation clinically [3-4] and administration of atorvastatin also reduce the occurrence of atrial fibrillation and increase of action potential duration in pericarditis animal model [5]. Moreover, statins may prevent atrial remodeling, reduce the occurrence of atrial
fibrillation and attenuate the down-regulation of L-type calcium currents in the long-term atrial pacing animal models. [6] Atrial remodeling due to atrial tachyarrhythmias can alter atrialelectrophysiology and promote AF, and these alterationsare believed to contribute to both the occurrence and the persistence of the arrhythmia. [7-10] These findings suggest the potential antiarrhythmic effects of statins. However, knowledge about the electrophysiological effects of statins on cardiomyocytes and the mechanisms of anti- fibrillation were limited. Moreover, it is not clear whether different statins may have different cardiac effects.
Pulmonary veins (PVs) were known to be important sources of ectopic beats with the initiation of paroxysmal atrial fibrillation or the foci of ectopic atrial tachycardia and focal atrial fibrillation [11-13]. Other studies also suggested that PVs have a role in the maintenance of atrial fibrillation [14-15]. Previous anatomical and electrophysiological studies in isolated PV specimen have demonstrated that PVs contain a mixture of pacemaker cells and working myocardium [16-20]. Our previous studies have demonstrated the presence of spontaneous activities or high frequency irregular rhythms in isolated canine PVs, which may underlie the arrhythmogenic activity of these vessels [20]. After the isolation of single cardiomyocytes, PVs were found to have cardiomyocytes with distinct electrophysiological characteristics and arrhythmogenic activities [21-23]. In addition, long-term rapid atrial pacing could increase PV arrhythmogneic activity through the induction of triggered activities, shortening of action potential duration or enhancement of automaticity and contribute to the occurrence of atrial fibrillation [20-21] The administration of thyroid hormone also was demonstrated to increase PV arrhythmogenic activity [22]. All of these findings suggest the critical role of PVs in the genesis of maintenance of atrial
fibrillation. Our previous studies have demonstrated that TNF-α increases PV arrhythmogenic activity and was suggested to be underlying the mechanism of inflammation induced-atrial fibrillation. [24] In addition, nitric oxide was been demonstrated to reduce PV arrhythmogenic activity and could be a potential antiarrhythmogenic drugs. [25] Previous studies have shown that statins may increase nitric oxide bioavailability and also have anti-infllammation effects [26-29], therefore, it is possible that
the statins may reduce the occurrence of atrial fibrillation through the decrease of PV arrhythmogenic activity with the production of nitric oxide and anti-inflammation. Therefore, the purposes of the present study are to investigate the effects of statins on PV arrhythmogenic activity.
Methods
Rabbit PV Tissue Preparations
The investigation conformed to the institutional
Guide for the Care and Use of Laboratory Animals.
Rabbits (1-1.5 Kg) were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg). A mid-line thoracotomy was then performed and the heart with the lungs was removed. For dissection of the PVs, the left atrium was opened by an incision along mitral valve annulus extending from the coronary sinus to the septum in Tyrode＇s soluton with a composition (in mM) of 137 NaCl, 4 KCl, 15 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 2.7 CaCl2, and 11 dextrose. The PVs were separated from the atrium at the left atrium-PV junction and separated from the lungs at the ending of the PV myocardial sleeves. One end of the preparation, consisting of the PVs and atrium-PV junction, was pinned with needles to the bottom of a tissue bath. The other end was connected to a Grass FT03C force transducer with a silk thread. The adventitia of the PVs faced upwards. The tissue was superfused at a constant rate (3 ml/min) with Tyrode＇s solution which was saturated with a 97% O2-3% CO2 gas mixture.The temperature was maintained constant at 37oC and the preparations were allowed to equilibrate for 1 hour before the electrophysiological study.
Electrophysiological and pharmacological studies
The transmembrane action potential (AP) of the PVs was recorded by
means of machine-pulled glass capillary microelectrodes filled with 3M of KCl and the PV preparation was connected to a WPI model FD223
electrometer under tension with 150 mg. The electrical and mechanical events were displayed simultaneously on a Gould 4072 oscilloscope and Gould TA11 recorder. The signals were recorded with DC coupling and a 10-KHz low-pass filter cutoff frequency using a data acquisition system.
Signals were recorded digitally with 16-bit accuracy at a rate of 125 KHz.
An electrical stimuli with a 10-ms duration and suprathreshold strength (30% above the threshold) were provided by a Grass S88 stimulator through a Grass SIU5B stimulus isolation unit. Different concentrations of simvastatin (0.1, 1 µM) were sequentially superfused to test the
pharmacological responses. The 90% and 50% AP durations (APD90, APD50), AP amplitude (APA), and contractile force were measured during 2 Hz
electrical stimuli before and after the drug administration.
Results
As the tracing shown in Figure 1, in the PVs with spontaneous activity, simvastatin (0.1 µM) did not change the PV firing rates significantly.
However, the PV firing rates decreased from 1.7±0.1 to 1.5±0.1 Hz (n=3) after the administration of simvastatin (1 µM) for one hour. The firing rates were further decreased to 1.4±0.1 Hz after the administration of simvastatin (1 µM) for 2 hours. Figure 2 shows the tracing after the administration of simvastatin (1 µM).
In the PV without spontaneous activity, simvastatin (0.1 µM) did not change the AP duration in PV cardiomyocytes. However, simvastatin at the concentration of 1 µM prolonged the AP duration from 88±7 ms to 93±7 ms (n=5) after the administration for 3 hours. The simvastatin (0.1, 1 µM)
did not change the resting membrane potential, amplitudes of AP and contractility or vessel tone. Figure 3 shows the AP morphology before and after the administration of statin.
In order to evaluate the mechanism of statin on PV electrical activity, L-NAME was administrated in the PV treated with simvastatin (1 µM) for 3 hours. As the tracing show in Figure 4, the administration of L-NAME (100 µM) accelerated to the PV firings rates, which suggests that simvastatin may reduce PV electrical activity through the production of nitic oxide.
Discussion
Previous studies have shown that statin may have a beneficial effect on the prevention of atrial fibrillation [3-5]. Gaspo et al. have found that stain may attenuated the rapid atrial pacing-induced electrical remodeling, which suggested the anti-arrhythmic potential of statin [6].
However, vitamins C and vitamins E did not have effect on atrial electrical activity which suggested that the anti-arrhythmic potential of statin did not arise frome the anti-oxidants effects. In this study, for the first time, we demonstrated that statin may alter the PV electrical activity.
Not only prolonged the AP duration, stain also decreases the PV firing rates. All of these findings may reduce PV arrhythmogeneis to result in the decrease of atrial fibrillation.
Previous studies have shown that nitic oxide may reduce PV arrhythmogenesis through the decrease of transient inward currents [25].
Statin has been shown to induce the occurrence of nitric oxide [26].
Therefore, it is possible that statin may reduce the PV arrhythmogenesis through the production of nitric oxide. In this study, administration of
L-NAME may reverse the inhibitory effects of statin. which suggests that statin may reduce PV arrhythmogenesis due to the production of nitric oxide.
Conclusion
We demonstrated the simvastatin may decrease the PV arrhythmogenesis through the production of nitric oxide. These results may underlie the anti-arrhythmic potential of statin and result in the decrease of atrial fibrillation.
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Figure Legends
Figure. 1. The tracings showing the PV firing rates before and after the
administration of simvastatin (0.1 µM).
Figure 2. The tracings showing the PV firing rates before and after the
administration of simvastatin (1 µM) at different time..
Figure 3. Effects of simvastatin (0.1, 1 µM) on the AP morphology of PV tissue specimen.
Figure 4. Effect of L-NAME on simvastatin-altered PV electrical activity.
