Choline produces antiarrhythmic actions in animalmodels by cardiac M3 receptors: improvement ofintracellular Ca2+ handlingas a common mechanism

Yan Liu, Hong-li Sun, Dan-lu Li, Li-yan Wang, Yang Gao, Yu-ping Wang, Zhi-min Du,Yan-jie Lu, and Bao-feng Yang

Abstract: It is well known that choline has protective effects on ischemic arrhythmias. We designed the present study toevaluate the antiarrhythmic effects of choline and to detect its related mechanisms in aconitine-induced rat and ouabain-induced guinea pig models of arrhythmia. Laser scanning confocal microscopy and patch-clamp technique were utilized tostudy the action of choline on intracellular calcium concentration and L-type calcium current (ICa-L) of cardiac myocytes.M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) was applied preliminarily to evaluatethe role of the M3 receptor. Choline significantly increased the survival time of arrhythmic rats and guinea pigs, delayedthe onset of arrhythmias and ventricular tachycardia, and decreased the arrhythmia score. The overload of intracellularCa2+ induced by aconitine or ouabain was reduced in isolated myocytes pretreated with choline. Choline reduced theincreased density of ICa-L induced by aconitine or ouabain. Moreover, the beneficial effects of choline were reversed by4-DAMP. Choline produced antiarrhythmic actions on arrhythmia models by stimulating the cardiac M3 receptor. Themechanism may be related to the improvement of Ca2+ handling.

Key words: choline, 4-DAMP, M3 receptor, aconitine, ouabain, arrhythmias, Ca2+.

Resume : Il est bien connu que la choline a des effets protecteurs sur les arythmies ischemiques. Pour examiner si l’effetantiarythmique de la choline sur le myocarde ischemique pourrait etre reproduits dans d’autres modeles animaux arythmo-genes, nous avons concu la presente etude pour evaluer les effets antiarythmiques de la choline et identifier ses mecanis-mes. Nous avons observes les effets de la choline sur des modeles de rats presentant une arythmie induite par la choline etde cobayes presentant une arythmie induite par l’ouabaıne. Nous avons utilise la microscopie confocale a balayage laser etla technique du patch clamp pour examiner l’action de la choline sur la concentration de calcium intracellulaire et le cou-rant calcique de type L (ICa-L) des myocytes cardiaques. Nous avons d’abord applique l’antagoniste du recepteur M3,4-DAMP (4-diphenylacetoxy-N-methylpyridine-methiodide), pour evaluer le role du recepteur M3. La choline a augmentesignificativement le temps de survie des rats et des cobayes presentant une arythmie, retarde le declenchement des aryth-mies et de la tachycardie ventriculaire, et diminue le score d’arythmie. La surcharge de Ca2+ intracellulaire induite parl’aconitine ou l’ouabaıne a ete reduite dans les myocytes isoles pretraites a la choline. La choline a diminue l’augmen-tation de la densite de l’ICa-L induite par l’aconitine ou l’ouabaıne. Le 4-DAMP a renverse les effets benefiques de la cho-line. La choline a eu des actions antiarythmiques sur les modeles d’arythmie en stimulant le recepteur M3 cardiaque. Lemecanisme pourrait etre lie a l’amelioration du metabolisme du Ca2+.

Mots-cles : choline, 4-DAMP, recepteur M3, aconitine, ouabaıne, arythmies, Ca2+.

[Traduit par la Redaction]

IntroductionCholine, a precursor and metabolite of acetylcholine, is

known to be a necessary substrate of membrane lipids. Agrowing body of evidence indicates that choline is not onlya structural component but also a functional modulator ofthe cellular membrane. Several studies have shown thatcholine plays an important role in the prevention of manypathologic conditions, such as cirrhosis of the liver, arterio-sclerosis, certain deficiencies of brain function and memory,and the pathogenesis of Alzheimer’s disease (Haubrich andPflueger 1979; De Jesus Moreno Moreno 2003). Availableevidence also suggests that choline has protective effects onischemic arrhythmias: administration of choline reduced theincidence of ventricular premature beats and the duration ofventricular tachycardia (Yang et al. 2005). The mechanismby which choline modulates arrhythmias may be related to

Received 24 March 2008. Accepted 3 October 2008. Publishedon the NRC Research Press Web site at cjpp.nrc.ca on3 December 2008.

Y. Liu,1 H.L. Sun,1 D.L. Li, L.Y. Wang, and Y. Gao.Department of Pharmacology, Harbin Medical University,Baojian Road 157, Harbin, Heilongjiang 150081, PR China.Y.P. Wang and Z.M. Du.2 Institute of Clinical Pharmacology,The Second Hospital, Harbin Medical University, Harbin,Heilongjiang 150081, PR China.Y.J. Lu and B.F. Yang. Department of Pharmacology, HarbinMedical University, Baojian Road 157, Harbin, Heilongjiang150081, PR China; Bio-Pharmaceutical Key Laboratory ofHeilongjiang Province-Incubator of State, Key Laboratory,Harbin, Heilongjiang 150081, PR China.

1The first and second authors contributed equally to this study.2Corresponding author (e-mail: dzm1956@126.com).

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activation of IKM3, the delayed rectifier K+ current mediatedby M3-muscarinic acetylcholine receptor (mAChR), in theheart (Liu et al. 2005).

Aconitine and ouabain have been widely used to screenand evaluate antiarrhythmic drugs in many countries, andthey have been used to develop stable and ideal animalmodels of arrhythmias. Previous studies have demonstratedthat aconitine- and ouabain-induced arrhythmias are relatedto L-type calcium current (ICa-L) (Gong et al. 2004). To in-vestigate whether the antiarrhythmic efficacy of choline inischemic myocardium could be reproduced in other arrhyth-mogenic animal models and to delineate the potential ionicmechanisms, we performed studies to evaluate the effects ofcholine on ventricular arrhythmias induced by aconitine orouabain and to explore the possible role of the M3-mAChRin the disturbance of Ca2+ handling.

Materials and methods

Animals and reagentsIn the present study, animals were cared for in accordance

with the Guide for the Care and Use of Laboratory Animals.We also followed the principles and protocols of laboratoryanimal care of the animal care committee of Harbin MedicalUniversity, which is accredited by the Chinese Associationof Laboratory Animal Care. Wistar rats weighing 250–300 g (9–12 weeks old) and guinea pigs weighing 300–350 g (2 months old), provided by the Experimental AnimalCenter of Harbin Medical University, China, Grade II, wereused in the experiment. Sodium pentobarbital was purchasedfrom Shanghai Chemical Reagent, China. Choline,aconitine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide), ouabain, and amiodarone were prepared bySigma, USA. Verapamil was purchased from Shang Hai HeFeng, China.

Arrhythmia model induced by aconitine in ratsForty Wistar rats of either sex were randomly divided

into 5 groups: control, choline (10 mg/kg, i.v.), choline +4-DAMP (10 mg/kg and 0.12 mg/kg, respectively, i.v.), ve-rapamil (0.4 mg/kg, i.v.), and amiodarone (5 mg/kg, i.v.).All rats were anesthetized with sodium pentobarbital(40 mg/kg, i.p.), and electrocardiogram (ECG) leads wereconnected to their limbs for continuous ECG monitoringthroughout the experiment. After the drugs were given,aconitine (0.06 mg/min, i.v.) was administered to inducethe ventricular arrhythmias. In each group, the arrhythmiaseverity was observed until the death of the rats.

Arrhythmia model induced by ouabain in guinea pigsForty guinea pigs of either sex were randomly divided

into 5 groups: control, choline (10 mg/kg, i.v.), choline +4-DAMP (10 mg/kg and 0.12 mg/kg, respectively, i.v.), ve-rapamil (0.4 mg/kg, i.v.), and amiodarone (5 mg/kg, i.v.),and then anesthetized with pentobarbital (40 mg/kg, i.p.).The standard limb II ECG was continuously recorded. Fiveminutes after the administration of drugs, ouabain was ad-ministered i.v. at a rate of 3 mg/min to induce ventriculararrhythmias. In each group, the change of arrhythmias wasobserved until the death of the guinea pigs.

Arrhythmia scoresThe arrhythmias were quantitatively estimated according

to the method described by Mest (Mest and Forster 1979).The degree of severity was assessed according to the follow-ing scale: 0, sinus rhythm; 20, first-degree atrial–ventricularblock, supraventricular arrhythmias; 40, ventricular prema-ture bigeminy, trigeminy, second-degree atrial–ventricularblock; 60, polyphyletic ventricular premature, paroxysmalventricular tachycardia; 80, ventricular fibrillation; 100,death.

Cell isolationSingle rat and guinea pig ventricular myocytes were iso-

lated as previously described in detail (Dong et al. 2004).Briefly, adult Wistar rats and guinea pigs were killed by ablow to the head. Their hearts were cannulated on a Langen-dorff perfusion apparatus and retrogradely perfused via theaorta for 2 min with Tyrode solution containing 1 mmol/LCaCl2 (pH 7.4) at 37 8C. Then the heart was perfused withnormal Ca2+-free Tyrode solution for 5 min, followed byperfusion with the same solution containing collagenase(type II, 100–150 kU/L) and 1% bovine serum albumin for10 min. The free wall of the ventricle was cut and trituratedfor 2 min and then incubated in Kraft–Bruhe (KB) solutionconsisting of the following (in mmol/L): KCl 25, KH2PO410, MgCl2 3, potassium glutamate 70, egtazic acid 0.5, glu-cose 10, Hepes 10, taurine 20, and KOH (pH adjusted to7.4) at 4 8C. Only Ca2+-tolerant, quiescent, and rod-shapedmyocytes were selected for measurement of intracellular cal-cium ([Ca2+]i) and electrophysiologic recording.

Measurement of [Ca2+]iFluorescence measurements in cardiomyocytes have been

described previously (Sun et al. 2006). Briefly, single cardi-omyocytes were attached to the cover slips of chamber withconcanavalin A (ConA) and incubated for 50 min at 37 8Cin 5 mmol/L fluo-3 AM (acetoxymethyl ester form, Molecu-lar Probes) working solution containing 0.02% Pluronic F-127. The fluorescent intensity was then detected by an in-verted Olympus confocal system (FluoView FV300, Japan)equipped with a 40� objective, 488-nanometre argon ion la-ser for excitation and 530 nm for emission at room temper-ature. The fluorescent images of [Ca2+]i were collected fromthe KCl, ouabain or aconitine, choline, and choline + 4-DAMP groups. Increases of [Ca2+]i were expressed as theratio of fluorescence intensity of fluo-3 AM over baseline(Fmax/F0).

Whole-cell patch-clamp techniqueThe cardiac myocytes were transferred to a chamber

mounted for electrophysiologic recording. The cells werebathed at room temperature in an extracellular solution ofthe following composition (in mmol/L): Tris–Cl 136, CsCl5.4, CaCl2 2, MgCl2 1, Hepes 10, and glucose 5, pH 7.4with Tris–OH). The whole-cell patch-clamp recording tech-nique that was used has been described in detail elsewhere(Yang et al. 2007). Calcium currents were recorded undervoltage-clamp configuration by using pCLAMP 9.02 soft-ware. Borosilicate glass electrodes with tip resistance of 2–4 MU were filled with pipette solution (in mmol/L: CsCl20, EGTA 10, MgCl2 1, MgATP 5, aspartate 110, CsOH

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110, and Hepes 10, pH 7.2 with CsOH). Na+ currents were in-activated at the holding potential of –50 mV and blocked bytetrodotoxin (TTX). Under this condition, an inward currentwas recorded and inhibited by verapamil. This inward currentwas ICa-L. The current was elicited by 300-millisecond depo-larizing steps from –40 mV to 60 mV with increments of10 mV. The magnitude of ICa-L was measured to be thepeak inward current. Under our experimental conditions,the run-down of ICa-L in the control was less than 5%throughout the observation period. The changes of ICa-Lwere measured after exposure to aconitine, ouabain, andcholine.

Data analysisAll data were expressed as means ± SE, and ANOVA was

used to evaluate the statistical significance. A two-tailed p <0.05 was considered to be statistically significant.

Results

Effects of choline on the risk of arrhythmias induced byaconitine in rats

Ventricular arrhythmias appeared 23.3 ± 2.8 min after ad-ministration of aconitine in rats. Intravenous administrationof choline delayed occurrence of arrhythmia (Fig. 1A), de-creased ventricular tachycardia duration (Fig. 1B), decreasedthe arrhythmia score, and increased the survival time of rats(Figs. 1C, 1D). The effects of choline were reversed by theM3 receptor antagonist 4-DAMP.

Effects of choline on the risk of arrhythmias induced byouabain in guinea pigs

The time to appearance of arrhythmia was 19.7 ± 1.3 minin the ouabain-induced arrhythmia model. Generally, ven-tricular premature and bigeminy occurred at first, followedby aggravating ventricular tachycardia and ventricular fibril-lation. Administration of choline substantially depressed theoccurrence of ventricular premature and ventricular tachy-cardia. Meanwhile, the survival time of guinea pigs wasalso prolonged (p < 0.05). 4-DAMP abolished the above ef-fects (p < 0.05). Verapamil and amiodarone notablydepressed the occurrence of arrhythmias, ventricular prema-ture and ventricular tachycardia, which prolonged the sur-vival time of guinea pigs (Fig. 2).

Effects of choline on the increase of [Ca2+]i induced byaconitine in rat cardiomyocytes

For illuminating the effects of choline on myocardial[Ca2+]i, the [Ca2+]i mobilization was observed by means oflaser scanning confocal microscopy (LSCM). In normalTyrode solution or Ca2+-free solution, 1 mmol/L aconitinewas used to determine whether aconitine was able to modify[Ca2+]i at resting levels in the rat cardiomyocytes. It wasfound that aconitine did not induce any detectable altera-tions of [Ca2+]i in the basal level compared with controlgroup (p > 0.05, data not shown). Then we examinedwhether aconitine could affect KCl-mediated Ca2+ mobiliza-tion. [Ca2+]i was elevated gradually after application of30 mmol/L KCl in the presence of 1.8 mmol/L Ca2+, and

Fig. 1. Protective effects of choline on the arrhythmia induced byaconitine in rats. (A) Onset of arrhythmias. (B) Duration of ventri-cular tachycardia (VT). (C) Arrhythmia score. (D) Survival time.Values are means ± SE, n = 8. *, Significant at p < 0.05 and**, p < 0.01 vs. Aco group. +, p < 0.05 and ++, p < 0.01 vs. cholinegroup. Aco, aconitine 0.06 mg/min; choline, an M3 receptor agonist,10 mg/kg; 4-DAMP, an M3 receptor antagonist, 0.12 mg/kg. Positivecontrols were Ver, verapamil 0.4 mg/kg, and amiodarone 5 mg/kg.

Fig. 2. Protective effects of choline on the arrhythmias induced byouabain in guinea pigs. (A) Onset of arrhythmias. (B) Duration ofventricular tachycardia (VT). (C) Arrhythmia score. (D) Survivaltime. Values are means ± SE, n = 8. *, Significant at p < 0.05 and**, p < 0.01 vs. Oua group. +, p < 0.05 and ++, p < 0.01 vs. cholinegroup. Oua, ouabain 3 mg/min; choline, 4-DAMP, Ver (verapamil),and amiodarone are the same as given in Fig. 1.

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the ratio of fluo-3 AM fluorescence (Fmax/F0) was in-creased 2.6 times ± 0.5 times at peak value (n = 6, p <0.01) compared with resting value. After pretreatment for5 min with Tyrode solution containing 1 mmol/L aconitine,the KCl-induced elevations of [Ca2+]i were markedly en-hanced (n = 6 from 4 rats, p < 0.01) compared with theKCl group, whereas aconitine-evoked elevations of [Ca2+]iwere inhibited by co-application of choline (1 mmol/L)(n = 6 from 4 rats, p < 0.01). The effects of cholinewere reversed by 4-DAMP applied 5 min before co-administration of choline and aconitine (Figs. 3A (panela) and 3B).

Effects of choline on the increase of [Ca2+]i induced byouabain in guinea pig cardiomyocytes

As illustrated in (Fig. 3A, panel b), 30 mmol/L KCl alsosubstantially increased [Ca2+]i in myocytes of guinea pig.After pretreatment for 5 min with Tyrode solution contain-ing 10 mmol/L ouabain, the KCl-induced elevations of[Ca2+]i were not markedly enhanced, but our data showedan increasing trend. In contrast, the effect of ouabain wassignificantly abrogated in the cells pretreated with choline.The effect of choline was completely reversed by 4-DAMPapplied 5 min before co-administration of choline and oua-bain.

Effects of choline on the density of ICa-L of aconitine inrat cardiomyocytes

Aconitine (1 mmol/L) increased the ICa-L in rat cardiomyo-cytes from –1009 pA ± 84 pA to –1772 pA ± 125 pA at 10mV (n = 6 from 4 rats, p < 0.05). Choline (1 mmol/L) inhib-ited the increase of ICa-L induced by aconitine to –1236 pA ±139 pA (n = 6 from 4 rats, p < 0.05) (Fig. 4A).

Effect of choline on the density of ICa-L of ouabain inguinea pig cardiomyocytes

Ouabain (10 mmol/L) had no obviously effect on ICa-L, butour data showed an increasing trend. Choline (1 mmol/L)significantly inhibited the density of ICa-L of ouabain (n = 6from 4 guinea pigs, p < 0.05) (Fig. 4B).

DiscussionThe main finding of the present study was that choline

substantially depressed ventricular arrhythmia in 2 differentanimal models of arrhythmia with overload of intracellularCa2+ as a common mechanism. The confocal microscopy ex-periment showed that choline effectively inhibited the in-crease of [Ca2+]i promoted by aconitine or ouabain, whichwere adopted to induce ventricular arrhythmia in various an-imal models. In the whole-cell patch-clamp experiment,

Fig. 3. Effects of choline on the increase in [Ca2+]i induced by aconitine and ouabain in rat and guinea pig cardiomyocytes. (A) Pooled dataof the effect of choline on [Ca2+]i. All data are means ± SE (n = 6 from 4 animals). (a) Effects of choline on the increase in [Ca2+]i inducedby aconitine in rat cardiomyocytes. # #, Significant at p < 0.01 vs. KCl. **, p < 0.01 vs. Aco. +, p < 0.05 vs. choline. (b) Effects of cholineon the change in [Ca2+]i induced by ouabain in guinea pig cardiomyocytes. *, p < 0.05 vs. Oua; +p < 0.05 vs. choline. (B) Representativefluorescent images of rat ventricular myocytes loaded with fluo-3 AM under KCl (a) at the peak value state, (b) treated with aconitine be-fore application of KCl at the peak, (c) pretreated with choline before application of aconitine and KCl at the peak, and (d) co-application of4-DAMP, choline, aconitine, and KCl at the peak. KCl, 30 mmol/L; Aco, aconitine 1 mmol/L; choline, 1 mmol/L; 4-DAMP, 3 nmol/L;Oua, ouabain 10 mmol/L.

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choline effectively suppressed the increase of ICa-L densitypromoted by aconitine. In other words, the beneficial effectsof choline are most likely mediated by stimulation of thecardiac M3-mAChR, which leads to reduction of intracellularCa2+ overload.

In this study, 5 min pretreatment with the M3 receptor ag-onist choline offered significant protective actions on ar-rhythmia induced by aconitine and ouabain in intactanesthetized rat and guinea pig. The M3 receptor antagonist4-DAMP abolished the beneficial effects of choline on ven-tricular arrhythmias, suggesting that such effects wereachieved via the selective activation of the cardiomyocyteM3 receptor. It is well established that choline has protectiveeffects on ischemic arrhythmias of rats induced by coronaryartery occlusion. A relevant example is that administrationof choline before ischemia not only attenuated the ische-mia-induced arrhythmias but also partially restored thesuppression of the association between connexin 43 andM3-mAChR (Yue et al. 2006).

Ca2+ is an important messenger, and movement of intra-cellular Ca2+ critically regulates normal cardiac excitation–

contraction (E-C) coupling in the heart. It is reported that aco-nitine induced disruption of intracellular Ca2+ homeostasis inthe cardiac excitation–contraction coupling, which is a crucialfactor of arrhythmic toxicity (Fu et al. 2007; Wada et al.2005). In ouabain-induced arrhythmias, an increase in intra-cellular Ca2+ was involved in arrhythmogenesis (Moroz andLipnitskii 2006). Above all, intracellular Ca2+ overload as acommon pathway plays a role in the initiation or maintenanceof ventricular arrhythmias induced by aconitine and ouabain.

Therefore, to identify the mechanism underlying the pro-tective effects of the M3 receptor, we examined the [Ca2+]imobilization and ICa-L of isolated rats and guinea pig myo-cytes under the influence of aconitine and ouabain. Our pre-vious experiments indicated that pretreatment of cardiacmyocytes with choline inhibited the increase in the fluores-cent intensity (FI) value of [Ca2+]i in H2O2-stimulated car-diac myocyte apoptosis (Liu et al. 2004). The present studyshowed that choline substantially depressed the intracellularCa2+ overload induced by aconitine or ouabain. The effect ofcholine was also partially reversed by 4-DAMP.

From these results, we speculate that choline plays an im-portant role in aconitine- and ouabain-induced arrhythmiavia stimulation of the M3 receptor and activation of IKM3,which may greatly affect K+ efflux and accelerate cardiacrepolarization, thus influencing Ca2+ influx on phase 2 pla-teau. Podzuweit has shown that choline can abolish the ven-tricular tachycardia induced by subepicardial infusion ofnorepinephrine and Ca2+ in open-chest pigs (Podzuweit1982). We conclude that the modulating mechanisms ofcholine on calcium abnormality induced by aconitine andouabain may be mediated by a decrease in Ca2+ entry.

In summary, choline produced antiarrhythmic actions onthe aconitine-induced rat arrhythmia model and the ouabain-induced guinea pig arrhythmia model by stimulating the car-diac M3 receptor. The mechanism may be related toimprovement of Ca2+ handling. A limitation of the presentstudy is that the possible effects of other subtypes ofmAChRs should not be overlooked. Although 4-DAMP isconsidered to be a selective antagonist of M3-mAChR, itmay affect other subtypes of mAChRs. Unfortunately, spe-cific antagonists of any of the mAChR subtypes are not cur-rently available. Therefore, 4-DAMP has been widely usedin the research of M3-mAChR. More conclusive data are re-quired to definitely establish the link between M3-mAChRsand downregulation of Ca2+ overload in aconitine- orouabain-induced arrhythmia.

AcknowledgementsThis work was supported by the National Natural Science

Foundation of China (30672462) and the Specialized Re-search Fund for the Doctoral Program of Higher Education(20060226019, 20050226010). The authors declare no con-flicts of interest.

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