arrhythmia models for drug research: classification of

333

Journal of Pharmacological Sciences

©2007 The Japanese Pharmacological Society

Critical Review

J Pharmacol Sci 103, 333 – 346 (2007)

Arrhythmia Models for Drug Research: Classification of Antiarrhythmic

Drugs

Keitaro Hashimoto1,*

1Professor Emeritus, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan

Received December 31, 2006

Abstract. The aim of this study was to classify antiarrhythmic drugs based on their effective-

ness on 6 in vivo arrhythmia models, mainly using dogs. The models were produced by two-stage

coronary ligation, digitalis, halothane-adrenaline, programmed electrical stimulation in old myo-

cardial infarction dogs, coronary artery occlusion / reperfusion, or chronic atrioventricular block.

Na+-channel–blocking drugs suppressed two-stage coronary ligation and digitalis arrhythmias.

Ca2+-channel blockers and β-blockers suppressed halothane-adrenaline arrhythmia. Positive

inotropic drugs aggravated halothane-adrenaline arrhythmia, but did not aggravate digitalis

arrhythmia. K+-channel blockers suppressed programmed electrical stimulation induced

arrhythmia, but induced torsades de pointes type arrhythmia in chronic atrioventricular block

dogs and aggravated halothane-adrenaline arrhythmia. Na+/H+-exchange blockers suppressed

coronary artery occlusion / reperfusion arrhythmias. This classification may be useful for predict-

ing the clinical effectiveness in the preclinical stage of drug development.

Keywords: arrhythmia model, antiarrhythmic drug, proarrhythmic potential of drugs, ion channel,

classification

1. Introduction

Currently, interest in arrhythmia treatment is turning

towards new devices and ablation techniques; and it is,

of course, certain that a failed sinus or Tawara’s (AV)

node should be replaced by appropriate electronic pace-

makers and that cardiac collapse by ventricular fibrilla-

tion (VF) or potentially lethal torsades de pointes (TdP)

should be stopped by electrical external or implantable

defibrillators. To implant pacemakers, only evidence of

malfunction of pacemaking and atrioventricular conduc-

tion is necessary. To install a defibrillator, the genetic

preponderance of the occurrence of these arrhythmias, or

evidence of existence of ventricular arrhythmia types

likely to develop into fatal sudden death or other precip-

itating factors should be known. Knowledge of pace-

making or precise mechanisms of generation of arrhyth-

mias in cellular and molecular levels is no longer neces-

sary. Also for applying the ablation technique to elimi-

nate substrates of arrhythmias, only the proper area,

automatic foci, or a part or the entire re-entry circuit

need to be known. But what about pharmacological

treatments? Is there no room for developing new anti-

arrhythmic drugs? As Christ and Ravens discussed in

her review (1), the relative importance of antiarrhythmic

drugs might have decreased, but they still are relied on

for prevention of atrial fibrillation and sudden cardiac

death by cardiologists world wide. More importantly, for

developing antiarrhythmic drugs, it has been necessary

to know the precise mechanisms of the generation of

action potentials, the different forms of action potentials,

and the abnormalities of normal action potentials that are

responsible for development into arrhythmias, and so on.

Thus introduction of newer drugs or understanding of

drug actions developed in parallel with the advances in

the knowledge about cardiac electrophysiology. Electri-

cal mechanisms of the heart are generated by complex,

multi-factorial phenomena simultaneously occurring

with channel- or receptor-related events. Molecular

research aims to elucidate the single function of a single

molecule and the action of drugs on ion channels, trans-

porters, or extracellular or intracellular receptors, but

*Corresponding author. [email protected]

Published online in J-STAGE: April 4, 2007

doi: 10.1254 / jphs.CRJ06013X

Invited article

K Hashimoto334

how these drugs work on arrhythmias is only known

from experimental or clinical arrhythmias. After the

CAST study (2) revealed the possible proarrhythmic

effects of Na+-channel–blocking antiarrhythmic drugs,

pharmacologists and cardiologists tried to make a new

classification or guide for choosing proper drugs to treat

clinical arrhythmias, namely the Sicilian Gambit, by

defining drug actions on cardiac tissues based on newer

molecular aspects of cardiac excitation and classifying

clinical arrhythmias to correlate or match the drugs and

the arrhythmias (3). However since the molecular

mechanisms of arrhythmias are still not well known, and

also comparative effectiveness of drugs on clinical

arrhythmias are not fully known, we have been examin-

ing drugs on various experimental animal arrhythmias

models. It is obvious that the present antiarrhythmic

drugs are not ideal ones, and also for many different

arrhythmias, many different types of drugs are neces-

sary. Drugs are still used to maintain sinus rhythm after

conversion from atrial defibrillation, to stop the transi-

tion to VF clinically, and to target pathophysiological

alterations to prevent the occurrence of lethal arrhyth-

mias. It may be worthwhile to review our studies and try

to classify drugs by their effectiveness on arrhythmias of

dogs, rats, guinea pigs, and so on. Our previous results

on classical and investigational antiarrhythmic drugs and

our recent results on cardioprotective drugs have been

summarized here.

Antiarrhythmic drugs have been classified by

Vaughan Williams mainly based on their effects on

cardiac action potentials into classes I to IV and later

correlated to their effects on Na+ channel, β-receptors,

and K+ and Ca2+ channels (4). Here our animal experi-

mental arrhythmia models, both the automaticity in-

duced and re-entry induced arrhythmias are presented,

which we used to quantitatively compare drug effects on

the arrhythmias. There have been many studies of anti-

arrhythmic drug efficacies using animal arrhythmias, but

most of them showed the effectiveness of a single drug

or made comparisons of that drug with one or two

standard drugs. To make a preclinical prediction of the

usefulness of the drug, comparisons among many drugs

were almost impossible. We used standard and repro-

ducible models that can occur clinically and tried to

study the antiarrhythmic drugs’ effects by comparing

them quantitatively and qualitatively to find common

pharmacological properties and predict antiarrhythmic

mechanisms of action of drugs. We have already written

several summary review papers (5 – 8), but in this

review, we have added new data obtained in recent

years to compare a wide range of drugs.

2. Drug effects on arrhythmia models

2-1. Canine two-stage coronary ligation arrhythmia

2-1-1. Characteristics

Ischemia, being a serious disorder of the myocardium,

causes pump and electrical failure; thus it is used often to

produce experimental arrhythmias. Ischemia is a time-

dependent phenomenon and starts with reversible

damage finally reaching the stage of irreversible damage

or scar formation; thus it is no wonder that ischemia

related arrhythmias also show a variety of electrical

abnormalities. The two-stage coronary ligation tech-

nique was originally reported by Harris (9) and is

characterized by consistent and stable sub-acute to

chronic ischemic arrhythmias, showing severe ventri-

cular tachycardia (VT), but never deteriorating to VF.

This arrhythmia is thought to be an automaticity

arrhythmia because overdriving the atria or ventricle, by

electrical stimulation at rates higher than the rate of VT,

suppressed the VT to conducted sinus rhythm (5).

2-1-2. Methods

“Two-stage” means that the first stage is the start of

30 min of ischemia, followed by the second stage of a

permanent occlusion of the left anterior descending

coronary artery (LAD) of the dog. In the first stage,

isolated LAD soon after bifurcation with the left circum-

flex coronary artery (LCX) was surgically isolated from

the neighboring coronary veins or connective tissues and

then tied completely with one of double threads together

with a needle of about 1 mm outer diameter, which was

immediately withdrawn to make the needle size patency

to produce a state of ischemia, but not result in complete

occlusion. This usually produces ischemic changes in

the electrocardiogram (ECG) and produces only prema-

ture ventricular contractions (PVC) lasting for a few min

followed by a quiescent 30 min, and then another suture

is used to completely occlude the LAD. After the final

ligature, the thoracotomized chest is closed, and the

animal is left to recover from the anesthesia for use 24 or

48 h after coronary ligation. Though cardiac surgery

under anesthesia in a sterile environment is needed, and

the technique of LAD isolation is not always easy, very

stable arrhythmias, PVC and VT (defined by more than

3 continuous PVC), can be obtained which usually

appear several hours later, and the VT becomes fulmi-

nant 24 h later in a conscious state, lasting up to 48 h;

thus this arrhythmia can be used to evaluate and compare

drug effects. The arrhythmias are continuous, and at the

peak of about 24 h after ligation, the PVC consists of

almost all the beats. To judge whether the beats are sinus

beat or PVC, atrial electrodes were sutured on the

epicardial surface of the right atrial appendage during

Arrhythmia Models and Drugs 335

the operation, and ECG and atrial electrogram were

simultaneously obtained. Preferably a His electrogram

must be obtained to judge whether a beat is of a

conducted sinus rhythm or not, but fixing intracardiac

His electrodes usable in a conscious state is quite

difficult and invasive, so we chose the alternate atrial

electrogram recordings. We used the arrhythmic ratio for

expressing the severity of ventricular arrhythmias by the

number of PVC over a certain time, in our case usually

10 sec, divided by the total number of beats including

the PVC and the normal sinus beats. This arrhythmic

ratio cannot differentiate the most severe VF from

severe VT, but as drugs are used usually before VF

occurs, it is a good measure to express the severity of

arrhythmia for quantitative comparison. We found that

24 h after LAD ligation, the arrhythmic ratio of the VT is

almost 1.0 in the two-stage coronary ligation arrhythmia

model, and we calculated the effective plasma concen-

tration of a drug to decrease the control arrhythmic ratio

to 50% from the arrhythmic ratio-drug plasma concen-

tration curve (EC50). For the drug plasma concentration

determinations, intra-arterial or intravenous catheters

were introduced, and the blood samples were drawn

without the conscious animal being aware of the proce-

dure. Another advantage of this arrhythmia model is that

the drugs are administered in a conscious state, so that it

is easy to evaluate the antiarrhythmic efficacy of drugs

while simultaneously evaluating their central nervous

system (CNS) side effects. Thus intravenous lidocaine

was not effective without CNS stimulant effects, even

though there has been a widespread belief that lidocaine

is the most effective drug in the clinical setting of acute

ischemic arrhythmias (5, 10). We gave drugs either by

an intravenous or oral route. We tested many Na+-

channel–blocking drugs and other drugs. When a drug

was effective, the time-dependent drug plasma concen-

tration curve, either after a bolus intravenous injection or

the curve at the rising phase after a constant infusion,

gave almost the same effective plasma concentration

values; namely, there was almost no hysteresis in the

antiarrhythmic effect–EC50 relationships (5).

This two-stage coronary ligation arrhythmia was

stable and the success rate to obtain arrhythmias for drug

evaluation is high, nearly 90%, in dogs. Pigs are prone to

develop VF after coronary ischemia, and smaller rats

and mice do not show such stable VT. Guinea pigs never

develop ischemia by in vivo coronary ligation (11). It

may still be the one of the best models for a new Na+-

channel–blocking antiarrhythmic drug to be tested pre-

clinically for further development.

2-1-3. Antiarrhythmic drugs

Drug effects can be summarized as follows: Na+-

channel–blocking drugs suppress this two-stage coro-

nary ligation arrhythmias and the effective plasma

concentrations are closely related to the intraarterial

doses to suppress intraventricular conduction (12),

suggesting that they suppress arrthythmia through a

mechanism involving Na+-channel block (5, 10, 12 – 40).

Figure 1 shows the correlation between the reported

Na+-channel–blocking concentration in in vitro experi-

ments mainly obtained in the isolated canine cardiac

ventricular muscle preparations and their effective

plasma concentrations (EC50) for suppressing the two-

stage coronary ligation arrhythmia models of the effec-

tive 29 drugs; earlier data (before 1991) of Na+-channel–

blocking concentration were those listed in Hashimoto

et al. (5), those after 1991 are from the respective paper

(12, 34, 35), and data on disopyramide isomers were

those of Vanhoutte et al. (36). These canine effective

plasma concentrations thus determined were also shown

to be very close to their clinical effective concentrations

(5, 12, 34 – 37), thus from this series of experiments, we

could predict clinically effective drug plasma concentra-

tion levels before these drugs went to clinical trials.

Though Na+-channel blockers are now classified accord-

ing to their mode and kinetics of Na+-channel inhibition,

our conclusion was that any drug that blocks Na+

channels can suppress this two-stage coronary ligation

arrhythmia, but by using this arrhythmia model, we

could demonstrate the interaction between two Na+-

channel blockers with different binding affinities (41)

used in combination in an in vitro preparation (42). In

the study, the actions of mexiletine and disopyramide

were additive, but those of mexiletine and aprindine

were not. As shown in Table 1, TJN-505 is a chemically

aconitine-like drug acting on Na+ channels and was

effective on the two-stage coronary ligation arrhythmia,

but there is no data on this drug as a Na+-channel–block-

ing drug in the in vitro cardiac tissue (38). HNS-32 is

also such a drug with Na+-channel–blocking activity

and was shown to be effective on this arrhythmia (39).

Unlike most of the Ca2+-channel blockers, AH-1058,

suppressed two-stage coronary ligation arrhythmia

possibly by inhibiting Ca2+ overload (43).

2-1-4. Ineffective and proarrhythmic drugs

Two-stage coronary ligation arrhythmias differ from

acute coronary occlusion / reperfusion arrhythmias,

described later, in that they almost never turn into VF or

result in animal deaths, and there were no drugs aggra-

vating the two-stage coronary ligation VT to VF. Among

classical antiarrhythmic drugs, the Ca2+-channel–block-

ing drugs verapamil (10) and gallopamil (44) did not

suppress these arrhythmias and also β-blockers were not

effective unless high doses to produce Na+-channel–

K Hashimoto336

blocking effects were given (10, 13, 14, 17, 19, 45). Pure

K+-channel–blocking drugs that we tested did not

suppress these arrhythmias (6, 46 – 49), but amiodarone

given intravenously suppressed the two-stage coronary

ligation arrhythmia, but the plasma concentration was

not determined in this experiment (50). Since there

was no prolongation of QT interval by intravenous

amiodarone injection, this antiarrhythmic effect was

thought to be due to amiodarone’s Na+-channel–block-

ing action (51). Positive inotropic drugs, catechola-

mines, phosphodiesterase (PDE) inhibitors (52 – 54), the

forskolin derivative, colforsin daropate (55) or the Ca2+-

sensitizer, MCI-154 (56), neither showed antiarrhythmic

effects nor aggravated the arrhythmia, unless used in

extreme doses. Zatebrazine, an If-blocking specific

negative chronotropic agent, did not suppress this

arrhythmia (57). As stated above, this arrhythmia was

suppressed by overdriving, so isoproterenol given at

1 – 3 µg /kg produced sinus tachycardia and transiently

suppressed this VT (our unpublished observation). A

cardioselective M2-receptor blocker, AF-DX 116, was

also examined on this arrhythmia, but it had no effect

(58).

2-2. Digitalis-induced ventricular arrhythmias


The cardiotonic steroid digitalis is known to be

arrhythmogenic and its effect can be explained by intra-

cellular Ca2+ overload due 1) to intracellular Na+

accumulation by Na+-K+-ATPase inhibition, followed by

2) intracellular Ca2+ accumulation by enhanced Na+/Ca2+

exchange (5, 59, 60). Ca2+ overload, thus produced,

can induce abnormal automaticity or delayed after-

depolarization-induced arrhythmia or cause re-entry

arrhythmia and conduction block by uncoupling the cell-

to-cell connections and /or stimulating vagal influence

on the AV nodal conduction (60). However the precise

mechanism of Ca2+ overload by the Na+/Ca2+ exchanger

and the resultant increase in the inward current, leading

to the development of delayed afterdepolarization is

still not known, and emergence of new chemicals to

selectively inhibit the Na+/Ca2+ exchanger may solve the

problem and may also be useful for clinical digitalis

toxicity arrhythmias. The automaticity mechanism can

be demonstrated by the overdrive suppression of this

arrhythmia (5).

Fig. 1. Correlation of drug plasma effective concentrations (EC50) determined using canine two-stage coronary ligation

arrhythmia and its Na+-channel blocking in vitro concentrations of ventricular muscle. Although Na+-channel–blocking

concentration determinations were done in various institutions and drug protein binding is neglected, there is roughly a good

relationship.


2-2-2. Methods

Digitalis can induce toxicity characterized by VT, VF,

and death both by intermittent intravenous bolus injec-

tion or continuous intravenous infusion in various

animals except in rats, which are known to need thou-

sands of times higher doses of digitalis than man and

other animals (61). Test drugs are usually administered

before digitalis administration by intravenous bolus

Table 1. Classification of drugs using in vivo arrhythmia models

Effective drugs Ineffective drugsAggravating drugs or procedure

Two-stage coronary ligation arrhythmia

I: A-2545, AFD-19, AFD-21 (NS-2), AHR 10718, AN-132, aprindine, AP-792, bidisomide, bisaramil, cibenzoline, disopyramide, d-disopyramide, l-disopyramide, etacizin, E-0747, flecainide, HNS-32, KT-362, mexiletine, nicainoprol, OPC88117, penticainide, phenytoin, pirmenol, propafenone, SD-3212, tocainide, NS-2, procainamide, pilsicainide, TJN-505, TYB-3823, YUTAC

II: pindolol, N-696III: amiodarone (i.v.)M: dilazep

I: lidocaineII: betaxolol, bupranolol, Ko 1400,

OPC-1427, oxprenolol, propranololIV: gallopamil, verapamilIII: dofetilide, E-4031, KCB-328,

sematilide, d-sotalolM: colforsin daropate, milrinone,

OPC-18790, vesnarinone, MCI-154, zatebradine, AF-DX 116

Digitalis arrhythmia I: A-2545, AFD-19, AFD-21 (NS-2), AHR 10718, AN-132, AP-792, aprindine, bidisomide, bisaramil, cibenzoline, disopyramide, E-0747, etacizin, flecainide, HNS-32, KT-362, mexiletine, nicainoprol, NS-2, OPC88117, penticainide, phenytoin, pirmenol, procainamide, pilsicainide, propafenone, SD-3212, TJN-505, tocainide, trapidil, TYB-3823, YUTAC

II: atenolol, carvedilol, pindolol, propranololIII: amiodarone (i.v.)M: magnesium sulphate, zadebradine, SEA0400,

amrinone, colforsin daropate, sulmazole

II: atenolol, betaxolol, N-696III: E-4031, dofetilide, sematilide,

KCB-328M: bepridil, nicorandil, nitroglycerin,

trimetazidine, cariporide, KB-R7943, milrinone, vesnarinone, MCI-154

Halothane-adrenaline arrhythmia

II: atenolol, betaxolol, Ko 1400, N-696, oxprenolol, pindolol, propranolol

IV: AH-1058, AP-792, diltiazem, gallopamil, nifedipine, nisoldipine, verapamil

III: amiodarone (i.v.)I: AFD-19, aprindine, cibenzoline, E-0747,

flecainide, KT-362, mexiletine, nicainoprol, OPC88117, penticainide, phenytoin, pirmenol, propafenone, SD-3212, TJN-505, TYB-3823, YUTAC

M: dilazep, NCO-700, nitroglycerin, trapidil, trimetazidine, l-carnitine

I: AHR 10718, AN-132, tocainideM: nicorandil, MCI-154, zatebradine

I: disopyramide, etacizin, pilsicainide, procainamide

III: azimilide, dofetilide, E-4031, KCB-328, nifekalant, sematilide

M: amrinone, milrinone, OPC-18790, sulmazole, vesnarinone

PES-induced arrhythmia

III: azimilide, dofetilide, KCB-328, nifekalant, sematilide

I: A-2545, aprindine, disopyramide

I: flecainide

Occlusion /reperfusion arrhythmia

Halothane-anesthetized dogsIII: E-4031, nifekalant

Pentobarbital-anesthetized dogsIII: KCB-328, nifekalant, d-sotalolM: cariporide, KB-R9032

RatsM: cariporide, FR 186666, KB-R9032, DIDS,

pravastatin

Halothane-anesthetized dogsIII: amiodarone (i.v., p.o.), dofetilide,

KCB-328, sematilide, d-sotalolPentobarbital-anesthetized dogs

III: dofetilide, sematilideM: KB-R7943, SEA0400, pravastatin

RatsM: SITS, fluvastatin

M: pacing-induced heart failure

Chronic AV block dog (TdP induction)

III: nifekalant, sematilideM: cisapride, sparfloxacin,

terfenadine

I: Na+-channel–blocking drugs; NIK-244 = etacizin, ME3202 (CM7857) = penticainide, SUN 1165 = pilsicainide. II: β-blocking drugs. III: K+-

channel–blocking drugs; MS-551 = nifekalant. IV: Ca2+-channel–blocking drugs. M: Miscellaneous drugs with different mechanisms of action;

AR-L115 = sulmazole, OPC8212 = vesnarinone, HOE642 = cariporide, NKH477 = colforsin daropate.

K Hashimoto338

injection, and a simple test to record the occurrence of

PVC, VT, and VF using small animals, especially the

guinea pig, is often used for screening possible anti-

arrhythmic effects of drugs, but precise quantitative data

are difficult to obtain from these experiments. We used

dogs, and they were anesthetized with intravenous

pentobarbital, and then intravenous catheter atrial

electrodes were introduced from the femoral vein (15,

62). Cumulative administration of digitalis, initially

40 µg /kg intravenous ouabain followed by a 10 µg /kg

every 20 min were administered to produce continuously

occurring VT, lasting more than 1 h, and then the test

drugs were administered and the blood to determine

drug plasma concentrations were drawn. As the drug

concentration decreased, VT reappeared after effective

drug administration, thus we could determine the canine

effective plasma concentrations of drugs (5, 15).


Drugs effective on our digitalis arrhythmias are seen

in Figure 2 with plasma EC50 values and in vitro Na+-

channel–blocking concentrations. The pattern of drug

effects are similar to that obtained using the two-stage

coronary ligation arrhythmias, namely, that the Na+-

channel–blocking drugs suppressed these digitalis

arrhythmias and the effective plasma concentrations are

closely related to their in vitro Na+-channel–blocking

concentrations (5, 15, 16, 18 – 40). Zatebradine did

not suppress the two-stage coronary ligation arrhythmia,

but digitalis arrhythmia was suppressed by a high dose

of intravenous 1.5 mg /kg bolus injection (57). The

mechanism of this effect might be either due to If block

or other channel effects, but there was no supporting

data of plasma concentration determination and also

there were no other If blockers available for our study.

Other drugs effective on digitalis arrhythmias without

Na+-channel–blocking effect, but for which the exact

mechanisms of action are unknown, are intravenous

magnesium sulfate (63), a possible Ca2+-channel

blocker, and the Na+/Ca2+-exchange inhibitor SEA0400,

reported to be relatively selective when suppressing

inward Ca2+ movement (reverse mode) (64). Also

positive inotropic drugs with prominent tachycardic

action, amrinone, colforsin daropate, and sulmazole,

suppressed this arrhythmia probably by overdriving the

increased automaticity (53, 55).


Ca2+-channel–blocking drugs did not suppress this

arrhythmia (44, 65 – 67) even though the mechanism of

generation is thought to be increased intracellular Ca2+

concentration. The exception was magnesium sulfate,

Fig. 2. Correlation of drug plasma effective concentrations (EC50) determined using canine digitalis arrhythmia and its Na+-

channel blocking in vitro concentrations of ventricular muscle. There is a roughly a good relationship.


which acts as a Ca2+-channel blocker in vivo, and which

suppressed digitalis arrhythmia (63). As for the ineffec-

tiveness of Ca2+-channel blockers, its hypotensive effect

might have made it impossible to raise the doses high

enough to decrease influx of Ca2+ to the myocardium.

We therefore tried intracoronary administration of a

high-dose verapamil in digitalis-induced VT, and as a

result, rapid pacing-induced and probably DAD-

induced PVC was suppressed by verapamil (68). In this

model, Na+-channel blockers were tested, including

bisaramil, disopyramide, lidocaine, and flecainide,

which also suppressed this DAD-induced PVC. β-

Blockers were not effective unless high doses to

suppress Na+ channels were given (17, 19, 45, 69). K+-

channel–blocking drugs did not suppress this arrhythmia

(6, 46 – 49), but intravenous amiodarone, probably by

its Na+-channel–blocking effect, suppressed this digitalis

arrhythmia (50). Also non-Ca2+-channel–blocking coro-

nary vasodilators such as bepridil, nicorandil, nitro-

glycerin, trimetazidine (65 – 67), the Na+/H+-exchange

inhibitor cariporide (70), and the Na+/Ca2+-exchange

inhibitor KB-R7943, which has multichannel effects

and is not selective concerning reverse mode Na+/Ca2+

exchange as compared to SEA0400 (71), were not

effective on the digitalis arrhythmia.

As for proarrhythmic drugs, adding more digitalis

changed the VT to lethal VF, so catecholamines must

theoretically aggravate digitalis-induced arrhythmia

because they add intracellular Ca2+ by opening Ca2+

channels via β-receptor stimulation; thus, we did not

systematically examine catecholamines. However we

examined PDE-inhibiting inotropic drugs in order to

obtain safety pharmacology data taking into consider-

ation the possible combined use of different types of

positive inotropic drugs. Milrinone, and vesnarinone

increased intracellular cyclic AMP concentration and

increased intracellular Ca2+ concentration, but actually

had no aggravating effects (53). As compared to

amrinone and sulmazole, milrinone and vesnarinone

may induce less tachycardia and might not have reached

a level of atrial rate sufficient to overdrive digitalis-

induced ventricular automaticity. Also MCI-154, a

positive inotropic drug having Ca2+ sensitizing and PDE

inhibiting action, did not aggravate digitalis arrhythmia

(56).

2-3. Halothane-adrenaline arrhythmia


Another automaticity arrhythmia is the classical cate-

cholamine-induced arrhythmia. Normal pacemaker

activity of the heart is under the control of sympathetic

and vagal influences and sympathetic stimulation and

sympathomimetic drugs are known to increase pace-

maker activity both of the normal and latent pacemakers,

thus catecholamines in higher doses induce atrial and

ventricular arrhythmias along with sinus tachycardia.

However some anesthetics, such as cyclopropane,

chloroform and halothane, are known to highly sensitize

the myocardium to catecholamines and under those

anesthetics, catecholamines in very low doses induce

severe VT (72). We used halothane and adrenaline in

dogs and guinea pigs to produce halothane-adrenaline

arrhythmia. It is an automaticity arrhythmia because

electrical overdrive suppresses the VT (5, 72, 73).

2-3-2. Methods

Dogs are initially anesthetized with thiopental sodium

and after intubation, 1% halothane vaporized by 100%

oxygen is used for anesthesia and intravenous catheter

and intravenous atrial electrodes were introduced from

the femoral vein (74). In the case of the guinea pig, under

intraperitoneal thiopental anesthesia, intubation was

performed to inhale halothane, but due to its small size

the atrial electrodes were not used (73). For determining

canine effective drug plasma concentrations, conti-

nuously occurring adrenaline arrhythmia was produced

by intravenous infusion of adrenaline, adjusting the

infusion speed enough to produce stable VT lasting

about 20 min, but not producing VF (74). The infusion

rate usually employed was around 2 – 3 µg /kg /min.

Most of the drugs were injected by an intravenous bolus

and the plasma concentration-dependent decrease of the

arrhythmic ratio was examined for effective drugs. For

guinea pigs, a bolus adrenaline injection was used at

higher halothane concentrations of 2% – 4%, and drugs

were injected before adrenaline challenges (73).


Since adrenaline stimulates cardiac β receptors, all the

β blockers were thought to be and actually were effective

in low β-blocking doses at low plasma concentrations

(13, 17, 19, 45, 69). Other important drugs that were

effective were Ca2+-channel blockers. All Ca2+-channel

blockers, verapamil, gallopamil, diltiazem, or even so-

called vaso-selective dihydropyridine Ca2+-channel

blockers, such as nifedipine, nisoldipine, AP-792, and

AH-1058, suppressed adrenaline arrhythmia at Ca2+-

channel–blocking concentrations (5, 40, 43, 44, 65 – 67,

69). Gallopamil, given into LAD, also suppressed intra-

coronary adrenaline induced triggered rhythm (44).

Some of the so-called coronary vasodilators, such as

dilazep, NCO-700, nitroglycerine, trapidil and trimetazi-

dine also suppressed adrenaline arrhythmias (5), but

this may not be due to their hypotensive effect, since

vasodilators with other mechanisms of action, such as

the K+-channel opener nicorandil, did not suppress the

K Hashimoto340

adrenaline arrhythmia (65). Many other drugs also

suppressed adrenaline arrhythmia by undetermined

mechanisms whether due to additional Ca2+-channel– or

β-receptor–blocking action or due to other mechanisms.

These include the Na+-channel blockers (5), AFD-19,

aprindine, cibenzoline, E-0747, flecainide, KT-362,

mexiletine, nicainoprol, OPC88117, penticainide,

phenytoin, pirmenol, propafenone, SD3212, TJN-505,

TYB-3823, and YUTAC, and other miscellaneous

drugs, such as l-carnitine (66) and colforsin daropate

(55).


Some of the Na+-channel blockers (5), such as AHR

10718, AN-132, and tocainide, required higher concen-

trations than needed to suppress two-stage coronary

ligation and digitalis arrhythmias; and since those

concentrations are not thought to be attainable in clinical

use, those drugs could be said to be ineffective.

Furthermore, some Na+-channel blockers even aggra-

vated the adrenaline-induced VT to VF, namely,

worsened this arrhythmia. They include commonly used

Na+-channel blockers (5), such as disopyramide,

etacizin, pilsicainide, and procainamide (29). K+-

channel blockers, azimilide (75), dofetilide (48), E-4031

(46), KCB-328 (49), nifekalant (76), and sematilide

(47), did not suppress, but instead aggregated this

arrhythmia (6, 76). To demonstrate K+-channel blockers’

proarrhythmic effect, we added another adrenaline

arrhythmia experiment (77, 78). We constructed a dose-

response relationship by administering bolus intra-

venous adrenaline and plotted the maximum arrhythmic

ratio attained against the doses. Then after bolus injec-

tion of K+-channel blockers, we again constructed

adrenaline dose-response curves. The K+-channel block-

ers we tested, nifekalant and KCB-328 (77) shifted the

curve to the left, showing that the proarrhythmic effect

of adrenaline was intensified. However Na+-channel

blockers with an action potential prolonging effect, such

as class 1A drugs like disopyramide, did not shift the

curve, showing that the proarrhythmic effect of the K+-

channel block seems to be suppressed by Na+-channel

block (79). Since Ca2+-channel activation must be the

mechanism of the generation of adrenaline arrhythmia,

not only β-receptor–mediated activators, catecholamine-

type positive inotropic drugs, but also phospho-

diesterase-inhibiting positive inotropic drugs also aggra-

vated the adrenaline arrhythmia (52 – 54). Among the

positive inotropic drugs, MCI-154, a phosphodiesterase

inhibitor with a Ca2+-sensitizing property (56), showed

the least proarrhythmic effect on this arrhythmia.

Zatebradine had no suppressing effect on the adrenaline

arrhythmia (57).

2-4. Programmed electrical stimulation induced re-

entry arrhythmia in old myocardial infarction dogs


This arrhythmia is produced in dogs given surgery to

produce myocardial infarction by two-stage coronary

ligation more than a week before, when there was no

spontaneous PVC (47). Since arrhythmias were induced

only by applying premature extrastimuli, the mechanism

of generation is thought to be re-entry around the scar

tissue of the old infarction. Drug effects can be demon-

strated by the change of the severity of induced arrhyth-

mias before and after the drug administration. The rank

of arrhythmias was used to evaluate drug effects, and

the order by their severity was VF as the most severe,

followed by sustained VT, non-sustained VT, PVC, and

no arrhythmia.

2-4-2. Methods

Dogs with old myocardial infarction were anesthe-

tized with pentobarbital and after reopening the thoracic

incision, epicardial stimulating bipolar electrodes were

sutured on the non-infarcted left ventricle, and up to

trains of 3 premature extrastimuli were applied to induce

non-sustained VT (lasting less than 30 s), sustained VT

(lasting more than 30 s), or VF (47). If sustained VT or

VF occurred, epicardial defibrillation was performed to

stop them. After 2 control runs to reproduce re-entry

arrhythmias, an intravenous bolus injection of a drug

was given and then the same protocol of arrhythmia

induction was applied.


Consistent effectiveness was observed for K+-channel

blockers, azimilide (75), dofetilide (48), KCB-328 (49),

nifekalant (80), and sematilide (47), with simultaneous

QT prolongation in ECG (6). Usually the control

arrhythmias were set up as severe ones by applying 3

premature extrastimuli. K+-channel blockers decreased

the severity of arrhythmias to PVC or not inducible (6).

For this arrhythmia, the procedure to induce arrhythmias

took some time, 2 – 3 min, so it was difficult to deter-

mine the effective plasma concentration for each drug so

only quantitative analysis was made by comparing the

intravenous doses. Besides K+-channel blockers, several

Na+-channel blockers, A-2545 (37), aprindine, and

disopyramide (81), have also been reported to be

effective.


After demonstration by CAST of proarrhythmic

potentials of Na+-channel blockers (2), this arrhythmia

model was used to demonstrate the potentials of these

side effects. The worst reputed drug flecainide has been


shown to aggravate EPS induced arrhythmias when the

control arrhythmias were set up as mild arrhythmias,

such as PVC (37, 82); however it is difficult to judge

whether this arrhythmia model is an appropriate and

sufficient one to differentiate or select dangerous Na+-

channel blockers.

2-5. Coronary artery occlusion /reperfusion arrhyth-

mia


This arrhythmia model mimics myocardial ischemia

related arrhythmias that are frequently observed in the

clinic. Coronary occlusion can be done by thread liga-

ture of the isolated coronary artery of the anesthetized

animal under thoracotomy, by inflating a balloon

occluder or ligature of a prepositioned thread around the

coronary artery in a conscious state, or by intracoronary

balloon inflation. For this model, various animals could

be used, such as dogs, cats, pigs, rabbits, rats, and so on,

with the exception of the guinea pig whose coronary

artery collateral circulation is too well developed (11). It

is known that there are predominant time zones for the

occurrence of acute coronary occlusion arrhythmias

(83). In the case of dogs, the first 5 and 30 min after

occlusion are those time zones (46, 84). The generation

mechanism of this arrhythmia is thought to be auto-

maticity induced ectopic beats and subsequent re-entry

movement of the excitation. The severity of arrhythmias

can be categorized by the type of arrhythmias, like PVC,

VT, or VF, or by the number of ectopic beats over time,

if VF does not occur. Reperfusion, after complete

coronary occlusion, often induced VF immediately. This

spontaneous arrhythmia occurs in almost 60% – 70% in

dogs and nearly 100% in pigs and rats, when the main

trunk of the LAD is occluded (83 – 86). The VF results

in death in dogs and pigs, but small animals like rats, VF

very often reverts to sinus rhythm (83 – 86).

2-5-2. Methods

Our canine coronary occlusion /reperfusion arrhyth-

mias were produced in halothane- or pentobarbital-

anesthetized animals (46). After the left thoracotomy,

the LAD was isolated and a string was placed for tying

the artery for usually 30 min, but we also used 20-min

occlusion in some studies (48). After stabilization

following the surgical procedures, a drug was given by

an intravenous bolus or by an infusion and then coronary

occlusion followed by reperfusion was performed. In

some experiments, chronic oral administration of drugs

for days or weeks before the experiment was performed

(87, 88). The occurrence of PVC or VF was monitored

by continuous ECG recordings. Normally, drug plasma

concentration determinations were not done because of

the normal variability of the time of occurrence of the

arrhythmias. In our rat model, LAD was not isolated,

instead a silk thread with a curved needle was placed

under the myocardium together with the coronary artery

and vein and both the artery and vein were tied for coro-

nary occlusion under intraperitoneal pentobarbital

anesthesia; in this method, the ischemia period ranged

from 5 – 120 min for occlusion arrhythmia studies and

usually 5 min for reperfusion studies (85, 86). In the case

of rat experiments, the number of VF occurrence was

counted along with the number of PVC.


Some Na+-channel and K+-channel blockers have

been reported to be effective in suppressing coronary

occlusion /reperfusion arrhythmias, but in the canine

model, the control occurrence of reperfusion VF was

variable and differed by anesthesia, about 60% in

halothane anesthetized dogs (our unpublished data of

43 /76 dogs) and about 70% in pentobarbital anesthe-

tized dogs (our data of 146 /217 dogs); we always

compared drug effects with the vehicle-treated control

group using the same number of dogs (6). We examined

several K+-channel blockers (6, 46 – 50, 87) because

ischemia must activate the ATP-dependent K+ channel

and shorten action potential duration, so the K+-channel

blocker with the opposite effect to prolong action

potential duration should be effective. Actually E-4031

(46) and nifekalant (84) suppressed coronary occlu-

sion /reperfusion arrhythmia in halothane-anesthetized

dogs, and KCB-328 (49), nifekalant (84) and d-sotalol

(84) suppressed those in pentobarbital anesthetized

dogs. Also ischemia stops the action of Na+/K+ ATPase

followed by increase in the intracellular Na+ concentra-

tion by Na+/H+ exchange, which is then followed by

Na+/Ca2+ exchange, inevitably resulting in intracellular

Ca2+ overload (7). Thus the two important exchange

system blockers also are expected to overcome this

intracellular Ca2+ overload. Among these drugs, only

the Na+/H+-exchange inhibitors cariporide (70) and

KB-R9032 (89) showed a consistent antifibrillatory

effect in pentobarbital anesthetized dogs (7) and cari-

poride (70, 90 – 92), FR 186666 (93), and KB-R9032

(94), in rats. These Na+/H+ exchange inhibitors have

been reported to be protective in various animal models

of coronary occlusion /reperfusion injuries, and the

antiarrhythmic effects are one of the common effects (7).

Other effective drugs in rat models were DIDS (95) and

long-term pravastatin administration (88), but whether

the effects were due to Cl /HCO3 exchange inhibition or

HMG-CoA reductase inhibition is unknown. Our recent

unpublished data on halothane anesthetized dogs, short

or long term pravastatin administration did not demon-

K Hashimoto342

strate the favorable rat results of suppressing the

coronary occlusion /reperfusion arrhythmias.


As stated above, some K+-channel blockers were

effective, but others, amiodarone (50, 87), dofetilide

(48), KCB-328 (49), sematilide (47) and, d-sotalol

(84), were not effective in halothane-anesthetized dogs;

and dofetilide (48) and sematilide (47) were not effec-

tive in pentobarbital-anesthetized dogs (6). Also in the

halothane anesthetized low heart rate open chest dogs,

some of the K+-channel blockers, amiodarone (50),

dofetilide (48), E-4031 (46), KCB-328 (49), nifekalant

(84), and sematilide (47), showed a proarrhythmic effect

by spontaneously inducing PVC and VT before applying

coronary occlusion, but these arrhythmias before

applying coronary occlusion were not observed in high

heart rate pentobarbital-anesthetized dogs by dofetilide

(48), KCB-328 (49), nifekalant (84), sematilide (47),

and d-sotalol (84). Not like Na+/H+-exchange inhibitors,

the Na+/Ca2+-exchange inhibitors KB-R7943 (71) and

SEA0400 (64) had no antiarrhythmic effect on the

coronary occlusion /reperfusion arrhythmia in pentobar-

bital-anesthetized dogs. It may be that the Na+ overload

by inhibition of the Na+/H+ exchange may be more

arrhythmogenic than Ca2+ overload by inhibition of the

Na+/Ca2+ exchange, but there is no proof for the in vivo

heart to support this idea. Other ineffective drugs were

SITS, a Cl /HCO3 inhibitor (95), and long-term fluvasta-

tin, a lipid soluble HMG-CoA reductase inhibitor (88),

administration in rat models. Because of the well known

fact that this arrhythmia is severe and often fatal, it is

difficult to evaluate or reveal proarrhythmic effects of

drugs using this model, except in the pacing-induced

heart-failure dogs, where all halothane-anesthetized

dogs fibrillated just after reperfusion (96).

2-6. Animal models to reveal proarrhythmic poten-

tials of drugs for safety pharmacological studies

Arrhythmia models are not only useful and essential

to find a means to suppress arrhythmias, but recent

demonstration of proarrhythmic effects of drugs, such as

a CAST study showing more deaths in patients receiving

Na+-channel blockers (2) and in patients receiving QT-

prolonging non-cardiovascular drugs, highlighted needs

to demonstrate proarrhythmic effects of drugs (97). Most

of the lethal QT-prolonging drugs produce tachyarrhyth-

mias, thus put simply, animals at slower basal heart rates

help in the detection of proarrhythmia. So anesthetized

with bradycardic agents, such as halothane (6, 98),

unanesthetized animals (99), sinus node–crushed, or AV

node–crushed animals (100 – 102) have been used.

However, it was difficult to observe TdP arrhythmias

with K+-channel blockers in normal animals anesthe-

tized with halothane or in a conscious state and also in

the old myocardial infarction dogs in a conscious state

(99). For finding the proarrhythmic effects of QT-

prolonging K+-channel blockers, many laboratories

demonstrated AV block dogs are suitable (100 – 102)

and probably other animals such as monkeys seem to be

useful. Already it has been demonstrated that in chronic

AV block dogs, bradycardia plus myocardial remodeling

increased the baseline QT prolongation and QT-prolong-

ing cardiac and non-cardiac drugs induced TdP and VF

in high incidences (100 – 107). Since the occurrence of

K+-channel blockers’ induced TdP and death occur

rarely in man, may be one in 10,000 or more, whether

this very high incidence of TdP in chronic AV block

dogs really mimics the human situation is still open for

discussion.

Other models already mentioned are halothane (not

pentobarbital) anesthetized open chest dogs, and sensiti-

zation in halothane-adrenaline arrhythmia models (6,

46 – 49, 75 – 77). Since QT prolongation per se is

beneficial in increasing contractile force and prevention

from re-entry arrhythmias, to identify high risk patients

among the QT-prolonging drug users, the human gene

analysis for the long QT syndrome using inquiry of

hereditary channelopathy may be an alternative

approach to prevent cardiac accidents by drugs with

properties to alter cardiac excitation and contraction.

This problem is still a central issue for developing

new drugs, and the International Conference on

Harmonization (ICH) has been working successfully to

establish guidelines for finding QT-prolonging pro-

perties of drugs in preclinical and early clinical experi-

ments (108). Future efforts are needed to differentiate

good or non-lethal QT-prolonging drugs from possible

dangerous ones and also identifying precipitating factors

making safe QT prolongation to TdP induction, such as

sex hormones, electrolyte imbalance such as hypopotas-

semia, hereditary abnormalities, and so on.

3. Limitations

The present review mainly summarizes our own

experimental results on drugs using various animal

arrhythmia models. Already, there have been many

excellent reviews on antiarrhythmic drugs that made

pharmacologically and pharmacokinetically sound use

of the presently available antiarrhythmic drugs, but there

have been few experiments to compare drugs using

several different animal models and applying the same

protocol for quantitative comparisons. Presently, the

pharmacological and non-pharmacological treatments

for arrhythmias have reached a mature state, where


dramatically different new drugs may not appear and

effective electronic devices are being refined, down-

sized, and becoming less expensive. Additionally the

resistance to use whole animals in medical research is

making arrhythmia model studies difficult and expen-

sive, so summarizing our previous results and others

may help decrease the useless repetition of experiments

and sacrifice of precious animals and hopefully promote

the refining of antiarrhythmic drug research and as a

result promote the emergence of some dramatically

different new drugs in the future.

Acknowledgments

Many thanks to my former colleagues, secretaries

who devoted their efforts to help fulfill my research

interests, and Ms Yasuko Hashimoto, who always

helped to improve the English of my papers including

the present one.

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57 Furukawa Y, Xue YX, Chiba S, Hashimoto K. Effects of

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58 Naito H, Furukawa Y, Hashimoto K. Effects of a cardioselective

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59 Hashimoto K. Electrophysiological mechanisms for digitalis

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63 Sugiyama A, Xue YX, Hagihara A, Saitoh M, Hashimoto K.

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69 Hashimoto K. Electrophysiological correlates of antiarrhythmic

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70 Xue YX, Aye NN, Hashimoto K. Antiarrhythmic effects of

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71 Miyamoto S, Zhu B-M, Kamiya K, Nagasawa Y, Hashimoto K.

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72 Hashimoto K, Hashimoto K. The mechanism of sensitization of

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73 Noda Y, Hashimoto K. Development of a halothane-adrenaline

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79 Miyamoto S, Zhu B-M, Teramatsu T, Aye NN, Hashimoto K.

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108 U.S. Department of Health and Human Services, Food and Drug

Administration, Center for Drug Evaluation and Research,

Center for Biologics Evaluation and Research. S7B Nonclinical

Evaluation of the Potential for Delayed Ventricular Repolariza-

tion (QT Interval Prolongation) by Human Pharmaceuticals.

October 2005 ICH.

arrhythmia models for drug research: classification of

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