arrhythmia models for drug research: classification of
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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. keitaroh@yamanashi.ac.jp
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
2-2-1. Characteristics
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.
Arrhythmia Models and Drugs 337
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).
2-2-3. Antiarrhythmic drugs
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).
2-2-4. Ineffective and proarrhythmic drugs
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.
Arrhythmia Models and Drugs 339
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
2-3-1. Characteristics
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).
2-3-3. Antiarrhythmic drugs
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).
2-3-4. Ineffective and proarrhythmic drugs
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
2-4-1. Characteristics
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.
2-4-3. Antiarrhythmic drugs
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.
2-4-4. Ineffective and proarrhythmic drugs
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
Arrhythmia Models and Drugs 341
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
2-5-1. Characteristics
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.
2-5-3. Antiarrhythmic drugs
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.
2-5-4. Ineffective and proarrhythmic drugs
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
Arrhythmia Models and Drugs 343
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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108 U.S. Department of Health and Human Services, Food and Drug
Administration, Center for Drug Evaluation and Research,
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October 2005 ICH.
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