antiarrhythmic effects of ranolazine in a guinea...

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Antiarrhythmic Effects of Ranolazine in a Guinea Pig

in vitro Model of Long-QT Syndrome

LIN WU, JOHN C. SHRYOCK, YEJIA SONG, YUAN LI, CHARLES

ANTZELEVITCH, and LUIZ BELARDINELLI

Pharmacological Sciences, CV Therapeutics, Inc., Palo Alto, California (L.W., Y.L., L.B.);

Department of Medicine, University of Florida, Gainesville, Florida (J.C.S., Y.S.); Masonic

Medical Research Laboratory, Utica, New York (C.A.)

JPET Fast Forward. Published on March 18, 2004 as DOI:10.1124/jpet.104.066100

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 18, 2004 as DOI: 10.1124/jpet.104.066100

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Wu et al: Antiarrhythmic effects of ranolazine Correspondence to:

Lin Wu or Luiz Belardinelli CV Therapeutics, Inc. 3172 Porter Drive Palo Alto, CA, 94304, USA Tel: ++1-650-384-8624 Fax: ++1-650-475-0393 E-mail: [email protected] E-mail: [email protected]

Number of text pages: 15 Number of:

Table: 0 Figures: 6

Reference: 32

Words in: Abstract: 224 Introduction: 668 Discussion: 1036

Abbreviations:

APD: action potential duration ATX: anemone toxin A-V: atrioventricular CPA: N6-cyclopentyladenosine CPP: coronary perfusion pressure EAD: early afterdepolarizations ECG: electrocardiogram VT: ventricular tachycardia LQT3: long QT syndrome 3 LVMAPD90: left ventricular monophasic action potential duration at 90% of

repolarization MAP: monophasic action potential MAPD: monophasic action potential duration TdP: torsades de pointes

Recommended section: Cardiovascular


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Abstract

Prolongation of the QT interval of the electrocardiogram (ECG) is associated with increased risk

of torsades de pointes ventricular tachycardia. Ranolazine, a novel antianginal agent, is reported

to decrease the delayed-rectifier potassium current IKr and to increase action potential duration

(APD) and the QT interval. However, ranolazine is also reported to reduce late sodium current

(late INa), a depolarizing current that contributes to prolongation of the plateau of the ventricular

action potential. We hypothesized that ranolazine would decrease APD and the occurrence of

arrhythmias when late INa is increased. Therefore, we measured the effects of ranolazine alone

and in the presence of anemone toxin (ATX)-II, whose action mimics the sodium channelopathy

associated with long-QT3 syndrome, on epicardial monophasic action potentials and ECGs

recorded from guinea pig isolated hearts. Ranolazine (0.1-50 µM) prolonged monophasic APD at

90% repolarization (MAPD90) by up to 22% but did not cause either early afterdepolarizations

(EADs) or ventricular tachycardia (VT). ATX-II (1-20 nM) markedly increased APD and caused

EADs and VT. Ranolazine (5-30 µM) significantly attenuated increases in MAPD90 and reduced

episodes of EADs and VT produced by ATX-II. Ranolazine also attenuated the synergistic

effect of MAPD90 increase caused by combinations of ATX-II and blockers of IK (E-4031).

Thus, although ranolazine alone prolonged APD, it reduced APD and ventricular arrhythmias

caused by agents that increased late INa and decreased IK.

Key words: ranolazine, ATX-II, E-4031, tachyarrhythmias, action potential duration


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Introduction

Prolongation of the duration of the ventricular action potential (indicated on the surface

electrocardiogram as an increase of the QT interval) due to inhibition of the rapid delayed-

rectifier potassium current, IKr, is caused by drugs from many therapeutic classes, and is

associated with an increased risk of ventricular tachyarrhythmias such as torsades de pointes

(TdP) (Belardinelli et al., 2003). However, QT interval prolongation per se may not be

proarrhythmic (Hondeghem et al., 2001). Ranolazine is a novel anti-anginal drug (Louis et al.,

2002, Pepine and Wolff, 1999, Chaitman et al., 2004) and it prolongs the QT interval but is not

known to increase the incidence of ventricular tachycardias (VT), and may reduce the incidence

of ischemia-related arrhythmias (Gralinski et al., 1996). At a cellular level, ranolazine has been

found to reduce the peak outward current conducted by the delayed-rectifier potassium channel

IKr with a potency (IC50 value) of 11.5 µM (Zygmunt et al., 2002), and to reduce the late inward

sodium current (late INa) with an IC50 of 5-21 µM, depending on pacing frequency and

membrane potential (Zygmunt et al., 2002; Song et al., 2004).

Inhibitions of IKr and late INa by ranolazine would be expected to have opposite effects on the

duration of the QT interval. Whereas inhibition of IKr prolongs action potential duration (APD),

inhibition of late INa decreases APD. Thus, the effect of ranolazine on the QT interval may

depend on the relative magnitudes of ranolazine-induced changes in IKr and late INa, and the

contributions of these ion currents to ventricular repolarization. We speculated that ranolazine

may be useful to reduce the QT interval and the incidence of early afterdepolarizations (EADs)

and TdP when late INa is elevated, as in heritable and/or acquired forms of the long-QT3 (LQT3)

syndrome. To test this hypothesis, the electrophysiological effects of ranolazine on the ventricle


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of the guinea pig isolated heart were determined in the absence and presence of agents that

increase late INa and/or decrease repolarizing potassium currents, thereby prolonging the

durations of the ventricular action potential and the QT interval. To increase late INa, hearts were

exposed to Anemonia sulcata toxin II (ATX-II). ATX-II prevents full inactivation of the inward

sodium current (Isenberg and Ravens, 1984), and therefore mimics in exaggerated fashion

mutations of the cardiac sodium channel gene SCN5A that are implicated as the mechanism for

the LQT3 form of long-QT syndrome (Bennett et al., 1995; Wang et al., 1995; Priori, 1996).

Two drugs that are reported to mimic forms 1 and 2 of the long-QT syndrome were used to

further prolong the duration of the ventricular action potential in the presence of low

concentrations of ATX-II: chromanol 293B (LQT1) and E-4031 (LQT2). Chromanol 293B is a

relatively selective blocker of IKs (Sun et al., 2001), the slowly-activating delayed-rectifier

potassium current (Barhanin et al., 1996). Mutations in the gene KCNQ1 (KvLQT1) may result

in a decrease of IKs, prolongation of action potential duration, and the LQT1 form of long-QT

syndrome (Shalaby et al., 1997). E-4031 was shown (Sanguinetti and Jurkiewicz, 1990) to

decrease the rapidly-activating potassium current IKr, a change that mimics the effect of

unfavorable mutations of the potassium channel gene KCNH2 (HERG) that are the molecular

mechanism of LQT2 (Curran et al., 1995). Class III antiarrhythmic drugs, female gender,

hypokalemia, and electrical remodeling in cardiac hypertrophy and failure may reduce the

magnitude of repolarizing potassium currents and increase the risk of TdP (Marban, 2002, Vos et

al., 2001). In addition, late INa appears to be increased in left ventricular myocytes from failing

hearts (Undrovinas et al., 2002). Regardless, ATX-II in combination with either the IKr blocker

E-4031 or the IKs blocker chromanol 293B were used in the present study to mimic concurrent

channelopathies (Marban, 2002) that may be present in clinical situations in which


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antiarrhythmic drugs are used. The guinea pig isolated heart was used in this study because both

Ikr and Iks are present in guinea pig, as in human, ventricular myocytes (Zicha et al., 2003).


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Methods

Animal model. Hartley guinea pigs of both sexes and weighing 250-300 g were purchased

from Simonsen Laboratories (Gilroy, CA). Animal use in this study was reviewed and approved

by the Institutional Animal Care and Use Committee of CV Therapeutics, Inc. (Palo Alto, CA).

Animals were anesthetized with isoflurane. Hearts were excised and perfused by the Langendorff

method at 10 ml/min with Krebs-Henseleit solution (NaCl 118, KCl 2.8, KH2PO4 1.2, CaCl2 2.5,

MgSO4 0.5, pyruvate 2.0, glucose 5.5, Na2EDTA 0.57, NaHCO3 25 mM, pH adjusted to 7.4)

warmed to 33°C. Isoflurane was chosen as the anesthetic agent because it does not appear to

inhibit the induction of TdP or to reduce the transmural dispersion of repolarization in the

ventricle, and may actually facilitate these phenomena (Antzelevitch et al., 1999; Weissenburger

et al., 2000). To facilitate exit of fluid from the left ventricle, the leaflets of the mitral valve

were trimmed with fine scissors. The right atrial wall was partially removed and a bipolar

Teflon-coated electrode was placed on the left atrium to pace the heart. Electrical stimuli of 3-ms

width and 3-fold threshold amplitude were delivered to the pacing electrode at a frequency of 3.5

Hz. The A1-adenosine receptor agonist N6-cyclopentyladenosine (CPA, 0.1 µM) was added to

the perfusion medium to block atrioventricular (A-V) nodal conduction and facilitate pacing of

the ventricle at the rate of 1.5 Hz. In preliminary experiments, we found that CPA (0.1-10 µM)

did not have a significant effect on ventricular MAPD in ventricular-paced hearts. When block

of A-V conduction was achieved, the pacing electrode was moved to the left ventricle inferior

wall. An equilibration period of 10-15 min after onset of left ventricular pacing was sufficient to

allow a regular capture by pacing stimuli of the ventricles, and recording of the monophasic

action potential (MAP). The coronary perfusion pressure of each isolated perfused heart was


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measured by a pressure transducer (Biopac, Goleta,CA) connected to the perfusion line about 4

cm from the aorta cannula. Hearts that did not maintain a minimum coronary perfusion pressure

of 50 mm Hg during the equilibration period were not used for experiments.

MAP and ECG measurements. MAPs were recorded using a pressure-contact Ag-AgCl

electrode applied to the left ventricular epicardial surface. The electrode was adjusted during an

experiment both to maintain stable MAP signals and to prevent damage to the ventricular

surface. Electrode signals were amplified (Biopac MP 150, Goleta, CA) and displayed

continuously on a computer screen in real-time for visual monitoring. In each protocol, to ensure

that a response to a given drug concentration had achieved a steady state before the drug

concentration was changed, the duration of either MAPD100 or QT interval was measured using

an on-screen caliper during the drug infusion period.

To record an electrocardiogram (ECG), a 1-cm thick sponge ring soaked in saline was

placed on the right ventricular free wall. One end of a Teflon-coated tungsten unipolar electrode

was inserted in the middle of the sponge and the opposite end plugged into the input of an ECG

amplifier. The reference electrode was placed directly into the ventricular epicardial wall close to

the A-V valves. ECG, MAP, and coronary perfusion pressure signals were collected in real time

and stored on a computer for subsequent analysis. MAP signals were exported to a specially

designed Excel template. Signals were used for analysis only when MAPD was stable at pacing

rates for at least 10 sec and after signal artifacts were removed.

Drug concentration-response relationships. Increasing concentrations of drug were

infused in a cumulative manner, allowing 7-15 min between changes of concentration. To

investigate the rate dependence of effects of drugs on the duration of the ventricular MAP, the

ventricular pacing rate was increased stepwise from 1 to 1.5, 2, and 2.5 Hz. Each pacing rate was


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maintained for 2 min and MAPs were measured during the last 5 sec of each period. The pacing

rate was then reduced to 1 Hz, and a 15-min infusion of either ATX-II (7 nM), chromanol 293B

(1 µM), E-3041 (1 µM), or ranolazine (5 µM) was begun. When MAPD90 prolongation attained

an apparent steady-state in the presence of drug, the ventricular pacing rate was again increased

progressively and MAPs were recorded.

Determination of antiarrhythmic effects of ranolazine. VT was defined as a sequence of

3 or more ventricular depolarizations at a rate >1.5 Hz. VT that terminated spontaneously was

defined as transient or nonsustained VT. VT that did not subside spontaneously unless

interrupted by a treatment (ranolazine) was defined as sustained VT. Polymorphic VT (TdP-like

VT) was defined as VT with much faster rate and different morphology in both MAP and ECG

recordings. A positive depolarization during phase 2 and/or 3 of an MAP was defined as an EAD

when associated with T wave changes on ECG recording, and as a ventricular extrasystolic beat

when associated with a QRS complex in the ECG. Confirmation of the spontaneous origin of

VT was obtained by its persistence and recurrence when delivery of pacing stimuli was

suspended for a minimum of 10 sec.

Statistical analysis

All data are reported as means ± SEM. Concentration-response curves were analyzed using

Prism version 3.0 (GraphPad, San Diego, CA). Repeated measure one-way ANOVA was used

to compare values of measurements obtained from the same heart before and after treatment.

When ANOVA revealed the existence of a significant difference among values, the Student-

Newman-Keuls test was applied to determine the significance of a difference between a selected


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pair of group means. A p value < 0.05 was taken as an upper limit to indicate a significant

difference.

Sources of drugs

Ranolazine (ranexa, (±)-N-(2,6-Dimethylphenyl)-(4[2-hydroxy-3-(2-

methoxyphenoxy)propyl]-1-piperazine) was synthesized at CV Therapeutics. CPA were

purchased from Sigma Chemical (St. Louis, MO), ATX-II (anemonia sulcata toxin-II) and E-

4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-methylsulfonylaminobenzoyl)piperidine) from

Alomone Labs (Jerusalem, Israel), and chromanol 293B (trans-N-[6-Cyano-3,4-dihydro-3-

hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-N-methyl-ethanesulfonamide) from Tocris

(Ellisville, MO).


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Results

Effects of ATX-II, chromanol 293B, E-4031 and ranolazine on MADP90, EADs and

arrhythmias

ATX-II, E-4031, chromanol 293B, and ranolazine each increased the duration of the MAP

recorded from the guinea pig heart in a concentration-dependent manner (Figure 1). E-4031 (10

µM), chromanol 293B (10 µM), and ranolazine (50 µM) increased the duration of the MAP by

50±4% (n=9), 12±3% (n=9), and 22±2% (n=13), respectively (Figure 1). The maximum effect of

ATX-II on MAPD90 could not be determined because ATX-II (>20 nM) caused frequent

premature ventricular beats or sustained episodes of VT that interfered with the calculation of

MAPD90. High concentrations (1-10 µM) of E-4031 also caused EADs that did not progress to

VT in the 6 hearts studied. Chromanol 293B and ranolazine each caused a moderate prolongation

of MAPD90, but did not cause either EADs or VT (Figure 1).

Attenuation by ranolazine of the effect of ATX-II on MAPD90

The concentration-response relationship for ATX-II to increase the duration of the MAP was

significantly flattened by 5 µM ranolazine (Figure 2). Ranolazine (5 µM) alone produced a

sustained, small (16 % above baseline) prolongation of MAPD90 from 202±9 to 235±4 ms (n=7)

but this prolongation was not additive to that caused by ATX-II. In contrast, the action of ATX-

II to lengthen MAPD90 was markedly reduced by ranolazine (Figures 2,3). An example of a

record from one of 7 hearts exposed to ranolazine in the presence of ATX-II is shown in Figure

3.


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Attenuation by ranolazine of ATX-II-induced EADs and VT

Ranolazine greatly reduced the occurrence of EADs, frequent premature ventricular beats,

and VT caused by ATX-II. Representative experimental recordings from two of 13 hearts

perfused with solution containing 20 nM ATX-II are shown in Figure 4. Perfusion of hearts with

20 nM ATX-II for 15 min led to significant prolongation of the MAPD and the QT interval,

EADs, and ventricular extrasystolic beats, which were followed by episodes of transient and

sustained polymorphic VT (Figure 4, panels A and B) in all hearts. These rhythm abnormalities

were not observed under control conditions in the absence of ATX-II. Administration of 5 µM

to 10 µM ranolazine in the continued presence of 20 nM ATX-II led to the suppression of EADs,

abolishment of polymorphic VTs and restoration of a regular ventricular response to pacing

(Figure 4, panel B, record c). EADs and polymorphic VT reappeared after termination of

ranolazine infusion (Figure 4, panel B, record d). Thus, ranolazine reversibly terminated

monomorphic or polymorphic (EAD-triggered) VT caused by ATX-II (20 nM).

Ranolazine not only reversed but also prevented the appearance of EADs, frequent

premature ventricular beats, and VT caused by ≥ 20 nM ATX-II. VT occurred in all 5 hearts in

Figure 2 that were treated with 20 nM ATX-II alone. In experiments wherein hearts (n=7, Figure

2) were pretreated with 5 µM ranolazine, no EADs or VTs were observed when hearts were

subsequently exposed to concentrations of ATX-II as high as 100 nM in the continued presence

of ranolazine.


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Antagonism by ranolazine of the MAPD90 prolongation caused by ATX-II plus E-4031

and chromanol 293B.

Ranolazine attenuated the actions of combinations of ATX-II with E-4031 (Figure 5A, B)

and chromanol 293B (not shown). In hearts paced at 1.5 Hz, perfusion of a low concentration of

either ATX-II (7 nM) or E-4031 (1 µM) produced moderate prolongations of MAPD90 (71±8 and

61±9 ms, n=3 and 4, respectively) (Figure 5B). Perfusion of ATX-II (7 nM) plus E-4031 (1 µM)

produced a marked synergistic increase of MAPD90 by 325±107 ms (Figure 5, panel B).

Ranolazine (5, 10 and 30 µM) significantly attenuated the effects of ATX-II plus E-4031 to

prolong MAPD90 (Figure 5 B). Similarly, ranolazine significantly (p<0.05, n=4) attenuated the

prolongation of MAPD90 caused by a combination of 7 nM ATX-II and 1 µM chromanol 293B.

In hearts (n=4) perfused with chromanol 293B (1 µM) alone or in combination with ATX-II (7

nM), MAPD90 was prolonged by 12±4 and 68±3 ms, respectively, above control. In the

continued presence of 1 µM chromanol 293B and 7 nM ATX-II, ranolazine (5, 10, and 30 µM)

reduced the prolongation of MAPD90 from 68±3 to 53±3, 45±3 and 38±5 ms, respectively, (n=4,

P<0.05). Interestingly, ranolazine neither increased nor decreased MAPD90 in the presence of E-

4031 alone (i.e., in the absence of ATX-II). E-4031 (1 µM) increased MAPD90 by 54±9 ms from

211±9 (control) to 265±9 ms (n=5, p<0.05). Values of MAPD90 in the presence of 1 µM E-4031

with 5, 10, and 30 µM ranolazine were 267±10, 266±11 and 263±14 ms, respectively (n=5,

p>0.05 vs E-4031 alone). In contrast, ranolazine increased MAPD90 in the presence of the IKs

blocker, chromanol 293B. Chromanol 293B (1 µM) increased MAPD90 by 14±6 (n=5) ms above

control. Values of MAPD90 in the presence of chromanol 293B alone and with 5, 10, and 30 µM


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ranolazine were 208±4, 220±5, 224±6 and 240±6 ms, respectively (n=5, p<0.01 for 30 µM

ranolazine vs. chromanol 293B alone).

Lack of rate dependence of action of ranolazine on duration of the MAP

Antiarrhythmic drugs must be effective at high heart rates; therefore, knowledge of the

dependence of drug action on heart rate is useful to predict efficacy of a prospective

antiarrhythmic agent (Hondeghem and Snyders, 1990). Reverse use dependence is considered to

be a proarrhythmic risk factor. The effect of a decrease in pacing cycle length (increase of pacing

frequency) on prolongations of MAPD90 by ranolazine, ATX-II, E-4031, and chromanol 293B is

shown in Figure 6. As expected, duration of the MAP decreased with a decrease in pacing cycle

length in control hearts (Figure 6, left panel). Ranolazine and chromanol 293B did not alter this

relationship, whereas E-4031 and ATX-II steepened it (Figure 6, left panel). This is shown more

clearly in the right panel of Figure 6, where the differences in values of MAPD90 between drug-

treated and control hearts are plotted as a function of pacing cycle length. An absence of rate

dependence is indicated by a zero slope of the relationship between cycle length and ∆MAPD90

(i.e., a line parallel to the abscissa). Prolongations of MAPD90 caused either by ranolazine or by

the IKs blocker chromanol 293 B were independent of rate, with slope values that were not

significantly different from zero. On the other hand, the frequency-response plots describing the

effects of E-4031 and ATX-II on MAPD90 had significantly positive slopes (0.018±0.003 and

0.026±0.07, respectively), indicative of a reverse rate-dependence of the effects of E-4031 and

ATX-II.


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Discussion

Drug-induced reduction of IKr, prolongation of the QT interval, and TdP are a potential risk

during treatment with a wide range of drugs from diverse therapeutic classes (Belardinelli et al.,

2003; Hondeghem et al., 2001). Therefore, we sought to determine whether the APD

prolongation caused by ranolazine was associated with proarrhythmic activity. In keeping with

its effect to reduce outward potassium current (IKr blockade), ranolazine prolonged left ventricle

MAPD90. However, as now acknowledged by many investigators, some drugs that prolong the

duration of the ventricular action potential and the QT interval do not increase the risk of TdP

(Hondeghem et al., 2001, Studenik et al., 2001, van Opstal et al., 2001). Thus, of particular

interest are the results that 1) ranolazine alone did not cause EADs, ectopic beats, or VT, 2)

ranolazine markedly reduced the proarrhythmic effects of ATX-II (Figures 2-4), and 3)

ranolazine reduced the duration of the MAP in the presence of a combination of two agents

(ATX-II and E-4031) that act by different mechanisms to increase MAPD90 (Figure 5). The

mechanism by which ranolazine decreased APD in the presence of ATX-II and 1 µM E-4031

(Figure 5) is likely to be a reduction of late INa. Because ranolazine decreased ventricular MAPD

in the presence of combinations of ATX-II with either E-4031 or chromanol 293B, but not in the

presence of the IK blockers alone (i.e., absence of ATX-II), the data suggest that ranolazine is

effective to reduce MAPD only when late INa contributes significantly to prolongation of the

action potential. However, it is also possible that ranolazine and E-4031 compete for the same

binding site in the HERG channel, because both ranolazine and E-4031 inhibit Ikr. The result that

ranolazine markedly reduced prolongation of the MAPD caused by ATX-II supports the findings

by Zygmunt et al. (2002) and Song et al (2004) that ranolazine decreased late INa. ATX-II is


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reported to inhibit inactivation of the cardiac Na+ channel, thus increasing both the inward

current entering the cardiomyocyte during the plateau of the action potential, and action potential

duration. This “gain of function” by the sodium channel delays repolarization and facilitates the

development of EADs (Isenberg and Ravens, 1984, Studenik et al., 2001). The actions of ATX-

II resemble those caused by mutations in the gene SCN5A (Bennett et al., 1995, Wang et al.,

1995) that lead to repeated openings of sodium channels during the action potential plateau.

Thus the guinea pig isolated heart exposed to ATX-II is a model for the human LQT3 syndrome,

which is characterized by a long QT interval and increased susceptibility to TdP induced by

drugs that reduce outward currents during the action potential plateau, namely blockers of IKr

(Makita et al., 2002, Roden, 2000). Patients with LQT3 syndrome are at increased risk of TdP

(Schwartz et al., 2001). In hearts pretreated with ATX-II, ranolazine not only did not further

prolong, but in contrast, shortened the duration of the MAP. Thus, it appears that the action of

ranolazine to inhibit late INa has more effect on action potential duration than its action to block

IKr under conditions that cause an increase of late INa.

A recent report of the effects of ranolazine to attenuate ATX-II-induced increases of late INa

and EADs in guinea pig isolated ventricular myocytes (Song et al., 2004) is consistent with the

results shown in the present study. Thus it is likely that the afterdepolarizations caused by ATX-

II (and abolished by ranolazine) noted in recordings of MAP in this study (Figure 4, panel A) are

indicative of the presence of EADs, as we have concluded. The effects of ranolazine to reduce

late INa, ventricular arrhythmias and prolongation of the MAP in the presence of ATX-II are

similar to those of mexiletine, a class IB antiarrhythmic agent (sodium channel blocker)(Shimizu

and Antzelevitch, 1997). Mexiletine was found to have a greater effect to block SCN5A mutant

sodium channels than to block wild-type sodium channels (Wang et al., 1997).


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Increased late INa and reductions in repolarizing K+ currents, whether caused by drugs or by

heritable ion channel dysfunction, prolong the QT interval and are major predisposing factors for

TdP in humans (Sasyniuk et al., 1989). Prolongation of the QT interval beyond a certain limit

may herald proarrhythmic events (Shaffer et al., 2002). If life-threatening ventricular

tachycardia episodes are almost invariably the consequence of a considerably prolonged QT

interval, then a drug like ranolazine with the property of limiting or reducing drug or disease-

induced prolongation of the QT interval may be of therapeutic value. However, much additional

research is required before the present results can be extrapolated to clinical practice. The

temporal and spatial patterns of electrical activity, ion channel expression, and

physiological/pathological regulation of ion channel function of the human heart ventricle are not

fully replicated in hearts of animals used for experimentation. In particular, as regards the present

study, the potassium currents of guinea pig ventricular myocytes differ in relative magnitude

from those of human ventricular myocytes; the magnitude of Ito is less, whereas the magnitude of

IKs is greater in the guinea pig compared to the human ventricular myocyte (Zicha et al., 2003).

In contrast to classical Class III antiarrhythmic agents and E-4031, which block IKr,

ranolazine prolonged cardiac repolarization in a way that did not wane when pacing rate was

increased. Ideally, ventricular tachycardias would be treated with an antiarrhythmic agent whose

effectiveness rose as heart rate increased (Hondeghem and Snyders, 1990). Unfortunately, many

Class III antiarrhythmic agents are less effective in prolonging action potential duration at high

than at low heart rates (Hondeghem and Snyders, 1990).

In conclusion, the results of this investigation indicate that the prolongation of action

potential duration by ranolazine appears not to be associated with an increased probability of

ventricular arrhythmias (EADs or VT). This finding lends credence to the proposal that


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prolongation of action potential duration per se is not proarrhythmic. Ranolazine reduced the

prolongation of cardiac repolarization caused by drugs that mimic ion channelopathies associated

with increased late INa or decreased IK and terminated episodes of ATX-II-induced non-sustained

and sustained ventricular tachycardia. The prolongation of MAPD caused by ranolazine was

independent of ventricular rate. Thus, under circumstances where late INa is increased,

ranolazine appears to act as an antiarrhythmic drug.


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Figure Legends

Figure 1. Concentration-response relationships for ATX-II, E-4031, chromanol 293B, and

ranolazine to increase duration of the ventricular monophasic action potential (MAPD90) in

guinea pig isolated hearts. Hearts were paced at a rate of 1.5 Hz. Baseline MADP90 values were

217±5 (n=5); 212±5 (n=9); 217±3 (n=9) and 217±7 ms (n=13) for ATX-II, E-4031, chromanol

293B, and ranolazine-treated hearts, respectively.

Figure 2. Attenuation by ranolazine of ATX-II induced increases of MAPD90. Shown are

concentration-response relationships for ATX-II to increase duration of the ventricular

monophasic action potential (MAPD90) in guinea pig isolated hearts, in the absence and presence

of 5 µM ranolazine. Hearts were paced at 1.5 Hz. Single and double asterisks indicate that

ventricular tachycardia occurred in 1 and all 5 hearts studied, respectively.

Figure 3. Effects of ranolazine to prolong the duration of the monophasic action potential

(MAPD)(panel A) and to attenuate the prolongation of MAPD caused by ATX-II (panel B).

Numbers on each panel indicate the sequence in which hearts were exposed to drugs.

Figure 4. Representative records showing effects of ATX-II and ranolazine on guinea pig left

ventricular monophasic action potentials (MAP) and electrocardiograms (ECG). A. MAP and

ECG signals recorded sequentially from a single heart in the absence of drug (a) and in the

presence of 20 nM ATX-II (b and c) and 20 nM ATX-II plus 10 µM ranolazine (d). EADs in

MAP signals corresponded to T wave changes in ECG without (b) or with (c) triggered extra


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ventricular beats. B. Records of MAP and ECG signals depicting the effect of ranolazine to

terminate ATX-II-induced polymorphic VT. MAP (top) and ECG (bottom) signals were

recorded sequentially from a guinea pig heart in the absence (a) and presence of drugs as

indicated (b,c,d) in each of the four panels.

Figure 5. Effect of ranolazine to attenuate prolongation of the ventricular monophasic action

potential caused by the combination of ATX-II and E-4031. A. MAP recorded from an isolated

guinea pig heart. Numbers indicate the sequence in which hearts were exposed to drugs. B.

Summary of effects of ATX-II (A, 7 nM, n=3) and E-4031 (E, 1 µM, n=4) alone and in

combination (n=7), and in the presence of ranolazine (R, n=7). An (*) indicates a significant

difference from A+E, p<0.001.

Figure 6. Rate dependence of actions of ranolazine, E-4031, ATX-II, and chromanol 293B on

the duration of the ventricular monophasic action potential (MAPD90) in guinea pig isolated

heart. Left panel. Relationship of MAPD90 to pacing cycle length during the absence (control)

and presence of either ranolazine (n=5), E-4031 (n=6), ATX-II (n=6), or chromanol 293B (n=5).

Right panel. Values of MAPD90 at different pacing cycle lengths using data shown in the left

panel, with drug effects expressed as differences from control responses obtained in the same

heart.


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antiarrhythmic effects of ranolazine in a guinea...

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