Ryanodine receptor inhibition potentiates the activity of Na channel blockers against spontaneous calcium elevations and delayed afterdepolarizations in Langendorff-perfused rabbit ventricles.

Abstract

Na channel blockers are effective in suppressing delayed afterdepolarizations (DADs) in isolated Purkinje fibers. However, in isolated mouse ventricular myocytes lacking calsequestrin, only those Na channel blockers that also inhibit type 2 ryanodine receptor channels were effective against spontaneous Ca elevation (SCaE) and DADs.
To test the hypothesis that combined Na channel and type 2 ryanodine receptor channel blocker ((R)-propafenone) is more effective than a Na channel blocker (lidocaine) in suppressing SCaE and DADs in the intact rabbit ventricles.
We compared (R)-propafenone (3 μmol/L) with lidocaine (50 μmol/L) on SCaE and DADs by using epicardial optical mapping of intracellular calcium (Ca(i)) and membrane voltage in Langendorff-perfused rabbit hearts. SCaE and DADs were induced by rapid pacing trains and isoproterenol (0.3 μmol/L) infusion. One arbitrary unit is equivalent to the Ca transient amplitude of paced beats.
SCaEs were observed at the cessation of rapid pacing in all hearts at baseline. (R)-Propafenone nearly completely inhibited DADs and SCaE (0.04 arbitrary units [95% confidence interval 0.02-0.06] vs 0.23 arbitrary units [95% confidence interval 0.18-0.28] at baseline; n = 6 hearts; P <.001). Lidocaine also significantly reduced the SCaE but was significantly (P <.05) less effective than (R)-propafenone. Both drugs increased the rise time of action potential upstroke and reduced conduction velocity to a similar extent, suggesting a significant inhibition of I(Na).
Both Na channel blockers significantly reduced tachycardia-induced SCaEs in the rabbit ventricles, but (R)-propafenone was significantly more effective than lidocaine. These data suggest that type 2 ryanodine receptor inhibition potentiates the activity of Na channel blockers against SCaE and DADs in the intact hearts.

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Ryanodine receptor inhibition potentiates the activity of Na
channel blockers against spontaneous calcium elevations and
delayed afterdepolarizations in Langendorff-perfused rabbit
ventricles
Young Soo Lee, MD, PhD,* Mitsunori Maruyama, MD, PhD,* Po Cheng Chang, MD,*
Hyung Wook Park, MD, PhD,* Kyoung-Suk Rhee, MD, PhD,* Yu-Cheng Hsieh, MD, PhD,*
Chia-Hsiang Hsueh, PhD,* Changyu Shen, PhD,†Shien-Fong Lin, PhD,* Hyun Seok Hwang, PhD,‡
Huiyong Yin, PhD,‡Björn C. Knollmann, MD, PhD,‡Peng-Sheng Chen, MD, FHRS*
From the *Department of Medicine, Krannert Institute of Cardiology and Division of Cardiology, Indiana University
School of Medicine, Indianapolis, Indiana,†Department of Biostatistics, Indiana University School of Medicine,
Indianapolis, Indiana, and‡Division of Clinical Pharmacology, Oates Institute for Experimental Therapeutics, Vanderbilt
University School of Medicine, Nashville, Tennessee.
BACKGROUND Na channel blockers are effective in suppressing
delayed afterdepolarizations (DADs) in isolated Purkinje fibers.
However, in isolated mouse ventricular myocytes lacking calse-
questrin, only those Na channel blockers that also inhibit type 2
ryanodine receptor channels were effective against spontaneous
Ca elevation (SCaE) and DADs.
OBJECTIVE To test the hypothesis that combined Na channel and
type 2 ryanodine receptor channel blocker ((R)-propafenone) is
more effective than a Na channel blocker (lidocaine) in suppress-
ing SCaE and DADs in the intact rabbit ventricles.
METHODS We compared (R)-propafenone (3 ?mol/L) with lido-
caine (50 ?mol/L) on SCaE and DADs by using epicardial optical
mapping of intracellular calcium (Cai) and membrane voltage in
Langendorff-perfused rabbit hearts. SCaE and DADs were induced
by rapid pacing trains and isoproterenol (0.3 ?mol/L) infusion.
One arbitrary unit is equivalent to the Ca transient amplitude of
paced beats.
RESULTS SCaEs were observed at the cessation of rapid pacing in
all hearts at baseline. (R)-Propafenone nearly completely inhibited
DADs and SCaE (0.04 arbitrary units [95% confidence interval
0.02–0.06] vs 0.23 arbitrary units [95% confidence interval 0.18–
0.28] at baseline; n ? 6 hearts; P ?.001). Lidocaine also signif-
icantly reduced the SCaE but was significantly (P ?.05) less
effective than (R)-propafenone. Both drugs increased the rise time
of action potential upstroke and reduced conduction velocity to a
similar extent, suggesting a significant inhibition of INa.
CONCLUSIONS Both Na channel blockers significantly reduced
tachycardia-induced SCaEs in the rabbit ventricles, but (R)-
propafenone was significantly more effective than lidocaine. These
data suggest that type 2 ryanodine receptor inhibition potentiates
the activity of Na channel blockers against SCaE and DADs in the
intact hearts.
KEYWORDS Depolarization; Action potentials; Calcium; Antiar-
rhythmic agents
ABBREVIATIONS AV ? atrioventricular; CI ? confidence interval;
CL ? cycle length; CPVT ? catecholaminergic polymorphic ven-
tricular tachycardia; CV ? conduction velocity; DAD ? delayed after-
depolarization; PCL ? pacing cycle length; RyR2 ? type 2 ryanodine
receptor; ScaE ? spontaneous Ca elevation; SR ? sarcoplasmic retic-
ulum; VEB ? ventricular escape beat; VF ? ventricular fibrillation
(Heart Rhythm 2012;9:1125–1132) © 2012 Heart Rhythm Society. All
rights reserved.
Introduction
Spontaneous Ca release from the sarcoplasmic reticulum
(SR) is thought to underlie the delayed afterdepolariza-
tion (DAD) and triggered action potentials that can be
observed in ventricular myocytes and Purkinje fibers.1,2
Studies in the 1980s have shown that INablockers, such
as tetrodotoxin and lidocaine, can suppress ouabain-in-
duced DADs in isolated Purkinje fiber,3likely by limiting
This manuscript was processed by a Guest Editor. This study was
supported in part by National Institutes of Health grants P01 HL78931,
R01 HL78932, R01 HL71140, R01 HL088635, R01 HL071670-08, and
R21 HL106554, a Nihon Kohden/St Jude Medical Electrophysiology fel-
lowship (to Dr Maruyama) and a Medtronic-Zipes Endowment (to Dr
Chen). Dr Lee is now with the Division of Cardiology, Department of
Internal Medicine, Catholic University of Daegu School of Medicine,
Daegu, Korea. CryoCath Technologies, Inc, donated the SurgiFrost probe
used in these experiments. Address for reprint requests and correspon-
dence: Dr Peng-Sheng Chen, MD, FHRS, Department of Medicine, Kran-
nert Institute of Cardiology and Division of Cardiology, Indiana University
School of Medicine,1801 N Capitol Ave, E475, Indianapolis, IN 46202.
E-mail address: chenpp@iupui.edu.
1547-5271/$ -see front matter © 2012 Heart Rhythm Society. All rights reserved.http://dx.doi.org/10.1016/j.hrthm.2012.02.031

Page 2

intracellular Na loading during rapid heart rates.4More
recently, Hwang et al5reported that among class I anti-
arrhythmic drugs, only flecainide and (R)-propafenone
also inhibit type 2 ryanodine receptor (RyR2) channels.
Propafenone is a racemic mixture of (S)-propafenone and
(R)-propafenone.6Both enantiomers are equally effective
Na channel blockers, but (R)-propafenone is a signifi-
cantly more potent RyR2 inhibitor than (S)-propafenone.5
(S)-Propafenone also blocks beta-adrenergic receptors,
whereas (R)-propafenone does not.6Consistent with
RyR2-blocking effects, flecainide and (R)-propafenone
both reduced arrhythmogenic Ca waves in cardiomyocytes
isolated from calsequestrin knockout mice, a genetic model of
catecholaminergicpolymorphic
(CPVT). In vivo, low-dose (R)-propafenone prevented ex-
ercise-induced ventricular tachycardia, while lidocaine was
significantly less effective. These results suggest that sup-
pressing the spontaneous SR Ca release and arrhythmogenic
Ca waves contributed to the antiarrhythmic efficacy of
propafenone and flecainide in patients with CPVT.5,7How-
ever, this hypothesis is supported only by studies in cells
isolated from mice. Whether or not flecainide and (R)-
propafenone suppress spontaneous SR Ca release, DAD,
and triggered activity in other species and in intact cardiac
muscle remains unknown. We recently reported that in
intact Langendorff-perfused rabbit ventricles, isoproterenol
infusion and the abrupt cessation of rapid pacing are con-
sistently followed by spontaneous Ca elevation (SCaE) and
DAD.2These hearts may also develop triggered activities
from 2 different competing ventricular foci, resulting in
bidirectional ventricular tachycardia.8The purpose of the
present study was to use this intact heart model of SCaE and
DAD to test the hypothesis that (R)-propafenone (which
blocks both RyR2 and INabut not beta-adrenergic receptor)
is more effective than lidocaine (which blocks only INa) in
suppressing SCaE and DAD in intact Langendorff-perfused
rabbit ventricles at the cessation of rapid pacing.
ventriculartachycardia
Methods
Langendorff-perfused rabbit ventricles
This study protocol was approved by the Institutional Ani-
mal Care and Use Committee of Indiana University School
of Medicine and the Methodist Research Institute and con-
formed to the Guide for the Care and Use of Laboratory
Animals. Details of this animal model have been reported
elsewhere.2Briefly, New Zealand White female adult rab-
bits (n ? 12) were anesthetized with Na pentobarbital (50
mg/kg). These hearts were rapidly excised and Langendorff
perfused at 25–30 mL/min with oxygenated Tyrode’s solu-
tion (NaCl 125 mmol/L, KCl 4.5 mmol/L, NaHCO324
mmol/L, NaH2PO41.8 mmol/L, CaCl21.8 mmol/L, MgCl2
0.5 mmol/L, and glucose 5.5 mmol/L) with a pH of 7.40.
We performed cryoablation of the atrioventricular (AV)
junction by using a 7-cm SurgiFrost probe (CryoCath Tech-
nologies, Inc, Quebec, Canada) with the probe temperature
of 135°C for 3 minutes. During the cryoablation, the epi-
cardium was protected by immersing the heart into warm
(37°C) Tyrode’s solution.
Optical mapping
The hearts were stained with Rhod-2 AM (1.48 ?mol/L) for
10 minutes for Caimapping and with RH237 for Vmmap-
ping. The double-stained hearts were excited with laser light
at 532 nm. The fluorescence was collected and recorded
with dual-CMOS cameras (BrainVision, Tokyo, Japan) at 2
ms/frame and 100 ? 100 pixels with a spatial resolution of
0.35 ? 0.35 mm2/pixel. The fluorescence obtained through
a common lens was separated with a dichroic mirror
(650-nm cutoff wavelength) and directed to the respective
camera with additional filtering (715-nm longpass for Vm
and 580 ? 20 nm for Cai). Optical signals were processed
with both spatial (3 ? 3 pixels Gaussian filter) and temporal
(3 frames moving average) filtering. The hearts were im-
mobilized with 20 ?mol/L blebbistatin (Tocris, Ellisville,
MO) and 5 ?mol/L cytochalasin D (Sigma, St Louis, MO)
during optical mapping.
Experiment protocol
We performed rapid pacing from the right ventricular
apex (150 or 200 ms, 400 beats) and induced ventricular
fibrillation (VF) followed by defibrillation. In addition to
rapid pacing and VF, we continuously infused isoproter-
enol (0.3 ?mol/L) throughout the experiment. The SCaE
and DADs, if any, were mapped at the cessation of rapid
pacing and VF. After obtaining baseline data, we re-
peated the same pacing and mapping protocol with (R)-
propafenone (3.0 ?mol/L) in 6 hearts and lidocaine (50
?mol/L) in 6 hearts. In contrast to racemic propafenone
or (S)-propafenone, (R)-propafenone does not inhibit
beta-adrenergic receptors and therefore does not block
the effects of isoproterenol. The concentrations of both
drugs are within the clinical therapeutic concentration
and are also comparable with that used by Hwang et al.5
Pseudoelectrocardiogram was monitored with 2 widely
spaced bipolar electrodes.
Data analysis
The amplitude of an SCaE was measured by an arbitrary
unit (AU), where 1 AU equals the Caiamplitude during
baseline pacing. The slope of an SCaE (AU/s) was mea-
sured at the steepest portion of Caiduring SCaE. Caimap
was constructed by the amplitude in Caiat each pixel in
the mapped region. The phase 0 duration was defined as
the time interval between 10% depolarization and 90%
depolarization on the phase 0 of action potential. It was
measured on the action potential of the last (400th) paced
beat at 200-ms pacing cycle length (PCL) and was used
as a measure of the Na channel–blocking effect of each
drug. We chose 200-ms PCL because it is short enough to
induce maximum use–dependent block of Na channel in
intact ventricles9but long enough to prevent the induc-
tion of VF halfway through the pacing train. The con-
duction velocity (CV) was also measured at that paced
1126Heart Rhythm, Vol 9, No 7, July 2012

Page 3

beat to serve as an additional measure of the Na channel–
blocking effect. Continuous variables were expressed as
mean and 95% confidence interval (CI). Paired Student’s
t tests were used to compare the SCaE and SCaE slopes
before and after the administration of drugs. Unpaired t
tests were used to compare the degree of SCaE between
drugs. All tests were performed at a 2-tailed significance
level of P ? .05. The 95% CI of the means of relevant
variables was calculated by using normal approximation.
The statistics were computed with PASW Statistics 19
(IBM, Chicago, IL).
Results
Induction of ScaE
As reported by Maruyama et al,2isoproterenol infusion
(0.3 ?mol/L) and rapid ventricular pacing at 150- or
200-ms PCL was always followed by SCaEs in the post-
pacing period (Figures 1–3). Rapid pacing repeatedly
induced VF in 1 heart. SCaE was observed at the termi-
nation of pacing-induced VF in that heart (Figure 1I).
The maximum amplitude and slope of SCaE and the time
to SCaE averaged 0.23 AU (95% CI 0.20–0.26), 1.70
AU/s (95% CI 1.34–2.05), and 314 ms (95% CI 296–
333), respectively (Table 1).
Effect of (R)-propafenone on ScaE
Figures 1 and 2 illustrate the effects of (R)-propafenone
on SCaEs and DADs. In Figures 1A, 1C, 1E, and 1G and
Figure 2B, SCaEs (arrows) and DADs (arrowhead) were
elicited at the cessation of rapid pacing (150 ms, 400
beats) in the presence of isoproterenol (0.3 ?mol/L). In
Figure 1I, the SCaE (arrow) was elicited by pacing-
induced VF-defibrillation episodes. In Figures 1B, 1D,
1F, 1H, and 1J and Figure 2C, the application of (R)-
propafenone (3.0 ?mol/L) nearly completely abolished
the SCaE and DAD (dotted arrows). The Caimaps at
baseline show the distribution of SCaE (green and yellow
colors) in each heart (Figure 1, left column). SCaEs were
almost completely suppressed by (R)-propafenone (Fig-
ure 1, right column and Table 1). The SCaE amplitudes
were too small to allow accurate measurements of the
slope of SCaE and the time to SCaE occurrence (Table
1). The earliest epicardial activation site (asterisk) of the
ventricular escape rhythm (Figure 2A) after the creation
of the AV block was in the left ventricular free wall
(Figure 2D). During isoproterenol infusion, both SCaE
(arrow) and DAD (arrowhead) were observed at the ear-
liest activation site in the base of the right ventricle (asterisk,
Figure 2E), suggesting that triggered activity occurred at a site
other than the ventricular escape beat (VEB). (R)-Propafenone
suppressed all SCaE and triggered activity (Figure 2C). The
Figure 1
asterisk in Caimaps. A, C, E, and G: SCaE (arrows) and DAD (arrowhead) were elicited at the cessation of rapid pacing. I: SCaE (arrow) was elicited after
defibrillating PIVF. B, D, F, H, and J: The application of (R)-propafenone (3.0 ?mol/L) abolished the SCaE and DAD (dotted arrows). In the Caimaps, SCaE
(green and yellow colors) was present at baseline but not after the application of (R)-propafenone. AU ? arbitrary unit; DAD ? delayed afterdepolarizations;
PIVF ? pacing-induced ventricular fibrillation; SCaE ? spontaneous Ca elevation; VEB ? ventricular escape beat.
Effects of (R)-propafenone on SCaE. The black and red lines indicate optical tracing of Vmand Cai, respectively, from the sites labeled by an
1127 Lee et al(R)-Propafenone Inhibits Spontaneous Ca Release

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earliest activation site of the VEB (Figure 2F) returned to the
same site as baseline (Figure 2D).
Effect of lidocaine on ScaE
Figure 3 shows effects of lidocaine on SCaE. In contrast to
(R)-propafenone, lidocaine did not nearly completely suppress
the SCaE, although the amplitude and slope of SCaE were
significantly decreased while the times to SCaE were signifi-
cantly increased compared with baseline values before lido-
caine infusion (Table 1). (R)-Propafenone reduced the ampli-
tudeofSCaEonaverageby84.6%(95%CI79.0–90.2),which
was significantly larger than the 21.5% (95% CI 11.3–31.6)
reduction produced by lidocaine (P ?.001).
Pacing threshold
Before the application of drugs, the pacing threshold was
less than 2.0 mA in all hearts studied. After (R)-
propafenone, it was still possible to achieve 1:1 capture with
Figure 2
earliest activation site (white asterisk) in the activation map of D, E, and F, respectively. The application of (R)-propafenone abolished SCaE, DAD, and
DAD-mediated triggered activity (C). AV ? atrioventricular; DAD ? delayed afterdepolarizations; SCaE ? spontaneous Ca elevation; VEB ? ventricular
escape beat.
Effect of (R)-propafenone on SCaE (arrows), DAD (arrowheads), and triggered activity. Optical tracings of A, B, and C were obtained at the
Figure 3
E: SCaE (arrows) and DAD (arrowheads) were elicited at the cessation of rapid pacing (150 or 200 ms, 400 beats) under isoproterenol infusion (0.3 ?mol/L).
B, D, and F: The application of lidocaine did not abolish the SCaE (dotted arrows) but effectively suppressed VEBs. In the Caimaps, SCaE (green or yellow
colors) under isoproterenol infusion was also observed after the application of lidocaine. AU ? arbitrary unit; DAD ? delayed afterdepolarizations; SCaE ?
spontaneous Ca elevation; VEB ? ventricular escape beat.
Effects of lidocaine on SCaE. Each optical tracing was recorded from the site labeled by an asterisk in the corresponding Caimap. A, C, and
1128Heart Rhythm, Vol 9, No 7, July 2012

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150 ms PCL for 400 beats with pacing output of 2.0 mA.
However, the pacing threshold after the application of lido-
caine was significantly increased to 4.5 mA (95% CI 3.6–
5.4) (P ? .001). Even with these outputs, we were not able
to achieve stable 1:1 capture at 150 ms in 1 lidocaine-treated
ventricle. In that ventricle, the shortest PCL was 200 ms.
Effects of (R)-propafenone and lidocaine on VEB
recovery time
The baseline P-wave cycle length (CL) and VEB CL
during AV block were 335 (95% CI 308–362) and 1030
ms (95% CI 797–1264), respectively, for all the 12 hearts
studied. We defined VEB recovery time as the time
interval between the 400th pacing artifact to the onset of
the first postpacing VEB. The post–rapid pacing VEB
intervals after the administration of (R)-propafenone
(1585 ms [95% CI 992–2177]) and lidocaine (2103 ms
[95% CI 1993–2213]) were significantly longer than at
baseline (631 ms, 95% CI 456– 806, P ? .013, and 527
ms, 95% CI 455–598, P ?.001, respectively). To further
characterize the effects of (R)-propafenone and lidocaine
on cardiac automaticity under adrenergic stimulation, we
analyzed the P-wave CL and VEB CL during AV block
and isoproterenol infusion, before and after drug admin-
istration. Figure 4 shows typical pseudoelectrocardio-
gram tracings of a rabbit heart during AV block, after
isoproterenol infusion and after adding (R)-propafenone
during continued isoproterenol infusion. The P-wave CL
and the VEB CL were shortened by isoproterenol and
lengthened after adding (R)-propafenone. For all the 6
hearts studied, the P-wave CL was 235 ms (95% CI
202–268) and the VEB CL was 407 ms (95% CI 350–
465) during isoproterenol infusion. (R)-Propafenone ad-
ministration during continued isoproterenol infusion sig-
nificantly increased P-wave CL to 256 ms (95% CI 238–
274) (P ? .035) and VEB CL to 782 ms (95% CI
425–1139) (P ? .042). In comparison, lidocaine admin-
Table 1
Effects of drug on SCaE
Amplitude of SCaE (AU)Slope of SCaE (AU/s)Time to SCaE (ms)
IsoAfter drug
P
Iso After drug
P
IsoAfter drug
P
(R)-Propafenone
(n ? 6)
Lidocaine (n ? 6) 0.24 (0.18–0.30)
Total (n ? 12)
0.23 (0.18–0.28) 0.04 (0.02–0.06)
?.001 1.86 (1.35–2.37)312 (278–348)
0.19 (0.15–0.22).010 1.45 (0.88–2.02) 0.67 (0.35–0.98)
1.64 (1.30–1.98)
0.002 316 (288–363)
314 (296–333)
379 (308–451).039
0.24 (0.20–0.27)
All data are expressed as mean and 95% confidence interval.
AU ? arbitrary unit; Iso ? during baseline isoproterenol infusion; SCaE ? spontaneous Ca elevation.
Figure 4
the creation of AV block, pECG showed the dissociation between P wave and VEB. B: The acceleration of both VEB and sinus rate after isoproterenol
infusion (0.3 ?mol/L). C: After the application of (R)-propafenone (3.0 ?mol/L), VEB and P-wave CL lengthened in spite of continuous isoproterenol
infusion. AV ? atrioventricular; pECG ? pseudoelectrocardiogram; VEB ? ventricular escape beat.
Effects of (R)-propafenone on the cycle length (CL) of ventricular escape beats (VEBs, arrows) and P waves (asterisks) on the pECG. A: After
1129Lee et al(R)-Propafenone Inhibits Spontaneous Ca Release

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istration during isoproterenol infusion also significantly
lengthened the VEB CL from 388 ms (95% CI 297–478)
to 1131 ms (95% CI 735–1527)(P ? .005) but did not
significantly change the P-wave CL (from 228 ms [95%
CI 205–251] to 245 ms [95% CI 203–287]; P ? .247).
Effect of (R)-propafenone and lidocaine on the
phase 0 duration and CV
Figure 5 shows effects of (R)-propafenone and lidocaine on
the phase 0 duration and CV. The phase 0 duration and CV
after the application of both drugs were significantly pro-
longed and decreased compared with control, respectively
(all P ?.05). These findings are consistent with Na channel–
blocking effects of the drugs tested.
Discussion
The primary finding of this study is that (R)-propafenone
and lidocaine both suppress SCaE and DADs in intact rabbit
heart. However, (R)-propafenone was much more effective
(85% reduction of SCaE amplitude), whereas the effect of
lidocaine was very modest (21% reduction), even though
the degree of Na channel block was similar by these 2 drugs.
This result suggests that the inhibition of RyR2 is very
important for drug efficacy against SCaE and DADs in the
intact ventricles.
Contribution of INablockade to the suppression
of SCaE in intact ventricles
Among the approved antiarrhythmic drugs, flecainide,
propafenone, and carvedilol appear to have significant in-
hibitory effects on RyR25,7,10,11and may be effective in the
treatment of CPVT and other arrhythmias induced by spon-
taneous SR Ca release. In addition to RyR2 inhibition,
flecainide and propafenone are effective INablockers. Sev-
eral reports indicate that RyR2 blockade is important for
suppressing SCaE and that it contributes to the antiarrhyth-
mic effects of flecainide.5,7,10–14In variance with these
studies, Liu et al15recently found no effects of flecainide on
SCaE in isolated myocytes and concluded that the antiar-
rhythmic activity of flecainide is primarily caused by the
reduction of INaand the concomitant increase in the thresh-
old for triggered activity. In contrast, the results of the
present study demonstrate that RyR2 block is important for
inhibiting SCaE in the intact heart. Our results further sug-
gest that a third factor contributes to the antiarrhythmic
efficacy: INablockers reduce Caiaccumulation during rapid
pacing and thereby indirectly prevent postpacing SCaE.
Rapid pacing can increase intracellular Na (Nai), which
slows the rate of action potential depolarization, allowing
time for greater ICa,Lactivation and enhancing the reverse
mode of Na-Ca exchanger (INCX).16It is possible that the
resulting increase in Caican facilitate the SR Ca accumu-
lation, leading to large SR Ca release at the cessation of
rapid pacing. A previous study by Rosen and Danilo3doc-
umented that lidocaine and tetrodotoxin are effective in
suppressing ouabain-induced DADs, especially at the ces-
sation of rapid pacing. The latter finding can be explained
by the effects of INainhibition on Caiaccumulation. More
recently, it has been proposed that INainhibition underlies
the mechanism by which combined therapy with droneda-
rone and ranolazine inhibits Ca-dependent arrhythmias from
the pulmonary veins.17In the present study, we show that
INainhibition by lidocaine prevented SCaE in the postpac-
Figure 5
(Iso) infusion, before drug administration, and the red lines show the Vmtracings after the administration of (R)-propafenone (R-propa) and lidocaine (Lido).
C and D: The activation map represents the CV during isoproterenol infusion and after drug administration. The duration of phase 0 and CV after the
administration of (R)-propafenone and lidocaine were significantly prolonged and decreased compared with control, respectively. *P ?.05.
Effects of drugs on the duration of phase 0 and conduction velocity (CV). A and B: The black tracings show Vmtracings during isoproterenol
1130 Heart Rhythm, Vol 9, No 7, July 2012

Page 7

ing period in intact rabbit ventricles. These findings provide
further insights into the antiarrhythmic actions of the INa
inhibitors in conditions of Caioverload and sympathetic
activation.
Drug dosage and antiarrhythmic activity
Clinically, active metabolites contribute importantly to
propafenone’s antiarrhythmic activity. For example, pro-
pafenone plasma concentrations of ?350 ng/mL (1 ?M)
result in a 70% inhibition of VEBs clinically, with concen-
trations of 1500 ng/mL (4.3 ?M) required in poor metabo-
lizers.18Therefore, the steady-state plasma concentrations
do not necessarily determine the drug efficacy. Furthermore,
protein binding of propafenone is ?95%, saturable and
enantiomer selective, with (S)-propafenone bound more
than (R)-propafenone in humans, whereas no stereoselec-
tivity for plasma protein binding was found in rabbits.19
Thus, while difficult to predict given that propafenone me-
tabolism is also enantiomer selective, the free concentration
of (R)-propafenone in humans would likely be around
10–20 ng/mL (0.03–0.06 ?M) during chronic dosing. At
the same time, micromolar propafenone concentrations are
required to block ventricular Na currents in isolated myo-
cytes.20A likely explanation for this discrepancy of free
clinical serum concentration and Na channel–blocking ef-
fect (as evidenced by QRS widening, reduced action poten-
tial duration upstroke velocity, increased pacing threshold)
is the observation that propafenone accumulates in cardiac
tissue. For example, in rabbit hearts, heart tissue concentra-
tions were ?100-fold higher than the perfusate concentra-
tions.21The same principles also apply to lidocaine, which
is 70%–80% protein bound and also accumulates in cardiac
tissue.18Thus, rather than using values of human free
plasma concentrations for our in vitro studies, we chose
experimental drug concentrations that achieve a measurable
Na channel block in the heart for both drugs (eg, reduced
upstroke velocity; see Figure 5).
Importance of RyR2 blockade
To control for the effect of INainhibition, lidocaine was
used in our experiments at a 16.6-fold higher concentrations
(50 ?M) than (R)-propafenone (3 ?M), given that lidocaine
is used clinically at 10- to 15-fold higher serum concentra-
tions than is propafenone.18At these concentrations, both
Na blockers significantly reduced tachycardia-induced
SCaEs in the rabbit ventricles, but (R)-propafenone was
significantly more effective than lidocaine in suppressing
SCaE. These findings are consistent with a previous study
that showed that propafenone was more effective than lido-
caine in suppressing ventricular arrhythmias in a canine
model22and that (R)-propafenone had the greatest effects on
spontaneous Ca waves among Na channel blockers.5,23The
report by Hwang et al5also showed the (R)-propafenone is
highly effective in preventing ventricular tachycardia in
mice or human patients with CPVT, a disease associated
with RyR2 mutation that causes abnormal spontaneous Ca
release from the SR during adrenergic stimulation.24–26Our
results suggest that (R)-propafenone is highly effective in
suppressing postpacing SCaE not only because it inhibits
INaand prevents Caiaccumulation but also because it in-
hibits RyR2 to prevent spontaneous SR Ca releases. The
latter antiarrhythmic property is not shared by lidocaine.
(R)-Propafenone and automaticity
Katoh et al27documented that propafenone (racemic mix-
ture of (S)-propafenone and (R)-propafenone) suppresses
Purkinje fiber automaticity. It also suppresses sinus node
automaticity in the presence of complete autonomic block-
ade. In the present study, we found that (R)-propafenone
lengthens the P-wave and VEB CL and prolonged postpac-
ing VEB recovery time during isoproterenol infusion. Mul-
tiple recent studies show that beta-adrenergic stimulation
accelerates the sinoatrial node and the AV node primarily
by activation of the Ca clock, which depends on both SR Ca
sequestration through Ca-ATP pump and spontaneous SR
Ca release through RyR2.28–31Because (R)-propafenone
significantly suppresses the SCaE in intact ventricles, it is
possible that (R)-propafenone also suppresses the SCaE in
sinus node and Purkinje fibers, hence reducing their firing
rate and prolonging the P-wave and VEB CL. However,
lidocaine had a similar effect as (R)-propafenone on VEB
CL, suggesting that INainhibition alone may be sufficient to
suppress VEB generation. More experiments in the sinus
node or Purkinje fibers will have to be done in future to test
the relative contribution of these mechanisms.
Clinical implication
A major clinical implication of the present study is that the
Na channel block alone only has modest efficacy for inhib-
iting SCaE and DAD in the intact ventricle. These effects
may be attributable to the prevention of Caiaccumulation
during tachycardia and isoproterenol infusion. In contrast,
(R)-propafenone, which inhibits both the Na channel and the
RyR2, is much more effective than lidocaine in suppressing
postpacing SCaE and DADs. These findings suggest that
(R)-propafenone may be a highly effective antiarrhythmic
drug for arrhythmias related to Caiaccumulation and sym-
pathetic stimulation.
Study limitation
We used blebbistatin (20 ?mol/L) and cytochalasin D (5
?mol/L) to suppress cardiac contraction during isoprotere-
nol infusion. Whether or not these electromechanical un-
couplers have affected the results of this study remain un-
known.
Conclusions
Both Na channel blockers significantly reduced tachycardia-
induced SCaEs in the rabbit ventricles, but (R)- propafenone
was significantly more effective than lidocaine. These data
suggest that type 2 ryanodine receptor inhibition potentiates
the activity of Na channel blockers against SCaE and DADs
in the intact hearts.
1131 Lee et al(R)-Propafenone Inhibits Spontaneous Ca Release

Page 8

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1132 Heart Rhythm, Vol 9, No 7, July 2012