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Inhibition of Cardiac Ca2+ Release Channels (RyR2) Determines

Efficacy of Class I Antiarrhythmic Drugs in Catecholaminergic

Polymorphic Ventricular Tachycardia

Hyun Seok Hwang, PhD, Can Hasdemir, MD, Derek Laver, PhD, Divya Mehra, BPharm,

Kutsal Turhan, MD, Michela Faggioni, MD, Huiyong Yin, PhD, and Björn C. Knollmann, MD,PhDOates Institute for Experimental Therapeutics (H.S.H., M.F., B.C.K.), Vanderbilt University School

of Medicine, Division of Clinical Pharmacology, Nashville, TN; the Department of Cardiology

(C.H.) and Department of Thoracic Surgery (K.T.), Ege University School of Medicine, Izmir,

Turkey; the School of Biomedical Sciences and Pharmacy (D.L., D.M.), University of Newcastle

and HMRI, Callaghan, Australia; and the Department of Pharmacology and Chemistry (H.Y.),

Vanderbilt University School of Medicine, Nashville, TN

Abstract

Background—Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by

mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin (Casq2) and can be difficult

to treat. The class Ic antiarrhythmic drug flecainide blocks RyR2 channels and prevents CPVT in

mice and humans. It is not known whether other class I antiarrhythmic drugs also block RyR2

channels and to what extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in

CPVT.

Methods and Results—We first measured the effect of all class I antiarrhythmic drugs

marketed in the United States (quinidine, procainamide, disopyramide, lidocaine, mexiletine,

flecainide, and propafenone) on single RyR2 channels incorporated into lipid bilayers. Only

flecainide and propafenone inhibited RyR2 channels, with the S-enantiomer of propafenone

having a significantly lower potency than R-propafenone or flecainide. In Casq2−/− myocytes, the

propafenone enantiomers and flecainide significantly reduced arrhythmogenic Ca2+ waves at

clinically relevant concentrations, whereas Na+ channel inhibitors without RyR2 blocking

properties did not. In Casq2−/− mice, 5 mg/kg R-propafenone or 20 mg/kg S-propafenone

prevented exercise-induced CPVT, whereas procainamide (20 mg/kg) or lidocaine (20 mg/kg)

were ineffective (n=5 to 9 mice, P<0.05). QRS duration was not significantly different, indicating

a similar degree of Na+ channel inhibition. Clinically, propafenone (900 mg/d) prevented ICD

shocks in a 22-year-old CPVT patient who had been refractory to maximal standard drug therapy

and bilateral stellate ganglionectomy.

Copyright © 2011 American Heart Association. All rights reserved.

Correspondence to Björn C. Knollmann, MD, PhD, Oates Institute for Experimental Therapeutics, Vanderbilt University School ofMedicine, Medical Research Building IV, Room 1265, 2215B Garland Ave, Nashville, TN [email protected].

Disclosures

None.

Drs Hwang, Hasdemir, and Laver contributed equally to this work.

The online-only Data Supplement is available at http://circep.ahajournals.org/cgi/content/full/CIRCEP.110.959916/DC1.

NIH Public AccessAuthor ManuscriptCirc Arrhythm Electrophysiol. Author manuscript; available in PMC 2013 May 29.

Published in final edited form as:

Circ Arrhythm Electrophysiol. 2011 April ; 4(2): 128–135. doi:10.1161/CIRCEP.110.959916.

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Conclusions—RyR2 cardiac Ca2+ release channel inhibition appears to determine efficacy of

class I drugs for the prevention of CPVT in Casq2−/− mice. Propafenone may be an alternative to

flecainide for CPVT patients symptomatic on β-blockers.

Keywords

class I antiarrhythmic drugs; propafenone; RyR2; catecholaminergic polymorphic ventricular

tachycardia; flecainide; ranolazine; tetrodotoxin; quinidine; procainamide; disopyramide;

lidocaine; mexiletine

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia

syndrome characterized by physical or emotional stress-induced bidirectional or

polymorphic ventricular tachycardia.1 The more common autosomal-dominant form has

been linked to mutations in the gene encoding the cardiac Ca2+ release channel (RYR2).2 A

less common but more severe autosomal-recessive form is caused by mutations in the gene

encoding cardiac calsequestrin (CASQ2),3,4 the major Ca2+-binding protein in the

sarcoplasmic reticulum (SR).5 Ventricular myocytes isolated from mouse models of both

forms of CPVT exhibit catecholamine-induced premature SR Ca2+ release and spontaneous

Ca2+ waves that trigger delayed afterdepolarizations (DADs) and premature beats.6,7 Thus,

the spontaneous opening of SR Ca2+ release channels elicited by catecholaminergic stress

are the likely culprit for triggering ventricular arrhythmias in CPVT.8

Although significantly reduced, cardiac events remain unacceptably high in CPVT patients

treated with β-blockers.9 Even implantable cardioverter-defibrillators (ICDs) are not

necessarily effective, because defibrillation shocks can cause catecholamine release and

electrical storm, and deaths have been reported in CPVT patients with ICDs.10-12 Thus,

there is a need for better drug therapy in CPVT. We recently found that the class Ic

antiarrhythmic drug flecainide directly targets the molecular defect in CPVT by open-state

block of RyR2 channels13 and prevents CPVT in mice and humans.14 However, it is not

known whether other class I antiarrhythmic drugs also block RyR2 channels and to what

extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in CPVT. Thus, we

tested the effect of all class I antiarrhythmic drugs currently marketed in the United States

on single RyR2 channels incorporated into lipid bilayers. We found that only propafenone

and flecainide inhibit RyR2 channels. The potency of RyR2 block determined

antiarrhythmic activity of Na+ channels blockers in vitro and in vivo in Casq2−/− mice, a

mouse model of CPVT. Clinically, propafenone prevented exercise-induced VT and

recurrent ICD shocks in a highly-symptomatic CPVT patient refractory to standard therapy.

Thus, RyR2 channel inhibition appears to be important for antiarrhythmic drug efficacy in

CPVT. Propafenone may be a promising therapeutic alternative to flecainide for CPVT

patients.

Methods

Animal Model and Experimental Procedures

All experiments were approved by the institutional animal care and use committees at

Animal Care and Use Committees of Vanderbilt University in the United States and

University of Newcastle in Australia and performed in accordance with National Institutes

of Health guidelines. Adult Casq2−/− mice (12 to 16 weeks old) were used for all

experiments. Casq2−/− mice consistently develop ventricular tachycardia during exercise or

after catecholamine challenge.6,14 All data were analyzed in blinded fashion regarding the

treatment groups. Quinidine hydrochloride monohydrate, procainamide hydrochloride,

disopyramide phosphate salt, lidocaine hydrochloride, mexiletine hydrochloride, flecainide

acetate, propafenone hydrochloride, ranolazine dihydrochloride, and tetrodotoxin were

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Circ Arrhythm Electrophysiol. Author manuscript; available in PMC 2013 May 29.

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obtained from Sigma (St Louis, MO). (+)-S-propafenone and (-)-R-propafenone were

separated on a ChiralPak AD column (25×0.46 cm, Chiral Technologies, Exton, PA) using

hexane and 2-propanol containing 0.4% diethylamine. The flow rate was 1 mL and UV

absorption was monitored at 247 nm. The purity of these 2 enantiomers was >99%.15

Single RyR2 Channel Measurements

SR vesicles containing RyR2 channels were obtained from sheep hearts and were

reconstituted into artificial lipid bilayers.16 During SR-vesicle incorporation, the cis

(cytoplasmic) bath contained (in mmol) 250 Cs+ (230 CsCH3O3S, 20 CsCl), 1.0 CaCl2, and

500 mannitol; the trans (luminal) solution contained 50 Cs+ (30 CsCH3O3S, 20 CsCl2) and

1.0 CaCl2. After detection of channels in the bilayer the [Cs+] of the trans solutions was

increased to 250 mmol/L by means of aliquot addition of 4 mol/L CsCH3O3S. The

cytoplasmic solution was exchanged to one containing 2 mmol/L ATP and 0.1 μmol/L free

Ca2+ (1 mmol/L CaCl2+4.5 mmol/L BAPTA) via bath perfusion. The perfusion system

allowed exposure of a single channel to multiple drugs and concentrations that could be

applied in any sequence. Solutions were pH-buffered with 10 mmol/L N-tris

[Hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES, ICN Biomedicals), and solutions

were titrated to pH 7.4 using CsOH (optical grade, ICN Biomedicals) and were redox-

buffered with 5 mmol/L glutathione (ICN Biomedicals).

Electric potentials are expressed using standard physiological convention (ie, cytoplasmic

side relative to the luminal side at virtual ground). Single-channel recordings were obtained

using bilayer potential difference of +40 mV. The current signal was digitized at 10 kHz and

low-pass filtered at 1 or 2 kHz with a gaussian digital filter. Open probability (Po) as well as

open and closed durations were measured by the 50% threshold detection method (Channel2

software by P.W. Gage and M. Smith, Australian National University, Canberra).

Cell Isolations and Ca2+ Fluorescence Recordings

Ventricular myocytes were isolated by a modified collagenase/protease method as

described.6 All the experiments were conducted in Tyrode solution containing (in mmol:

CaCl2 2, NaCl 134, KCl 5.4, MgCl2 1, glucose 10, and HEPES 10, pH 7.4. Final

concentration of Ca2+ was 2 mmol/L. After isolation, myocytes were loaded with Fura-2

acetoxymethyl ester (Fura-2AM).6,17 Briefly, myocytes were incubated with Fura-2AM (2

μmol/L) for 6 minutes at room temperature to load the indicator in the cytosol. Myocytes

were washed twice for 10 minutes with Tyrode solution. A minimum of 30 minutes was

allowed for deesterification before imaging the cells. Ca2+ fluorescence ratios (Fratio) were

recorded and normalized relative to the mean of vehicle group. The ratiometric fluorescent

records were analyzed using commercially available data analysis software (IonWizard,

IonOptix, Milton, MA). Spontaneous Ca2+ waves were measured using the following

protocol: Fura-2AM-loaded myocytes were field stimulated at 1 Hz for 20 seconds until they

reach a steady Ca2+ transient height. Then, stimulation was switched off and myocytes were

monitored for 40 seconds for the occurrence of spontaneous Ca2+ waves, followed by

application of caffeine (10 mmol/L) for 5 seconds using a rapid concentration clamp system.

Amplitudes of caffeine-induced Ca2+ transients were used as estimates of SR Ca2+ content.

Analysis was carried out as recently described by us.14 A spontaneous SR Ca2+ wave was

defined as any spontaneous increase of 0.07 ratiometric units or more from the diastolic

Fratio.

ECG Recordings of Exercise-Induced and Isoproterenol-Induced Ventricular Tachycardiain Mice

Treadmill Exercise Test—Exercise testing in conscious Casq2−/− mice was carried out

as previously described.6 Briefly, mice were initially anesthetized (pentobarbital, 70 mg/g),

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and an ECG transmitter (Data Sciences International, St Paul, MN) was implanted into the

abdominal cavity with subcutaneous electrodes in lead II configuration. Animals were

allowed to recover for at least 6 days after surgery before participating in the treadmill

exercise studies. Study drug or vehicle (DMSO) was injected intraperitoneally 30 minutes

before exercise. We previously established that a dose of 20 mg/kg flecainide results in a

serum flecainide serum concentration of 2.5 μmol/L 1 hour after intraperitoneal injection,

causes a 25% increase in QRS duration, and effectively suppressed exercise-induced CPVT

in mice.14 Because measurement of the QRS duration provides a rapid, noninvasive and

accurate biomarker of Na+ channel block18 and because the objective of our study was to

compare the efficacy of antiarrhythmic drugs at a similar degree of Na+ channel block, study

drugs were administered at doses that produce a 25% increase in QRS duration (online-only

Data Supplement Figure 1). Mice were placed individually into a special chamber of the

motorized rodent treadmill (Exer-6M, Columbus Instruments, Columbus, OH) and exercised

until they exhibited signs of exhaustion.6 For each mouse, treadmill testing was performed 4

times at an interval of >72 hours in randomized sequence. High-quality ECGs were recorded

and analyzed from 15 minutes before exercise until 12 hours after as previously reported.14

Isoproterenol Challenge—The β-adrenergic receptor agonist isoproterenol was used to

induce ventricular arrhythmia in anesthetized Casq2−/− mice as previously described.6

Briefly, mice were anesthetized with isoflurane vapor titrated to maintain the lightest

anesthesia possible. On average, 1.0% vol/vol isoflurane vapor was sufficient to maintain

adequate anesthesia. Loss of toe-pinch reflex and respiration rate was used to monitor levels

of anesthesia. Baseline ECG was recorded for 5 minutes, followed by an additional 10

minutes after administration of isoproterenol (3 mg/kg i.p.). R-propafenone (5 mg/kg) or

vehicle (DMSO) was injected intraperitoneally 30 minutes before administration of

isoproterenol. Isoproterenol challenge was performed 2 times in the same mouse at an

interval of >72 hours, with half the animals receiving first placebo, then R-propafenone, and

the other half receiving first R-propafenone, then placebo.

Human Studies

Propafenone is an approved antiarrhythmic drug and was administered as part of routine

clinical care. The patient’s legal guardian provided consent for retrospective data analysis,

left and subsequently right stellate ganglionectomy, and exercise stress testing. Patient data

collection was approved by the institutional ethics committee. ICD interrogations and

analysis were performed every 2 months on a regular basis or immediately if the patient

received ≥2 ICD shocks.

Statistical Analysis

Differences between the groups of conscious mice were assessed using a 1-way ANOVA

with repeated-measure analysis conducted with SPSS, version 17.0 (SPSS Inc, Chicago, IL),

followed by the Tukey post hoc test when appropriate. The incidence and rate of

spontaneous Ca2+ waves in myocytes and ventricular ectopy in mice were compared by

Fisher exact test and nonparametric Mann-Whitney test. Drug effects on single RyR2

channels were compared using 1-way ANOVA followed by the Tukey post hoc test when

appropriate. The single-channel concentration response curves for S- and R-propafenone

were compared by applying a t test to the differences between measured values and a

common Hill curve over the concentration range of 10 to 50 μmol/L. Results were

considered statistically significant if the probability value was <0.05.

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Results

Propafenone Inhibits RyR2 Ca2+ Release Channels by Open State Block

We first tested the effect of all class I antiarrhythmic drugs currently marketed in the United

States (quinidine, procainamide, disopyramide, lidocaine, mexiletine, flecainide, and

propafenone) on single RyR2 channels incorporated into artificial lipid bilayers. These

experiments were carried out using cytoplasmic and luminal [Ca2+] that mimic cellular

[Ca2+] during diastole (control condition: 2 mmol/L ATP, [Ca2+]cyt=0.1 μmol/L, and

[Ca2+]lum=1 mmol/L). Although class Ia and Ib drugs had no significant effect on RyR2

channels, both class Ic drugs flecainide and propafenone significantly inhibited RyR2

channels at a concentration of 20 μmol/L (Figure 1A).

Propafenone is used clinically as a racemic mixture of S- and R-enantiomeres.19 Both

enantiomers are equipotent Na+ channel inhibitors.20 Only S-propafenone has moderate β-

adrenergic receptor-blocking activity.20 Thus, we next examined the effects of R- and S-

propafenone on RyR2 channels. A representative example of RyR2 channel opening under

these control conditions is shown in Figure 1B (top trace). RyR2 open probability (Po) was

≈0.1. Addition of S-propafenone, R-propafenone, and flecainide to the cytoplasmic bath

caused similar effects on RyR2 channel gating (Figure 1B). The reduction in RyR2 Po was

primarily the consequence of reduced channel To, because all 3 drugs induced brief channel

closures to a subconductance state with ≈30% of the open state conductance (Figure 1B and

1C). The concentration dependencies of R-propafenone, S-propafenone, and flecainide on

To are shown in Figure 1C. Note that none of the 3 drugs significantly changed channel

closed times (Tc, Figure 1C), which is in contrast to the RyR2 channel inhibitor tetracaine13

that is not effective in CPVT myocytes.14 R-propafenone and flecainide reduced RyR2 Po

with similar potency, whereas S-propafenone was significantly less potent (Figure 1D).

Thus, RyR2 channel block by propafenone is stereoselective. The mechanism of

propafenone action is consistent with an open state block and similar to flecainide.

Only Na+ Channel Inhibitors With RyR2 Channel Blocking Properties ReduceIsoproterenol-Induced Ca2+ Waves in Casq2−/− Myocytes

We next tested the efficacy of class I antiarrhythmic drugs and other Na+ channel inhibitors

in ventricular myocytes isolated from a mouse model of CPVT, Casq2−/− mice. All study

drugs were applied for 30 minutes before measurements were obtained. As previously

reported,6 β-adrenergic stimulation with isoprotorenol causes spontaneous Ca2+ waves

(SCW) and triggered beats in Casq2−/− myocytes (Figure 2A). Compared with treatment

with vehicle, the rate of isoproterenol-stimulated Ca2+ waves was drastically reduced by

application of either R-propafenone or flecainide (Figure 2A and 2B), with an IC50 of

approximately 1.1±0.5 μmol/L and 2.0±0.2 μmol/L, respectively. Consistent with its lower

potency in single RyR2 channels (Figure 1C), S-propafenone was significantly less effective

compared to flecainide and R-propafenone in intact myocytes (S-propafenone, 6 μmol/L

0.41±0.12; P<0.05; Figure 2B). Class I agents that did not reduce RyR2 channel open

probability (Figure 1A) had also no significant effect on isoproterenol-induced SCW in

Casq2−/− myocytes (Figure 2A and 2B). Moreover, the selective Na+ channel blocker

tetrodotoxin and the late Na+ current inhibitor ranolazine also had no significant effect on

SCW rate (Figure 2B). At the same time, none of the drugs tested significantly changed

amplitude and decay rate of field-stimulated Ca2+ transients (data not shown) or SR Ca2+

content (Figure 2C). The latter is important because SR Ca2+ content is a determinant of the

incidence of spontaneous Ca2+ waves.21 Taken together, these results demonstrated that

RyR2 channel block is required to suppress arrhythmogenic Ca2+ waves, whereas Na+

channel block by itself has no significant effect.

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RyR2 Channel Block Is Required for Antiarrhythmic Efficacy of Class I Drugs in CPVT Mice

To test the therapeutic efficacy of R-propafenone in vivo, we first studied anesthetized

Casq2−/− mice challenged with isoproterenol (3 mg/kg). Casq2−/− mice exhibit

catecholamine-induced and exercise-induced polymorphic or bidirectional ventricular

tachycardia.6,13,14 Analogous to flecainide,14 pretreatment with R-propafenone (5 mg/kg

i.p.) prevented isoproterenol induced-ventricular ectopy (Figure 3). Ventricular extrasystoles

occurred in 9 of 14 Casq2−/− mice pretreated with vehicle (ventricular extrasystoles per

minute, 4.6±1.6), but only in 1 of 14 Casq2−/− mice pretreated with R-propafenone

(ventricular extrasystoles per minute, 0.3±0.3, Mann-Whitney test, P=0.01). Interestingly,

R-propafenone treatment significantly reduced the isoproterenol-induced heart rate response

(peak heart rate [bpm]: Veh, 558±11 versus R-prop, 476±10; P<0.05).

Next, we examined to what extent RyR2 and Na+ channel block contribute to antiarrhythmic

efficacy in vivo. To address this question, we compared the effect of 2 groups of drugs on

exercised-induced CPVT of conscious Casq2−/− mice: (1) class I antiarrhythmic drugs

without RyR2-blocking properties (procainamide and lidocaine, Figure 1A); and (2), class I

drugs with RyR2-blocking properties (S- and R-propafenone). Both propafenone

enantiomers are equipotent as Na+ channel blockers,20 but S-propafenone has lower potency

on RyR2 channels (Figure 1C) and correspondingly lower efficacy in suppressing SCW in

myocytes (Figure 2A). All drugs were dosed to produce a similar increase in QRS duration

(online-only Data Supplement Figure 1), an in vivo marker of Na+ channel inhibition.18

Pretreatment with a single injection of R-propafenone (5 mg/kg) completely prevented VT

during and for up to 4 hours after treadmill exercise, whereas procainamide (20 mg/kg) or

lidocaine (20 mg/kg) had no significant effect on exercise-induced VT (Figure 4). The time

course of R-propafenone reduction of VT is shown in online-only Data Supplement Figure

2. Consistent with its lower potency on RyR2 channels in vitro (Figure 1D), a 4-fold higher

dose of S-propafenone (20 mg/kg) than R-propafenone was required to suppress VT in vivo

(Figure 4). Collectively, these results indicated that RyR2 inhibition is required for

antiarrhythmic efficacy of class I agents in CPVT mice. Furthermore, the stereoselective

action of R-propafenone as RyR2 inhibitor translates into a higher potency of preventing

exercise or catecholamine-induced polymorphic ventricular tachycardia in vivo.

Propafenone Suppressed ICD Discharges and Exercise-Induced Ventricular Tachycardia ina CPVT Patient

We next tried propafenone in a 22-year-old CPVT patient (RyR2 missense mutation

p.L4105F)22 who was refractory to therapy. His medical history was remarkable for cerebral

palsy caused by birth hypoxia. Within 24 hours of admission, the patient had polymorphic

ventricular tachycardia associated with loss of consciousness requiring 4 direct-current

cardioversions. Holter monitoring showed frequent polymorphic and bidirectional VT. The

patient underwent placement of a single-chamber ICD in June 2007. Ventricular arrhythmias

were initially controlled with the combination of metoprolol (200 mg/d) and verapamil (120

mg/d).22 Starting in the fall of 2008, the patient had frequent appropriate ICD discharges (91

shocks during the 6-month period from November 2008 until April 2009) despite maximal

standard drug therapy and bilateral cardiac sympathetic denervation performed by stellate

ganglionectomy (Figure 5). Because flecainide is not available for clinical use in Turkey,

where the patient lives, propafenone (initially 300 mg/d, subsequently 600 mg/d, and finally

900 mg/d) was started in April 2009 and resulted in a dramatic reduction of ICD discharges

(Figure 5). Only 2 episodes of ICD discharges occurred during 12 months of follow-up, after

the propafenone dose was increased to 600 to 900 mg/d. Propafenone therapy also

completely prevented ventricular arrhythmias during treadmill exercise testing (online-only

Data Supplement Figure 3).

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Discussion

Only Class Ic Antiarrhythmic Drugs Propafenone and Flecainide Directly Target MolecularDefects in CPVT Patients

Over the last decade, the disease mechanism responsible for the CPVT has been well

established.8 Mutations in RyR2 or Casq2 cause a sensitization of the RyR2 Ca2+ release

channel complex to SR luminal Ca2+.23,24 As a result, RyR2 channels open prematurely

under conditions of high SR Ca2+ load, which typically occurs in conditions of β-adrenergic

stimulation and/or fast heart rates.25 The premature Ca2+ release from dyadic RyR2 channel

clusters triggers a propagated Ca2+ wave, which in turn activates the electrogenic NaCa

exchanger located on the cell membrane.26 The ensuing NaCa exchange produces a net

inward Na+ current, which depolarizes the cell membrane and generates a DAD.27 DADs of

sufficient amplitude activate voltage-gated Na+ channels and trigger full action potentials.

Given its unique pharmacological properties, propafenone has multiple modes of action in

CPVT: The RyR2 open channel block discovered here will reduce the likelihood of RyR2

opening triggering an arrhythmogenic Ca2+ wave.13 The class Ic-type tonic Na+ channel

inhibition during diastole will decrease the likelihood of any remaining DADs in triggering

premature ventricular extrasystoles.28 Although less potent as a RyR2 inhibitor, the β-

blocking activity of the S-enantiomere will also contribute to clinical efficacy of

propafenone racemate in CPVT. These multiple modes of propafenone action may explain

why propafenone was effective in a patient refractory to all currently known

pharmacological and surgical therapy in CPVT.

RyR2 Open Channel Block by Class Ic Antiarrhythmic Agents

It is interesting to note that local anesthetics and other drugs that inhibit Na+ channels often

also modulate RyR2 channels, albeit with a large variety of effects. In the present study, we

show that flecainide and propafenone effectively inhibit RyR2 by reducing channel mean

open durations via an open-state blocking mechanism. The RyR2 inhibitory action was not

shared by any of the clinically available class Ia or Ib antiarrhythmic agents. Other

anesthetics such as tetracaine, procaine, and QX314 also inhibit RyR2 channels29 but have a

significantly lower potency and a very different mode of action compared with that of

propafenone and flecainide.13 Tetracaine, unlike flecainide and propafenone, causes long

closures by binding to channels in their closed state, leading to substantially increased

closed-channel intervals.13 The prototypical class Ib agent lidocaine has no effects on RyR2

channels (Figure 1A) and activates RyR2 channels at higher concentrations.30 The tricyclic

antidepressant amitriptyline, which is a potent Na+ channel inhibitor, activates RyR2

channels at clinically relevant concentrations.16 Amitriptyline binds to RyR2 in their open

state and induces prolonged channel openings with ≈80% of the normal channel

conductance.16

The open-state RyR2 channel block probably contributes to the effectiveness of flecainide

and propafenone in suppressing DADs and triggered arrhythmias in experimental models.28

RyR2 channel inhibition may also explain the negative inotropic effects of propafenone and

flecainide, which exceed that expected from Na+ channel block alone31 and the significant

risk of aggravation of heart failure observed clinically with propafenone and flecainide.32

Implications for Drug Therapy in CPVT

Currently, β-blockers are the first line of therapy in CPVT.8 They are the only class of drugs

that has been shown to be effective in improving survival in CPVT.9 Because the incidence

of cardiac events remains considerable even on β-blockers9,33 and ICDs are not always

effective,10-12 there is a need for alternative therapies. Small case series suggest that Ca2+

channels blockers34 or stellate ganglionectomy35 may provide additional benefit. However,

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as illustrated by CPVT patients reported here and previously,14 neither therapy appears to be

completely effective in CPVT. Class Ia and Ib antiarrhythmic agents are not effective as

shown here and previously reported by others in CPVT mouse models36 and humans.33

These results are not surprising because lidocaine can activate RyR2 channels30 and would

be predicted to worsen the underlying molecular defect. We recently reported that

flecainide, a class Ic agent, appeared effective in a mouse model CPVT and in 2 patients

with drug-refractory CPVT, possibly because of its dual mode of action. Although a recent

study failed to find a significant effect of flecainide during an in vivo screen of

antiarrhythmic drugs in CPVT mice,36 this study was neither designed nor statistically

powered to examine flecainide action.37 Another group recently confirmed flecainide

efficacy in Purkinje cells.38 Our results with propafenone further support the hypothesis that

open-state block of RyR2 channels13 coupled with Na+ channel inhibition during diastole

presents a promising therapeutic approach in CPVT. Our finding that S-propafenone was

less effective than R-propafenone in reducing exercise-induced ventricular tachycardia in

CPVT mice suggests that the RyR2 blocking action may be more important than Na+

channel blocking activity for antiarrhythmic drug efficacy in CPVT. Because the

propafenone racemate used clinically has also β-blocking properties, it may be a promising

therapeutic option for CPVT patients who require life-long drug therapy.

Study Limitations

Our data do not exclude the possibility that in addition to the RyR2 inhibition discovered in

the present study, block of Na+ channels, and/or other ion channel also contribute to the

antiarrhythmic efficacy of propafenone in CPVT mice. The exact contribution of RyR2

channel block to propafenone action will have to be addressed by comparison with a

selective open channel RyR2 inhibitor, an agent that is currently not available. Although our

in vitro studies suggest that propafenone is a mechanism-based drug therapy in CPVT, our

clinical experience is limited to a single patient. Given the proarrhythmic effects of class Ic

agents in patients with ischemic heart disease or heart failure,39 the diagnosis of CPVT

should be clearly established and structural heart disease ruled out. As with any new

therapeutic regimen, future clinical studies have to be done to define the risks and benefits

of propafenone as treatment for CPVT.

Conclusion

In this study, we have shown that among clinically available class I antiarrhythmic drugs,

only flecainide and propafenone inhibit RyR2 channels, suppress arrhythmogenic Ca2+

waves, and prevent CPVT in Casq2−/− mice. Thus, the potency of RyR2 channel inhibition

rather than Na+ channel block appears to determine efficacy of class I agents for the

prevention of CPVT. Clinically, propafenone was effective in suppressing appropriate ICD

shocks in a CPVT patient refractory to standard therapy. Thus, propafenone and flecainide

both are promising mechanism-based drug therapies for CPVT patients. However, the

stereoselective action of propafenone discovered in the present study has to be considered

for its clinical use in CPVT patients, given the stereoselective metabolism of propafenone

racemate in humans.40,41

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank Paul Johnson and Meegan Jones at the University of Newcastle for assistance with the single-channel

recording and Melissa Ryan at Vanderbilt University for myocyte isolations.

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Sources of Funding

This work was supported by National Institutes of Health grants HL88635 and HL71670 (to Dr Knollmann),

American Heart Association postdoctoral fellowship grant 09POST2240022 (to Dr Hwang), and an NSW Health

infrastructure grant and a project grant from the University of Newcastle (to Dr. Laver).

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CLINICAL PERSPECTIVE

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare familial

arrhythmia syndrome characterized by emotional or physical stress-induced polymorphic

or bidirectional ventricular tachycardia. CPVT has been linked to mutations in genes that

regulate Ca2+ release from the sarcoplasmic reticulum (eg, RYR2, CASQ2). Although β-

blockers are first-line therapy, they are not always effective, and better drug therapy is

needed. We recently discovered that the class I antiarrhythmic drug flecainide directly

targets the molecular defect in CPVT by blocking RyR2 Ca2+ release channels and

prevented CPVT in mice and humans. In the present study, we extended this work and

tested the efficacy of all Food and Drug Administration-approved class I antiarrhythmic

drugs on RyR2 channels, in isolated myocytes, and in vivo using a mouse CPVT model.

We found that only propafenone and flecainide inhibit RyR2 channels and prevent

exercise-induced CPVT in mice, whereas all other class I drugs lack RyR2 inhibitory

properties and were ineffective. This result suggests that RyR2 channel inhibition

importantly contributes to antiarrhythmic efficacy in CPVT and should be considered

when selecting drug therapy for CPVT patients. As illustrated by the CPVT case report,

propafenone may be a promising alternative to flecainide for CPVT patients whenever

flecainide is not clinically available or not tolerated. However, RyR2 channel inhibition

by propafenone is stereoselective, with R-propafenone being significantly more potent

than S-propafenone. Because propafenone is available clinically only as racemate and its

metabolism is also stereoselective, large interindividual differences in clinical response

may be expected when treating CPVT patients with propafenone.

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Figure 1.

Among class I antiarrhythmic drugs, only flecainide, propafenone, and its enantiomers (S-

and R-propafenone) inhibit RyR2 activity. A, Relative change in channel open probability

caused by study drug application at a concentration of 20 μmol/L. B, Records are

representative examples of single channel activity of RyR2 in lipid bilayers. The baseline

current during channel closures is labeled “C” (dashed lines) and channel openings

correspond to upward current transitions. Control conditions were 1 mmol/L luminal Ca2+

(trans bath), 0.1 μmol/L cytoplasmic Ca2+ plus 2 mmol/L ATP (cis bath). Bilayer potential

was 40 mV (relative to trans bath as ground). Relatively high drug concentrations (50 μmol/

L) were used to better illustrate their effects on RyR2 channel gating. Drug addition

introduced short (≈1 ms) closures to a substrate, labeled “S” (dotted lines), at ≈30% of the

full channel conductance. The full durations of the long (≈1 second) closed periods that are

present in control and drug records are not seen on this time scale. C, Concentration-

response relationship of RyR2 channel inhibition by R- and S-propafenone and flecainide.

RyR2 channel open probability (Po), mean open time (To), and mean closed time (Tc) are

expressed relative to values in absence of drug. D, Comparison of IC50 values determined by

least-squares fitting of Hill curves to the Po and To concentration response data (n=7 to 13

channels per group). *P<0.05.

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Figure 2.

Efficacy of class I antiarrhythmic drugs and other Na+ channel inhibitors for suppressing

isoproterenol-induced spontaneous Ca2+ waves (arrow). A, Representative Ca2+

fluorescence records from Casq2−/− myocytes after 30 minutes’ exposure to vehicle

(DMSO), procainamide (15 μmol/L), lidocaine (50 μmol/L), R-propafenone (6 μmol/L),

and tetrodotoxin (TTX, 6 μmol/L). After 20 seconds pacing and in presence of isoproterenol

(1 μmol/L), SCWs were recorded during 40 seconds without pacing. SR Ca2+ contents were

quantified by rapid caffeine (10 mmol/L) application at the end of recording. Average rate of

SCW (B) and SR Ca2+ content (C) are expressed relative to vehicle. Quinidine (6 μmol/L),

disopyramide (6 μmol/L), mexiletine (6 μmol/L), flecainide (6 μmol/L), and ranolazine (15

μmol/L), *P<0.01 versus vehicle, #P<0.05 versus R-propafenone; n=28 to 38 myocytes per

group.

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Figure 3.

R-propafenone prevents catecholamine-induced ventricular arrhythmia in Casq2−/− mice.

Plotted is the heart rate of an anesthetized Casq2−/− mouse in response to isoproterenol (3

mg/kg) injection 30 minutes after subcutaneous injection with vehicle (DMSO, left) or R-

propafenone (n=14, 5 mg/kg, right). Note the complete suppression of ventricular ectopy

illustrated by the representative ECG records above the heart rate plot.

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Figure 4.

Efficacy of class I antiarrhythmic drugs in preventing exercise-induced VT in conscious

Casq2−/− mice. Procainamide (n=5, 20 mg/kg), lidocaine (n=5, 20 mg/kg), R-propafenone

(n=9, 5 mg/kg), or S-propafenone (n=5 per dose, 5 mg/kg, 20 mg/kg as indicated) was

injected intraperitoneally 30 minutes before exercise. A, Representative example of

nonsustained bidirectional VT episode. B and C, Incidence of VT during treadmill exercise

(B) and during the 4-hour period after exercise (C) for each treatment group. *Versus

vehicle, P<0.05.

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Figure 5.

Effect of propafenone treatment in a 21-year-old CPVT patient (RyR2 missense mutation)

with recurrent appropriate ICD shocks despite maximal conventional drug therapy and

bilateral cardiac sympathetic denervation. Single arrow indicates the date of left cardiac

sympathetic denervation and double arrow the date of right cardiac sympathetic denervation.

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