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Inhibition of cardiac Ca2+ release channels as therapy for arrhythmia

D. Mehra,1 D.F. van Helden,1 H.S. Hwang,2 B.C. Knollmann2 and D.R. Laver,1 1Department of Biomedical Sciences and Pharmacy, University of Newcastle and HMRI, Callaghan, NSW 2308, Australia and 2Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN 37240, USA.

The ryanodine receptors (RyR2) are the calcium release channels in sarcoplasmic reticulum (SR) which is the main Ca2+ store in the heart. Mutations in RyR2 or calsequestrin cause arrhythmias as a result of increased diastolic Ca2+ release via RyR2. Such an abnormality manifests in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a form of cardiac arrhythmia which is the focus of our research.

Flecainide, a sodium channel blocker of the Class I anti arrhythmic group was found to prevent arrhythmia in CPVT by inhibiting RyR2 (Watanabe et al., 2009). However, it is not clear how inhibition per se has an anti arrhythmic action because another inhibitor, tetracaine, has a pro-arrhythmic action (Watanabe et al., 2009). It was noted that flecainide decreased channel mean open time whereas tetracaine increased channel closed times (Hilliard et al., 2010). Therefore, we test the hypothesis that the therapeutic inhibition of RyR2 relies on reducing channel open times without affecting its closed times. Our approach is to measure the actions of a range of class 1 drugs on RyR2 open times in single channel recording and correlate this with their therapeutic action determined in isolated cardiomyocytes and in a CPVT mouse model.

RyR2 was isolated from sheep and human hearts as described previously (Laver et al., 1995). RyRs were incorporated into artificial lipid bilayers and channel gating was measured by single channel recording. RyR2 open and closed times were measured in the presence of diastolic [Ca2+] (100 nmol/l cytoplasmic and 0.1 mmol/l luminal). SR Ca2+ release was measured using confocal microscopy on intact ventricular cardiomyocytes isolated from hearts of calsequestrin knockout mice (CPVT mouse model). Fura2-AM was used to measure the effect of Class I anti arrhythmic drugs on isoproterenol-induced calcium waves.

RyR2 open time was decreased by the Class Ic anti-arrhythmic drugs whereas Classes Ia and 1b had no significant effect at concentrations up to 50 μmol/l (see Table). None of the Class Ic drugs had any significant effect on RyR2 closed durations. The ability of the drugs to reduce RyR2 open times correlated with their ability to prevent the Ca2+ waves in cardiomyocytes; an indicator for their anti-arrhythmic efficacy. Both flecainide and propafenone suppressed ventricular tachycardia in calsequestrin knockout mice.

The data suggest that potency of RyR2 open time reduction rather than just a reduction in open probability determines efficacy of class I agents for the prevention of CPVT. This may lead to a paradigm shift in drug development process by directing strategies away from discovering high affinity compounds to searching for low affinity compounds with short residence time in the channel.

Comparison of the potency (IC50) of class 1 drugs on the mean open time of cardiac RyRs and their IC50s for reduction in frequency of Ca2+ waves in intact cardiomyocytes. n.d. = not done. n in parentheses.

compound IC50 (μmol/l) in
RyR2 open time
IC50 (μmol/l)
Ca2+ waves
Class 1a quinidine >50 (5) >6 (28)
procainamide >50 (5) >15 (28)
disopyramide >50 (7) >6 (28)
Class 1b lidocaine >50 (5) >50 (28)
mexilitine >50 (6) >6 (28)
Class 1c flecainide 16.7 ± 4.0 (14) 2.0 ± 0.2 (36)
R-propafenone 8.7 ± 0.6 (13) 1.1 ± 0.5 (36)
S-propafenone 17.3 ± 1.6 (14) 5 ± 1 (36)
encainide 22.5 ± 1.2 (9) n.d.

Hilliard FA, Steele DS, Laver D, Yang Z, Le Marchand SJ, Chopra N, Piston DW, Huke S, Knollmann BC. (2010) Journal of Molecular and Cellular Cardioliology, 48: 293-301.

Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Journal of Membrane Biology 147: 7-22.

Watanabe H, Chopra N, Laver D, Hwang HS, Davies SS, Roach DE, Duff HJ, Roden DM, Wilde AA, Knollmann BC. (2009) Nature Medicine 15: 380-383.