Bannister, ML

George, CH

MacLeod, KT

Maxwell, C

Mukherjee, S

Sikkel, MB

Thomas, NL

Williams, AJ

The Journal of Physiology

English

PubMed

Spiral - Imperial College Digital Repository

J Physiol 594.21 (2016) pp 6431–6432 6431TheJournalofPhysiologyLETTERQuestioning flecainide’smechanism of action in thetreatment of catecholaminergicpolymorphic ventriculartachycardiaFlecainide attenuates cardiac Ca2+ cyclingabnormalities in malignant catecholamine-triggered arrhythmias but its mechanism ofaction remains highly contentious. We readwith interest the study of Yang et al. (2016)that used in silico predictions in an attemptto determine the relative contribution ofblock by flecainide of ryanodine receptor2 (RyR2) and the Na+ channel to theoverall therapeutic effect of this drug incatecholaminergic polymorphic ventriculartachycardia (CPVT). Flecainide’s actionswere modelled by considering its inhibitionof the Na+ current (INa) and RyR2 incombination and individually. The authorsconclude that the effects of the drug onNa+ channels are insufficient to explain itsefficacy in CPVT, while its block of RyR2alone was as effective as the combined blockof INa and RyR2.Our concerns about the usefulness of thisapproach are twofold:(1) Purported block of RyR2 by flecainideThe blocking parameters used in Yanget al. (2016) are based on values reportedin Hilliard et al. (2010) and subsequentpublications from the same group. Themajor problem with the use of theseparameters to model open channel blockby flecainide of RyR2-mediated Ca2+ releasefrom the sarcoplasmic reticulum (SR)is that Hilliard et al. (2010) did notdemonstrate block of this current, ratherthey reported partial block of K+ movementin the opposite (cytosol to luminal)direction.To fully understand the problem it isnecessary to outline some basic features ofRyR2 channel structure and function.RyR2 provides a ligand-regulated pathwayfor the release of Ca2+ from the cardiacSR to initiate myocyte contraction. In itsopen conformation Ca2+ flows through thepore-forming region (PFR) of RyR2, downits concentration gradient, from the SRlumen to the cytosol. The key structuralfeatures of the RyR2 PFR, based on recenthigh-resolution cryo-electron microscopyinvestigations (Yan et al. 2014), are shownschematically in Fig. 1. Ca2+ leaving the SRenters the luminal mouth of the open PFRbefore passing through a region equivalentto the selectivity filter of the more discri-minating monovalent-selective channelsand eventually into a large, water-filled,lumen lumenCa2+Ca2+Ca2+K+K+ K+lumencytosolcytosolSR lumenAB C DF GEcytosol cytosollumen lumen lumencytosol cytosol cytosolFigure 1. Ionic fluxes and interaction of flecainide in RyR2A, schematic representation of the location and orientation of RyR2 in the cardiac myocyte SRmembrane system. The trans-membrane domain contains the pore-forming region (PFR) and thelarge cytosolic domain abuts transverse tubule invaginations of the sarcolemma. B–G, schematicdiagrams depicting the open RyR2 PFR (B, indicated by circle) together with relevant cation fluxesand interactions of flecainide (green octagon). Only two of the four PFR monomers are shownfor clarity. C, to initiate contraction Ca2+ is released from the SR, flowing down its concentrationgradient through the open RyR2 PFR. D, to dissipate the diffusion potential generated by Ca2+release, a counter current of K+ flows from the cytosol to the SR lumen, at least in part throughRyR2. E, the RyR2 blocking parameters used in Yang et al. (2016) are based on data reported inHilliard et al. (2010) that describe the effects of flecainide on the cytosolic to luminal flux of K+.F, under these conditions flecainide enters the cytosolic cavity of RyR2 and partially occludesthe pore. G, the physiologically relevant flux of Ca2+ from the lumen to the cytosol displacesflecainide (Bannister et al. 2015). As a consequence open channel block of RyR2 by flecainidecan play no part in the effectiveness of this drug in the treatment of CPVT.cytosolic cavity contiguous with the bulkcytosol, lined with trans-membrane helices(Fig. 1C). RyR2-mediated Ca2+ release isan electrogenic process during which acharge-compensating K+ counter currentflows from the cytosol into the SR, at leastin part, through RyR2 (Fig. 1D).C© 2016 The Authors The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society DOI: 10.1113/JP272497This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distributionand reproduction in any medium, provided the original work is properly cited.6432 Letter J Physiol 594.21To monitor flecainide’s actions on RyR2Hilliard et al. (2010) incorporated channelsinto phospholipid bilayers and measuredK+ flux under voltage clamp conditions.RyR2 incorporates into the bilayer in afixed orientation so that its configurationis defined. In these investigations, the netdirection of K+ flux through RyR2 was fromthe cytosolic to luminal side of the channel,driven by a trans-membrane potential withthe solution at the cytosolic side held at+40 mV relative to that at the luminalside of the channel (Fig. 1E). Flecainidewas added to the cytosolic solution and,from here, interacts with RyR2 (Fig. 1F).When bound, flecainide partially occludesthe RyR2 PFR and individual blockingevents are resolved with dwell-times in themillisecond range. A residual current of20% of the full open current continuesto flow with flecainide bound. The keypoint of our comment on the work ofYang et al. (2016) is that the experimentsof Hilliard et al.(2010) merely demonstrateblock of cytosolic to luminal flux of K+through RyR2; they provide no informationon the ability of flecainide to block theflux of cations, and in particular Ca2+,in the physiologically relevant directionthrough RyR2, that is, from the SR lumento the cytosol. For this reason the use ofparameters of block determined by Hilliardet al. (2010) in the models described inYang et al. (2016) is inappropriate andirrelevant and their conclusion that theaction of flecainide on RyR2 is sufficientto explain flecainide efficacy in CPVT, isunsound.The possibility that block by flecainidecould influence RyR2-mediated Ca2+release was examined in Bannister et al.(2015) using individual recombinanthuman RyR2 channels in planar bilayers. Inthese investigations we demonstrated thatflecainide limits cytosolic to luminal K+ fluxthrough RyR2 by interacting at a site withinthe cytosolic cavity of the PFR. Flecainideonly has access to this site from the cyto-sol and, crucially, the affinity with whichflecainide is bound is insufficient to pre-vent it being displaced by cation flux inthe physiologically relevant, luminal to cyto-solic, direction. This is the case for either K+flux driven by a trans-membrane potentialof −40 mV or by Ca2+ flux driven by aluminal to cytosolic concentration gradient(the flux equivalent to Ca2+ release from theSR) (Fig. 1G). Further, flecainide did notinfluence RyR2 gating and had negligibleeffect on the charge-compensating K+current carried collectively by RyR2 and theSR K+ channel during Ca2+ release. Basedon these observations we concluded thatthe efficacy of flecainide in the treatmentof CPVT is not the result of a direct inter-action with RyR2. Given this, we believethat the inclusion of parameters describingblock of RyR2 by flecainide in the modelsused in Yang et al. (2016) is unjustifiedand will inevitably lead to spuriousconclusions.(2) Block of Na+ channel by flecainideA key factor in the conclusion of Yanget al. (2016) that block of the Na+ channelalone is insufficient to explain flecainideefficacy in CPVT was provided by modellingflecainide’s action in the absence of RyR2block. However, by their own admission,the IC50 used in the model to describeflecainide’s inhibition of Ca2+ waves ‘doesnot separate out external contributions tothis parameter, such as lowered junctionalCa2+ brought about by INCX activity vialowered [Na+]’ resulting from block of INa,as proposed by Sikkel et al. (2013). It wouldbe very interesting to see if inclusion of thispotential mechanism of action of flecainidein the model could provide an alternativeexplanation for the efficacy of flecainide inCPVT.Alan J. Williams1, Mark L. Bannister1,N. Lowri Thomas1, Markus B. Sikkel2,Saptarshi Mukherjee1, Chloe Maxwell1,Kenneth T. MacLeod2and Christopher H. George11School of Medicine, Cardiff University,Cardiff CF14 4XN, UK2Myocardial Function Section, NationalHeart and Lung Institute, Imperial CollegeLondon, London W12 0NN, UKEmail: williamsaj9@cardiff.ac.ukReferencesBannister ML, Thomas NL, Sikkel MB,Mukherjee S, Maxwell C, MacLeod KT,George CH & Williams AJ (2015). Themechanism of flecainide action in CPVT doesnot involve a direct effect on RyR2. Circ Res116, 1324–1335.Hilliard FA, Steele DS, Laver D, Yang Z, LeMarchand SJ, Chopra N, Piston DW, Huke S& Knollmann BC (2010). Flecainide inhibitsarrhythmogenic Ca2+ waves by open stateblock of ryanodine receptor Ca2+ releasechannels and reduction of Ca2+ spark mass.J Mol Cell Cardiol 48, 293–301.Sikkel MB, Collins TP, Rowlands C, Shah M,O’Gara P, Williams AJ, Harding SE, Lyon AR& MacLeod KT (2013). Flecainide reducesCa2+ spark and wave frequency via inhibitionof the sarcolemmal sodium current.Cardiovasc Res 98, 286–296.Yan Z, Bai X-C, Yan C, Wu J, Li Z, Xie T, PengW, Yin C-C, Li X, Scheres SHW, Shi Y & YanN (2014). Structure of the rabbit ryanodinereceptor RyR1 at near-atomic resolution.Nature 517, 50–55.Yang P-C, Moreno JD, Miyake CY,Vaughn-Behrens SB, Jeng M-T, Grandi E,Wehrens XHT, Noskov SY & Clancy CE(2016). In silico prediction of drug therapy incatecholaminergic polymorphic ventriculartachycardia. J Physiol 594, 567–593.Additional informationCompeting interestsNone declared.FundingWork described in this letter was funded by theBritish Heart Foundation (PG14/34/30835).C© 2016 The Authors The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society

Questioning flecainide's mechanism of action in the treatment of catecholaminergic polymorphic ventricular tachycardia

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Questioning flecainide's mechanism of action in the treatment of catecholaminergic polymorphic ventricular tachycardia

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