Reply from Pei‐Chi Yang, Jonathan D. Moreno, Mao‐Tsuen Jeng, Xander H. T. Wehrens, Sergei Noskov and Colleen E. Clancy

We appreciate Williams et al. (2016) taking the time to comment on our recently published study (Yang et al. 2016). In their letter, the authors question the ‘usefulness’ of the computational modelling and simulation approaches that we used in part because as they state, ‘The blocking parameters used in Yang et al. (2016) are based on values reported in Hilliard et al. (2010) and subsequent publications from the same group.’ This statement does not reflect the careful process that we actually used in building our modelling approaches, where we rather considered the full range of experimentally measured IC50 values for flecainide interaction that have been reported in multiple studies. In addition to the assumption of IC50 = 0 μm (i.e. no interaction with RyR) as reported by the Williams group (Bannister et al. 2015), we reported the following in our paper (Yang et al. 2016): ‘Isoproterenol-stimulated Ca2+ waves in CASQ2 knockout (KO) CASQ2(−/−) mice were inhibited by flecainide with an IC50 of 2.0 ± 0.2 μm (Hwang et al. 2011), while other experimental preparations measured an IC50 range from 2 to 17 μm (Brunton et al. 2010; Hilliard et al. 2010; Hwang et al. 2011; Mehra et al. 2014) … We also predicted cases for variable flecainide IC50 = 3, 4, and 5 μm shown in Fig. 1.’ The model simulations led to the predictions that IC50 values above 5 μm are too low to show therapeutic benefit to normalize the catecholaminergic polymorphic ventricular tachycardia (CPVT) phenotype. An alternative interpretation is that the concentration of flecainide near the receptor is considerably higher than in the bulk water compartments, a possibility supported by our physics-based approach (Fig. 5 in Yang et al. 2016) that shows accumulation of flecainide on the membrane surface and very favourable conditions for neutral flecainide in the hydrophobic core of the membrane. Detailed investigations into membrane partitioning of drugs are ongoing in our group. The point of the simulations in our study was to make predictions about the necessary and sufficient targets of flecainide and the range of IC50 that would allow for normalization of the CPVT phenotype since the experimental literature has shown such variety in reported values. When we started the investigation reported in Yang et al. (2016), we had no preconceived intent or notion about the results. The predictions are the resulting outputs of the model, and suggest that Na+ channel block alone is not sufficient to prevent the CPVT phenotype. The critical point here is that the disparity in sensitivity of the dose–response for flecainide interaction with the RyR depends on the experimental approach being used. This issue has been the subject of discussion by others (Steele et al. 2013; Sikkel et al. 2013b; Smith & MacQuaide, 2015). Williams et al. describe their recent work in their letter. It is important to mention, however, the numerous other studies that report alternative data and explanations. Some in native myocytes show very clear effects of flecainide on spontaneous Ca2+ release (i.e. Ca2+ waves) under experimental conditions where cytosolic [Ca2+] and [Na+] are clamped, demonstrating a direct action of flecainide on RyR2-mediated sarcoplasmic reticulum (SR) Ca2+ release (Savio-Galimberti & Knollmann, 2015; Hilliard et al., 2010; Galimberti & Knollmann, 2011). Moreover, in native myocytes, flecainide does not inhibit physiological Ca2+ current-induced SR Ca2+ release but only inhibits spontaneous SR Ca2+ release, which occurs in the setting of diastolic [Ca2+] (i.e. 100 nm) (Hilliard et al. 2010). Such conditions are difficult to model using RyR2 channels incorporated into artificial bilayers and hence were never tested by the group of Williams et al. Other studies demonstrate a clear benefit of flecainide in the clinical CPVT setting, but not in experiments with other Na+ channel blockers (Watanabe et al. 2009; Hwang et al. 2011; van der Werf et al. 2011). Williams et al. performed single-channel experiments in an experimental model comprising phosphatidylethanolamine (PE) bilayers to show that flecainide does not block ion current by binding to a site within the cytosolic domain of the pore-forming domain of RyR2. However, other data and the physics-based computational approaches in our paper suggest that lipophilic drug access may be critical and is a vital component of drug interactions with membrane protein targets such as RyR2. The potential of mean force calculations we performed in our study suggest that flecainide concentration in the lipid phase could be substantially greater than what would be expected in the bilayer studies. Carvedilol is another example of a very hydrophobic/lipophilic drug that interacts with RyR2 without blocking unitary conductance in single-channel experiments. Liposome partitioning experiments suggest that up to 90% of carvedilol molecules are lipid-phase localized (Cheng et al. 1996). The lipophilic access mechanism would imply different dose–response ratios and use-dependent features of drug interaction with the RyR2 target in contrast to a single-site drug block mechanism endorsed by Williams et al. It is important to point out that lipophilic access mechanisms have been shown recently for various membrane targets found in the heart (Lees-Miller et al. 2015; Boiteux et al. 2014) and are likely to exist for RyR2 given the lipophilicity of many drugs interacting with this channel. Williams et al. have undertaken valuable biophysical studies using purified recombinant channels in artificial lipid bilayers. We argue, however, that such a system is far removed from the physiological reality and cannot unequivocally prove the absence of a flecainide interaction with RYR2 channels in a native cellular environment. For example, Cannon et al. (2003) reconstituted RyR2 into a bilayer composed by 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) showing that channel activity depends critically on the bilayer composition. Another study showed that the polyunsaturated fatty acid eicosapentanoic acid (EPA) exerts its antiarrhythmic effect by reducing the opening probability of RyR2 (Swan et al. 2003). This is important, because the artificial bilayer used by Williams et al. was composed of 100% (PE), but the actual SR lipid content from dog hearts showed the presence of triglycerides, cholesterol and other phospholipids like phosphatidylinositol (PI), phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS). Most of these lipids have been found to regulate the gating (and hence the activity) of other channels as well (Suh & Hille, 2008). Williams and coauthors also mention the potential for INa block to result in reduced junctional Ca2+ concentration through modulated INCX activity. As has been discussed previously (Steele et al. 2013), the experimental conditions used in Sikkel et al. (2013a) employed fast pacing that is well known to cause Na+ loading and resultant Ca2+ loading that can trigger sparks and waves. Early experimental and computational studies support this mechanism and showed that Na+ channel blockers are effective to suppress these events (Leblanc & Hume, 1990; Faber & Rudy, 2000). In our models, fast pacing rates also caused Na+ accumulation (Morotti et al. 2014, Shannon et al. 2004), but INa block only led to modest reduction in junctional [Na+] and thus [Ca2+]. In conclusion, the study by Williams et al. has shown that flecainide does not inhibit recombinant RyR2 channels in artificial bilayers by the pore channel block that they observed. Given the contrasting plethora of evidence from other experimental work and our modelling studies predicting that flecainide inhibition of Na+ currents alone is insufficient to explains its efficacy in CPVT, we contend that further studies are warranted to reveal the mechanism of flecainide action on RyR2, which thus far remains elusive and may not be discoverable using a reductionist approach alone. None declared. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

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