Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and Results We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands. Conclusions Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD. Translational Perspective Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD

Loss of function of hNav1.5 by a ZASP1 mutation associated with intraventricular conduction disturbances in left ventricular noncompaction

BACKGROUND: Defects of cytoarchitectural proteins can cause left ventricular noncompaction, which is often associated with conduction system diseases. We have previously identified a p.D117N mutation in the LIM domain-binding protein 3-encoding Z-band alternatively spliced PDZ motif gene (ZASP) in a patient with left ventricular noncompaction and conduction disturbances. We sought to investigate the role of p.D117N mutation in the LBD3 NM_001080114.1 isoform (ZASP1-D117N) for the regulation of cardiac sodium channel (Na(v)1.5) that plays an important role in the cardiac conduction system. METHODS AND RESULTS: Effects of ZASP1-wild-type and ZASP1-D117N on Na(v)1.5 were studied in human embryonic kidney-293 cells and neonatal rat cardiomyocytes. Patch-clamp study demonstrated that ZASP1-D117N significantly attenuated I(Na) by 27% in human embryonic kidney-293 cells and by 32% in neonatal rat cardiomyocytes. In addition, ZASP1-D117N rightward shifted the voltage-dependent activation and inactivation in both systems. In silico simulation using Luo-Rudy phase 1 model demonstrated that altered Na(v)1.5 function can reduce cardiac conduction velocity by 28% compared with control. Pull-down assays showed that both wild-type and ZASP1-D117N can complex with Na(v)1.5 and telethonin/T-Cap, which required intact PDZ domains. Immunohistochemical staining in neonatal rat cardiomyocytes demonstrates that ZASP1-D117N did not significantly disturb the Z-line structure. Disruption of cytoskeletal networks with 5-iodonaphthalene-1-sulfonyl homopiperazine and cytochalasin D abolished the effects of ZASP1-D117N on Na(v)1.5. CONCLUSIONS: ZASP1 can form protein complex with telethonin/T-Cap and Na(v)1.5. The left ventricular noncompaction-specific ZASP1 mutation can cause loss of function of Na(v)1.5, without significant alteration of the cytoskeletal protein complex. Our study suggests that electric remodeling can occur in left ventricular noncompaction subject because of a direct effect of mutant ZASP on Na(v)1.5

Time-Dependent Block and Resurgent Tail Currents Induced by Mouse β4154–167 Peptide in Cardiac Na+ Channels

Resurgent tail Na+ currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse β4 subunit, β4154–167, acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233–244). To explore these phenotypes in vitro, we characterized β4154–167 actions in inactivation-deficient cardiac hNav1.5 Na+ channels expressed in human embryonic kidney 293t cells. Intracellular β4154–167 from 25–250 μM elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents at 50 mV in a concentration-dependent manner. On and off rates for β4154–167 binding were estimated at 10.1 μM−1s−1 and 49.1 s−1, respectively. Upon repolarization, large tail currents emerged with a slight delay at −140 mV, probably as a result of the rapid unblocking of β4154–167. Near the activation threshold (approximately −70 mV), resurgent tail currents were robust and long lasting. Likewise, β4154–167 induces resurgent currents in wild-type hNav1.5 Na+ channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na+ channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na+ channels opened persistently but became resistant to β4154–167 and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na+ channels resistant to β4154–167. Together, our in vitro studies identify a putative S6-binding site for β4154–167 within the inner cavity of hNav1.5 Na+ channels. Such an S6 receptor readily explains (1) why β4154–167 gains access to its receptor as an open-channel blocker, (2), why bound β4154–167 briefly prevents the activation gate from closing by a “foot-in-the-door” mechanism during deactivation, (3) why BTX inhibits β4154–167 binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates β4154–167 binding

Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation

Cardiac voltage-gated sodium channels (Nav1.5) play an essential role in regulating cardiac electric activity by initiating and propagating action potentials in the heart. Altered Nav1.5 function is associated with multiple cardiac diseases including long-QT3 and Brugada syndrome. Here, we show that Nav1.5 is subject to palmitoylation, a reversible post-translational lipid modification. Palmitoylation increases channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation increases closed-state channel inactivation and reduces myocyte excitability. We identify four cysteines as possible Nav1.5 palmitoylation substrates. A mutation of one of these is associated with cardiac arrhythmia (C981F), induces a significant enhancement of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Our data indicate that alterations in palmitoylation can substantially control Nav1.5 function and cardiac excitability and this form of post-translational modification is likely an important contributor to acquired and congenital arrhythmias

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.Peer ReviewedPostprint (published version

Ranolazine as an Alternative Therapy to Flecainide for SCN5A V411M Long QT Syndrome Type 3 Patients

[EN] The prolongation of the QT interval represents the main feature of the long QT syndrome (LQTS), a life-threatening genetic disease. The heterozygous SCN5A V411M mutation of the human sodium channel leads to a LQTS type 3 with severe proarrhythmic effects due to an increase in the late component of the sodium current (INaL). The two sodium blockers flecainide and ranolazine are equally recommended by the current 2015 ESC guidelines to treat patients with LQTS type 3 and persistently prolonged QT intervals. However, awareness of pro-arrhythmic effects of flecainide in LQTS type 3 patients arose upon the study of the SCN5A E1784K mutation. Regarding SCN5A V411M individuals, flecainide showed good results albeit in a reduced number of patients and no evidence supporting the use of ranolazine has ever been released. Therefore, we ought to compare the effect of ranolazine and flecainide in a SCN5A V411M model using an in-silico modeling and simulation approach. We collected clinical data of four patients. Then, we fitted four Markovian models of the human sodium current (INa) to experimental and clinical data. Two of them correspond to the wild type and the heterozygous SCN5A V411M scenarios, and the other two mimic the effects of flecainide and ranolazine on INa. Next, we inserted them into three isolated cell action potential (AP) models for endocardial, midmyocardial and epicardial cells and in a one-dimensional tissue model. The SCN5A V411M mutation produced a 15.9% APD90 prolongation in the isolated endocardial cell model, which corresponded to a 14.3% of the QT interval prolongation in a one-dimensional strand model, in keeping with clinical observations. Although with different underlying mechanisms, flecainide and ranolazine partially countered this prolongation at the isolated endocardial model by reducing the APD90 by 8.7 and 4.3%, and the QT interval by 7.2 and 3.2%, respectively. While flecainide specifically targeted the mutation-induced increase in peak INaL, ranolazine reduced it during the entire AP. Our simulations also suggest that ranolazine could prevent early afterdepolarizations triggered by the SCN5A V411M mutation during bradycardia, as flecainide. We conclude that ranolazine could be used to treat SCN5A V411M patients, specifically when flecainide is contraindicated.This work was partially supported by Fondo Europeo de Desarrollo Regional (FEDER, "Union Europea, Una forma de hacer Europa") with the Ministerio de Economia y Competitividad (DPI2015-69125-R), Direccion General de Politica Cientifica de la Generalitat Valenciana (PROMETEO/2020/043) and Instituto de Salud Carlos III (La Fe Biobank PT17/0015/0043), as well as by Vicerrectorado de Investigacion, Innovacion y Transferencia de la Universitat Politecnica de Valencia with Ayuda a Primeros Proyectos de Investigacion (PAID-06-18), and by Memorial Nacho Barbera.Cano, J.; Zorio, E.; Mazzanti, A.; Arnau, MÁ.; Trenor Gomis, BA.; Priori, SG.; Saiz Rodríguez, FJ.... (2020). Ranolazine as an Alternative Therapy to Flecainide for SCN5A V411M Long QT Syndrome Type 3 Patients. Frontiers in Pharmacology. 11:1-19. https://doi.org/10.3389/fphar.2020.580481S11911Abdelsayed, M., Peters, C. H., & Ruben, P. C. (2015). Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants. The Journal of Physiology, 593(18), 4201-4223. doi:10.1113/jp270139Ackerman, M. J., Priori, S. G., Willems, S., Berul, C., Brugada, R., Calkins, H., … Zipes, D. P. (2011). HRS/EHRA Expert Consensus Statement on the State of Genetic Testing for the Channelopathies and Cardiomyopathies. Heart Rhythm, 8(8), 1308-1339. doi:10.1016/j.hrthm.2011.05.020Aliot, E., Capucci, A., Crijns, H. J., Goette, A., & Tamargo, J. (2010). Twenty-five years in the making: flecainide is safe and effective for the management of atrial fibrillation. Europace, 13(2), 161-173. doi:10.1093/europace/euq382Andrikopoulos, G. K. (2015). Flecainide: Current status and perspectives in arrhythmia management. World Journal of Cardiology, 7(2), 76. doi:10.4330/wjc.v7.i2.76Belardinelli, L., Liu, G., Smith-Maxwell, C., Wang, W.-Q., El-Bizri, N., Hirakawa, R., … Shryock, J. C. (2012). A Novel, Potent, and Selective Inhibitor of Cardiac Late Sodium Current Suppresses Experimental Arrhythmias. Journal of Pharmacology and Experimental Therapeutics, 344(1), 23-32. doi:10.1124/jpet.112.198887Bengel, P., Ahmad, S., & Sossalla, S. (2017). Inhibition of Late Sodium Current as an Innovative Antiarrhythmic Strategy. Current Heart Failure Reports, 14(3), 179-186. doi:10.1007/s11897-017-0333-0Blich, M., Khoury, A., Suleiman, M., Lorber, A., Gepstein, L., & Boulous, M. (2019). Specific Therapy Based on the Genotype in a Malignant Form of Long QT3, Carrying the V411M Mutation. International Heart Journal, 60(4), 979-982. doi:10.1536/ihj.18-705Bohnen, M. S., Peng, G., Robey, S. H., Terrenoire, C., Iyer, V., Sampson, K. J., & Kass, R. S. (2017). Molecular Pathophysiology of Congenital Long QT Syndrome. Physiological Reviews, 97(1), 89-134. doi:10.1152/physrev.00008.2016Britton, O. J., Bueno-Orovio, A., Van Ammel, K., Lu, H. R., Towart, R., Gallacher, D. J., & Rodriguez, B. (2013). Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology. Proceedings of the National Academy of Sciences, 110(23), E2098-E2105. doi:10.1073/pnas.1304382110Caballero, R., Dolz-Gaiton, P., Gomez, R., Amoros, I., Barana, A., Gonzalez de la Fuente, M., … Delpon, E. (2010). Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification. Proceedings of the National Academy of Sciences, 107(35), 15631-15636. doi:10.1073/pnas.1004021107Carrasco, J. I., Izquierdo, I., Medina, P., Arnau, M. Á., Salvador, A., & Zorio, E. (2012). Flecainide, a Therapeutic Option in a Patient With Long QT Syndrome Type 3 Caused by the Heterozygous V411M Mutation in the SCN5A Gene. Revista Española de Cardiología (English Edition), 65(11), 1058-1059. doi:10.1016/j.rec.2012.03.013Chadda, K. R., Jeevaratnam, K., Lei, M., & Huang, C. L.-H. (2017). Sodium channel biophysics, late sodium current and genetic arrhythmic syndromes. Pflügers Archiv - European Journal of Physiology, 469(5-6), 629-641. doi:10.1007/s00424-017-1959-1Chang, K. C., Dutta, S., Mirams, G. R., Beattie, K. A., Sheng, J., Tran, P. N., … Li, Z. (2017). Uncertainty Quantification Reveals the Importance of Data Variability and Experimental Design Considerations for in Silico Proarrhythmia Risk Assessment. Frontiers in Physiology, 8. doi:10.3389/fphys.2017.00917Chorin, E., Hu, D., Antzelevitch, C., Hochstadt, A., Belardinelli, L., Zeltser, D., … Viskin, S. (2016). Ranolazine for Congenital Long-QT Syndrome Type III. Circulation: Arrhythmia and Electrophysiology, 9(10). doi:10.1161/circep.116.004370Colquhoun, D., Dowsland, K. A., Beato, M., & Plested, A. J. R. (2004). How to Impose Microscopic Reversibility in Complex Reaction Mechanisms. Biophysical Journal, 86(6), 3510-3518. doi:10.1529/biophysj.103.038679Crumb, W. J., Vicente, J., Johannesen, L., & Strauss, D. G. (2016). An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA) proposed ion channel panel. Journal of Pharmacological and Toxicological Methods, 81, 251-262. doi:10.1016/j.vascn.2016.03.009Dutta, S., Chang, K. C., Beattie, K. A., Sheng, J., Tran, P. N., Wu, W. W., … Li, Z. (2017). Optimization of an In silico Cardiac Cell Model for Proarrhythmia Risk Assessment. Frontiers in Physiology, 8. doi:10.3389/fphys.2017.00616Elkins, R. C., Davies, M. R., Brough, S. J., Gavaghan, D. J., Cui, Y., Abi-Gerges, N., & Mirams, G. R. (2013). Variability in high-throughput ion-channel screening data and consequences for cardiac safety assessment. Journal of Pharmacological and Toxicological Methods, 68(1), 112-122. doi:10.1016/j.vascn.2013.04.007Ficker, E., Jarolimek, W., Kiehn, J., Baumann, A., & Brown, A. M. (1998). Molecular Determinants of Dofetilide Block of HERG K + Channels. Circulation Research, 82(3), 386-395. doi:10.1161/01.res.82.3.386Grandi, E., Pasqualini, F. S., & Bers, D. M. (2010). A novel computational model of the human ventricular action potential and Ca transient. Journal of Molecular and Cellular Cardiology, 48(1), 112-121. doi:10.1016/j.yjmcc.2009.09.019Guo, D., & Jenkinson, S. (2019). Simultaneous assessment of compound activity on cardiac Nav1.5 peak and late currents in an automated patch clamp platform. Journal of Pharmacological and Toxicological Methods, 99, 106575. doi:10.1016/j.vascn.2019.04.001Hegyi, B., Bányász, T., Izu, L. T., Belardinelli, L., Bers, D. M., & Chen-Izu, Y. (2018). β-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII. Journal of Molecular and Cellular Cardiology, 123, 168-179. doi:10.1016/j.yjmcc.2018.09.006Horne, A. J., Eldstrom, J., Sanatani, S., & Fedida, D. (2011). A novel mechanism for LQT3 with 2:1 block: A pore-lining mutation in Nav1.5 significantly affects voltage-dependence of activation. Heart Rhythm, 8(5), 770-777. doi:10.1016/j.hrthm.2010.12.041Horvath, B., Banyasz, T., Jian, Z., Hegyi, B., Kistamas, K., Nanasi, P. P., … Chen-Izu, Y. (2013). Dynamics of the late Na+ current during cardiac action potential and its contribution to afterdepolarizations. Journal of Molecular and Cellular Cardiology, 64, 59-68. doi:10.1016/j.yjmcc.2013.08.010Horváth, B., Hézső, T., Szentandrássy, N., Kistamás, K., Árpádffy-Lovas, T., Varga, R., … Nánási, P. P. (2020). Late sodium current in human, canine and guinea pig ventricular myocardium. Journal of Molecular and Cellular Cardiology, 139, 14-23. doi:10.1016/j.yjmcc.2019.12.015KAUFMAN, E. S. (2008). Use of Ranolazine in Long-QT Syndrome Type 3. Journal of Cardiovascular Electrophysiology, 19(12), 1294-1295. doi:10.1111/j.1540-8167.2008.01255.xLancaster, M. C., & Sobie, E. (2016). Improved Prediction of Drug-Induced Torsades de Pointes Through Simulations of Dynamics and Machine Learning Algorithms. Clinical Pharmacology & Therapeutics, 100(4), 371-379. doi:10.1002/cpt.367Li, Z., Dutta, S., Sheng, J., Tran, P. N., Wu, W., Chang, K., … Colatsky, T. (2017). Improving the In Silico Assessment of Proarrhythmia Risk by Combining hERG (Human Ether-à-go-go-Related Gene) Channel–Drug Binding Kinetics and Multichannel Pharmacology. Circulation: Arrhythmia and Electrophysiology, 10(2). doi:10.1161/circep.116.004628Liu, H., Atkins, J., & Kass, R. S. (2003). Common Molecular Determinants of Flecainide and Lidocaine Block of Heart Na+ Channels. Journal of General Physiology, 121(3), 199-214. doi:10.1085/jgp.20028723Liu, H., Tateyama, M., Clancy, C. E., Abriel, H., & Kass, R. S. (2002). Channel Openings Are Necessary but not Sufficient for Use-dependent Block of Cardiac Na+ Channels by Flecainide. Journal of General Physiology, 120(1), 39-51. doi:10.1085/jgp.20028558Lu, H. R., Vlaminckx, E., & Gallacher, D. J. (2008). Choice of cardiac tissue in vitro plays an important role in assessing the risk of drug-induced cardiac arrhythmias in human: Beyond QT prolongation. Journal of Pharmacological and Toxicological Methods, 57(1), 1-8. doi:10.1016/j.vascn.2007.06.005Makielski, J. C. (2016). Late sodium current: A mechanism for angina, heart failure, and arrhythmia. Trends in Cardiovascular Medicine, 26(2), 115-122. doi:10.1016/j.tcm.2015.05.006Makita, N., Behr, E., Shimizu, W., Horie, M., Sunami, A., Crotti, L., … Roden, D. M. (2008). The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome. Journal of Clinical Investigation. doi:10.1172/jci34057Maltsev, V. A., Sabbah, H. N., Higgins, R. S. D., Silverman, N., Lesch, M., & Undrovinas, A. I. (1998). Novel, Ultraslow Inactivating Sodium Current in Human Ventricular Cardiomyocytes. Circulation, 98(23), 2545-2552. doi:10.1161/01.cir.98.23.2545Moreno, J. D., & Clancy, C. E. (2012). Pathophysiology of the cardiac late Na current and its potential as a drug target. Journal of Molecular and Cellular Cardiology, 52(3), 608-619. doi:10.1016/j.yjmcc.2011.12.003Moreno, J. D., Lewis, T. J., & Clancy, C. E. (2016). Parameterization for In-Silico Modeling of Ion Channel Interactions with Drugs. PLOS ONE, 11(3), e0150761. doi:10.1371/journal.pone.0150761Moreno, J. D., Yang, P.-C., Bankston, J. R., Grandi, E., Bers, D. M., Kass, R. S., & Clancy, C. E. (2013). Ranolazine for Congenital and Acquired Late I Na -Linked Arrhythmias. Circulation Research, 113(7). doi:10.1161/circresaha.113.301971Moreno, J. D., Zhu, Z. I., Yang, P.-C., Bankston, J. R., Jeng, M.-T., Kang, C., … Clancy, C. E. (2011). A Computational Model to Predict the Effects of Class I Anti-Arrhythmic Drugs on Ventricular Rhythms. Science Translational Medicine, 3(98). doi:10.1126/scitranslmed.3002588Moss, A. J., Windle, J. R., Hall, W. J., Zareba, W., Robinson, J. L., McNitt, S., … Manalan, A. S. (2005). Safety and Efficacy of Flecainide in Subjects with Long QT-3 Syndrome (DeltaKPQ Mutation): A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Annals of Noninvasive Electrocardiology, 10(s4), 59-66. doi:10.1111/j.1542-474x.2005.00077.xMOSS, A. J., ZAREBA, W., SCHWARZ, K. Q., ROSERO, S., MCNITT, S., & ROBINSON, J. L. (2008). Ranolazine Shortens Repolarization in Patients with Sustained Inward Sodium Current Due to Type-3 Long-QT Syndrome. Journal of Cardiovascular Electrophysiology, 19(12), 1289-1293. doi:10.1111/j.1540-8167.2008.01246.xO’Hara, T., Virág, L., Varró, A., & Rudy, Y. (2011). Simulation of the Undiseased Human Cardiac Ventricular Action Potential: Model Formulation and Experimental Validation. PLoS Computational Biology, 7(5), e1002061. doi:10.1371/journal.pcbi.1002061Paul, A. A., Witchel, H. J., & Hancox, J. C. (2002). Inhibition of the current of heterologously expressed HERG potassium channels by flecainide and comparison with quinidine, propafenone and lignocaine. British Journal of Pharmacology, 136(5), 717-729. doi:10.1038/sj.bjp.0704784Penniman, J. R., Kim, D. C., Salata, J. J., & Imredy, J. P. (2010). Assessing use-dependent inhibition of the cardiac Na± current (INa) in the PatchXpress automated patch clamp. Journal of Pharmacological and Toxicological Methods, 62(2), 107-118. doi:10.1016/j.vascn.2010.06.007Postema, P. G., De Jong, J. S. S. G., Van der Bilt, I. A. C., & Wilde, A. A. M. (2008). Accurate electrocardiographic assessment of the QT interval: Teach the tangent. Heart Rhythm, 5(7), 1015-1018. doi:10.1016/j.hrthm.2008.03.037Priori, S. G., Blomström-Lundqvist, C., Mazzanti, A., Blom, N., Borggrefe, M., Camm, J., … Van Veldhuisen, D. J. (2015). 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death. European Heart Journal, 36(41), 2793-2867. doi:10.1093/eurheartj/ehv316Rivolta, I., Abriel, H., Tateyama, M., Liu, H., Memmi, M., Vardas, P., … Kass, R. S. (2001). Inherited Brugada and Long QT-3 Syndrome Mutations of a Single Residue of the Cardiac Sodium Channel Confer Distinct Channel and Clinical Phenotypes. Journal of Biological Chemistry, 276(33), 30623-30630. doi:10.1074/jbc.m104471200Romero, L., Cano, J., Gomis-Tena, J., Trenor, B., Sanz, F., Pastor, M., & Saiz, J. (2018). In Silico QT and APD Prolongation Assay for Early Screening of Drug-Induced Proarrhythmic Risk. Journal of Chemical Information and Modeling, 58(4), 867-878. doi:10.1021/acs.jcim.7b00440Romero, L., Trenor, B., Yang, P.-C., Saiz, J., & Clancy, C. E. (2015). In silico screening of the impact of hERG channel kinetic abnormalities on channel block and susceptibility to acquired long QT syndrome. Journal of Molecular and Cellular Cardiology, 87, 271-282. doi:10.1016/j.yjmcc.2015.08.015Rudy, Y., & Silva, J. R. (2006). Computational biology in the study of cardiac ion channels and cell electrophysiology. Quarterly Reviews of Biophysics, 39(1), 57-116. doi:10.1017/s0033583506004227Saint, D. A. (2008). The cardiac persistent sodium current: an appealing therapeutic target? British Journal of Pharmacology, 153(6), 1133-1142. doi:10.1038/sj.bjp.0707492Smallwood, J., Robertson, D., & Steinberg, M. (1989). Electrophysiological effects of flecainide enantiomers in canine Purkinje fibres. Naunyn-Schmiedeberg’s Archives of Pharmacology, 339(6). doi:10.1007/bf00168654Sobie, E. A. (2009). Parameter Sensitivity Analysis in Electrophysiological Models Using Multivariable Regression. Biophysical Journal, 96(4), 1264-1274. doi:10.1016/j.bpj.2008.10.056Soltis, A. R., & Saucerman, J. J. (2010). Synergy between CaMKII Substrates and β-Adrenergic Signaling in Regulation of Cardiac Myocyte Ca2+ Handling. Biophysical Journal, 99(7), 2038-2047. doi:10.1016/j.bpj.2010.08.016Trenor, B., Cardona, K., Gomez, J. F., Rajamani, S., Ferrero, J. M., Belardinelli, L., & Saiz, J. (2012). Simulation and Mechanistic Investigation of the Arrhythmogenic Role of the Late Sodium Current in Human Heart Failure. PLoS ONE, 7(3), e32659. doi:10.1371/journal.pone.0032659Yang, P.-C., DeMarco, K. R., Aghasafari, P., Jeng, M.-T., Dawson, J. R. D., Bekker, S., … Clancy, C. E. (2020). A Computational Pipeline to Predict Cardiotoxicity. Circulation Research, 126(8), 947-964. doi:10.1161/circresaha.119.316404Yang, P.-C., El-Bizri, N., Romero, L., Giles, W. R., Rajamani, S., Belardinelli, L., & Clancy, C. E. (2016). A computational model predicts adjunctive pharmacotherapy for cardiac safety via selective inhibition of the late cardiac Na current. Journal of Molecular and Cellular Cardiology, 99, 151-161. doi:10.1016/j.yjmcc.2016.08.011Yang, P., Moreno, J. D., Miyake, C. Y., Vaughn‐Behrens, S. B., Jeng, M., Grandi, E., … Clancy, C. E. (2015). In silico prediction of drug therapy in catecholaminergic polymorphic ventricular tachycardia. The Journal of Physiology, 594(3), 567-593. doi:10.1113/jp271282Zhu, W., Mazzanti, A., Voelker, T. L., Hou, P., Moreno, J. D., Angsutararux, P., … Silva, J. R. (2019). Predicting Patient Response to the Antiarrhythmic Mexiletine Based on Genetic Variation. Circulation Research, 124(4), 539-552. doi:10.1161/circresaha.118.31405