In silico assessment of drug safety in human heart applied to late sodium current blockers

Drug-induced action potential (AP) prolongation leading to Torsade de Pointes is a major concern for the development of anti-arrhythmic drugs. Nevertheless the development of improved anti-arrhythmic agents, some of which may block different channels, remains an important opportunity. Partial block of the late sodium current (INaL) has emerged as a novel anti-arrhythmic mechanism. It can be effective in the settings of free radical challenge or hypoxia. In addition, this approach can attenuate pro-arrhythmic effects of blocking the rapid delayed rectifying K+ current (IKr). The main goal of our computational work was to develop an in-silico tool for preclinical anti-arrhythmic drug safety assessment, by illustrating the impact of IKr/INaL ratio of steady-state block of drug candidates on “torsadogenic” biomarkers. The O’Hara et al. AP model for human ventricular myocytes was used. Biomarkers for arrhythmic risk, i.e., AP duration, triangulation, reverse rate-dependence, transmural dispersion of repolarization and electrocardiogram QT intervals, were calculated using single myocyte and one-dimensional strand simulations. Predetermined amounts of block of INaL and IKr were evaluated. “Safety plots” were developed to illustrate the value of the specific biomarker for selected combinations of IC50s for IKr and INaL of potential drugs. The reference biomarkers at baseline changed depending on the “drug” specificity for these two ion channel targets. Ranolazine and GS967 (a novel potent inhibitor of INaL) yielded a biomarker data set that is considered safe by standard regulatory criteria. This novel in-silico approach is useful for evaluating pro-arrhythmic potential of drugs and drug candidates in the human ventricle.This work was supported by (1) Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica, (2) Plan Avanza en el marco de la Accion Estrategica de Telecomunicaciones y Sociedad de la Informacion del Ministerio de Industria Turismo y Comercio of Spain (TSI-020100-2010-469), (3) Programa de Apoyo a la Investigacion y Desarrollo (PAID-06-11-2002) de la Universidad Politecnica de Valencia, (4) Programa Prometeo (PROMETEO/2012/030) de la Conselleria d'Educacio Formacio I Ocupacio, Generalitat Valenciana and (5) Gilead Sciences, Ltd.Trénor Gomis, BA.; Gomis-Tena Dolz, J.; Cardona Urrego, KE.; Romero Pérez, L.; Rajamani, S.; Belardinelli, L.; Giles, WR.... (2013). In silico assessment of drug safety in human heart applied to late sodium current blockers. Channels. 7(4):1-14. https://doi.org/10.4161/chan.2490511474Maltsev, V. A., Silverman, N., Sabbah, H. N., & Undrovinas, A. I. (2007). Chronic heart failure slows late sodium current in human and canine ventricular myocytes: Implications for repolarization variability. European Journal of Heart Failure, 9(3), 219-227. doi:10.1016/j.ejheart.2006.08.007Zaza, A., Belardinelli, L., & Shryock, J. C. (2008). Pathophysiology and pharmacology of the cardiac «late sodium current». Pharmacology & Therapeutics, 119(3), 326-339. doi:10.1016/j.pharmthera.2008.06.001Song, Y., Shryock, J. C., Wagner, S., Maier, L. S., & Belardinelli, L. (2006). Blocking Late Sodium Current Reduces Hydrogen Peroxide-Induced Arrhythmogenic Activity and Contractile Dysfunction. Journal of Pharmacology and Experimental Therapeutics, 318(1), 214-222. doi:10.1124/jpet.106.101832Milberg, P., Pott, C., Fink, M., Frommeyer, G., Matsuda, T., Baba, A., … Eckardt, L. (2008). Inhibition of the Na+/Ca2+ exchanger suppresses torsades de pointes in an intact heart model of long QT syndrome-2 and long QT syndrome-3. Heart Rhythm, 5(10), 1444-1452. doi:10.1016/j.hrthm.2008.06.017Jia, S., Lian, J., Guo, D., Xue, X., Patel, C., Yang, L., … Yan, G.-X. (2011). Modulation of the late sodium current by ATX-II and ranolazine affects the reverse use-dependence and proarrhythmic liability of IKrblockade. British Journal of Pharmacology, 164(2), 308-316. doi:10.1111/j.1476-5381.2010.01181.xUNDROVINAS, A. I., BELARDINELLI, L., UNDROVINAS, N. A., & SABBAH, H. N. (2006). Ranolazine Improves Abnormal Repolarization and Contraction in Left Ventricular Myocytes of Dogs with Heart Failure by Inhibiting Late Sodium Current. Journal of Cardiovascular Electrophysiology, 17(s1), S169-S177. doi:10.1111/j.1540-8167.2006.00401.xWu, L., Shryock, J. C., Song, Y., Li, Y., Antzelevitch, C., & Belardinelli, L. (2004). Antiarrhythmic Effects of Ranolazine in a Guinea Pig in Vitro Model of Long-QT Syndrome. Journal of Pharmacology and Experimental Therapeutics, 310(2), 599-605. doi:10.1124/jpet.104.066100MOSS, 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.xSong, Y., Shryock, J. C., Wu, L., & Belardinelli, L. (2004). Antagonism by Ranolazine of the Pro-Arrhythmic Effects of Increasing Late INa in Guinea Pig Ventricular Myocytes. Journal of Cardiovascular Pharmacology, 44(2), 192-199. doi:10.1097/00005344-200408000-00008Belardinelli, 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.112.198887Banyasz, T., Koncz, R., Fulop, L., Szentandrassy, N., Magyar, J., & Nanasi, P. (2004). Profile of IKs During the Action Potential Questions the Therapeutic Value of IKs Blockade. Current Medicinal Chemistry, 11(1), 45-60. doi:10.2174/0929867043456304Hopenfeld, B. (2006). A mathematical analysis of the action potential plateau duration of a human ventricular myocyte. Journal of Theoretical Biology, 240(2), 311-322. doi:10.1016/j.jtbi.2005.09.021Maleckar, M. M., Greenstein, J. L., Giles, W. R., & Trayanova, N. A. (2009). Electrotonic Coupling between Human Atrial Myocytes and Fibroblasts Alters Myocyte Excitability and Repolarization. Biophysical Journal, 97(8), 2179-2190. doi:10.1016/j.bpj.2009.07.054Nygren, A., Fiset, C., Firek, L., Clark, J. W., Lindblad, D. S., Clark, R. B., & Giles, W. R. (1998). Mathematical Model of an Adult Human Atrial Cell. Circulation Research, 82(1), 63-81. doi:10.1161/01.res.82.1.63Viswanathan, P. (1999). Pause induced early afterdepolarizations in the long QT syndrome: a simulation study. Cardiovascular Research, 42(2), 530-542. doi:10.1016/s0008-6363(99)00035-8Wu, R., & Patwardhan, A. (2007). Effects of rapid and slow potassium repolarization currents and calcium dynamics on hysteresis in restitution of action potential duration. Journal of Electrocardiology, 40(2), 188-199. doi:10.1016/j.jelectrocard.2006.01.001Zaniboni, M. (2011). 3D current–voltage–time surfaces unveil critical repolarization differences underlying similar cardiac action potentials: A model study. Mathematical Biosciences, 233(2), 98-110. doi:10.1016/j.mbs.2011.06.008Zaniboni, M. (2012). Late Phase of Repolarization is Autoregenerative and Scales Linearly with Action Potential Duration in Mammals Ventricular Myocytes: A Model Study. IEEE Transactions on Biomedical Engineering, 59(1), 226-233. doi:10.1109/tbme.2011.2170987Zaniboni, M., Riva, I., Cacciani, F., & Groppi, M. (2010). How different two almost identical action potentials can be: A model study on cardiac repolarization. Mathematical Biosciences, 228(1), 56-70. doi:10.1016/j.mbs.2010.08.007Belardinelli, L., Antzelevitch, C., & Vos, M. A. (2003). Assessing predictors of drug-induced torsade de pointes. Trends in Pharmacological Sciences, 24(12), 619-625. doi:10.1016/j.tips.2003.10.002Shah, R. R., & Hondeghem, L. M. (2005). Refining detection of drug-induced proarrhythmia: QT interval and TRIaD. Heart Rhythm, 2(7), 758-772. doi:10.1016/j.hrthm.2005.03.023Hondeghem, L. M., Carlsson, L., & Duker, G. (2001). Instability and Triangulation of the Action Potential Predict Serious Proarrhythmia, but Action Potential Duration Prolongation Is Antiarrhythmic. Circulation, 103(15), 2004-2013. doi:10.1161/01.cir.103.15.2004Mirams, G. R., Cui, Y., Sher, A., Fink, M., Cooper, J., Heath, B. M., … Noble, D. (2011). Simulation of multiple ion channel block provides improved early prediction of compounds’ clinical torsadogenic risk. Cardiovascular Research, 91(1), 53-61. doi:10.1093/cvr/cvr044Obiol-Pardo, C., Gomis-Tena, J., Sanz, F., Saiz, J., & Pastor, M. (2011). A Multiscale Simulation System for the Prediction of Drug-Induced Cardiotoxicity. Journal of Chemical Information and Modeling, 51(2), 483-492. doi:10.1021/ci100423zSuzuki, S., Murakami, S., Tsujimae, K., Findlay, I., & Kurachi, Y. (2008). In silico risk assessment for drug-induction of cardiac arrhythmia. Progress in Biophysics and Molecular Biology, 98(1), 52-60. doi:10.1016/j.pbiomolbio.2008.05.003Mirams, G. R., Davies, M. R., Cui, Y., Kohl, P., & Noble, D. (2012). Application of cardiac electrophysiology simulations to pro-arrhythmic safety testing. British Journal of Pharmacology, 167(5), 932-945. doi:10.1111/j.1476-5381.2012.02020.xSarkar, A. X., & Sobie, E. A. (2011). Quantification of repolarization reserve to understand interpatient variability in the response to proarrhythmic drugs: A computational analysis. Heart Rhythm, 8(11), 1749-1755. doi:10.1016/j.hrthm.2011.05.023CHANG, C., ACHARFI, S., WU, M., CHIANG, F., WANG, J., SUNG, T., & CHAHINE, M. (2004). A novel SCN5A mutation manifests as a malignant form of long QT syndrome with perinatal onset of tachycardia/bradycardia. Cardiovascular Research, 64(2), 268-278. doi:10.1016/j.cardiores.2004.07.007Weirich, J., & Antoni, H. (1998). Rate-dependence of antiarrhythmic and proarrhythmic properties of class I and class III antiarrhythmic drugs. Basic Research in Cardiology, 93(0), s125-s132. doi:10.1007/s003950050236O’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.1002061Wu, L., Ma, J., Li, H., Wang, C., Grandi, E., Zhang, P., … Belardinelli, L. (2011). Late Sodium Current Contributes to the Reverse Rate-Dependent Effect of I Kr Inhibition on Ventricular Repolarization. Circulation, 123(16), 1713-1720. doi:10.1161/circulationaha.110.000661Banyasz, T., Barandi, L., Harmati, G., Virag, L., Szentandrassy, N., Marton, I., … P. Nanasi, P. (2011). Mechanism of Reverse Rate-Dependent Action of Cardioactive Agents. Current Medicinal Chemistry, 18(24), 3597-3606. doi:10.2174/092986711796642355Noble D, Tsien RW. The repolarization process of heart cells. In: DeMello WC, ed. Electrical Phenomena in the Heart. New York: Academic Press, 1972:133-161.Fink, M., Noble, D., Virag, L., Varro, A., & Giles, W. R. (2008). Contributions of HERG K+ current to repolarization of the human ventricular action potential. Progress in Biophysics and Molecular Biology, 96(1-3), 357-376. doi:10.1016/j.pbiomolbio.2007.07.011Goineau, S., Castagné, V., Guillaume, P., & Froget, G. (2012). The comparative sensitivity of three in vitro safety pharmacology models for the detection of lidocaine-induced cardiac effects. Journal of Pharmacological and Toxicological Methods, 66(1), 52-58. doi:10.1016/j.vascn.2012.06.001Wu, L., Guo, D., Li, H., Hackett, J., Yan, G.-X., Jiao, Z., … Belardinelli, L. (2008). Role of late sodium current in modulating the proarrhythmic and antiarrhythmic effects of quinidine. Heart Rhythm, 5(12), 1726-1734. doi:10.1016/j.hrthm.2008.09.008Diness, J. G., Hansen, R. S., Nissen, J. D., Jespersen, T., & Grunnet, M. (2009). Antiarrhythmic effect of IKr activation in a cellular model of LQT3. Heart Rhythm, 6(1), 100-106. doi:10.1016/j.hrthm.2008.10.020Laursen, M., Olesen, S.-P., Grunnet, M., Mow, T., & Jespersen, T. (2011). Characterization of cardiac repolarization in the Göttingen minipig. Journal of Pharmacological and Toxicological Methods, 63(2), 186-195. doi:10.1016/j.vascn.2010.10.001HONDEGHEM, L. M. (2006). Thorough QT/QTc Not So Thorough: Removes Torsadogenic Predictors from the T-Wave, Incriminates Safe Drugs, and Misses Profibrillatory Drugs. Journal of Cardiovascular Electrophysiology, 17(3), 337-340. doi:10.1111/j.1540-8167.2006.00347.xHondeghem, L. M., & Snyders, D. J. (1990). Class III antiarrhythmic agents have a lot of potential but a long way to go. Reduced effectiveness and dangers of reverse use dependence. Circulation, 81(2), 686-690. doi:10.1161/01.cir.81.2.686Bril, A. (1998). Combined potassium and calcium channel antagonistic activities as a basis for neutral frequency dependent increase in action potential duration: comparison between BRL-32872 and azimilide. Cardiovascular Research, 37(1), 130-140. doi:10.1016/s0008-6363(97)00216-2Jurkiewicz, N. K., & Sanguinetti, M. C. (1993). Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent. Specific block of rapidly activating delayed rectifier K+ current by dofetilide. Circulation Research, 72(1), 75-83. doi:10.1161/01.res.72.1.75O’Hara, T., & Rudy, Y. (2012). Quantitative comparison of cardiac ventricular myocyte electrophysiology and response to drugs in human and nonhuman species. American Journal of Physiology-Heart and Circulatory Physiology, 302(5), H1023-H1030. doi:10.1152/ajpheart.00785.2011Stengl, M., Volders, P. G. A., Thomsen, M. B., Spatjens, R. L. H. M. G., Sipido, K. R., & Vos, M. A. (2003). Accumulation of slowly activating delayed rectifier potassium current (IKs) in canine ventricular myocytes. The Journal of Physiology, 551(3), 777-786. doi:10.1113/jphysiol.2003.044040Shah, R. R. (2002). The significance of QT interval in drug development. British Journal of Clinical Pharmacology, 54(2), 188-202. doi:10.1046/j.1365-2125.2002.01627.xNuyens, D., Stengl, M., Dugarmaa, S., Rossenbacker, T., Compernolle, V., Rudy, Y., … Carmeliet, P. (2001). Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome. Nature Medicine, 7(9), 1021-1027. doi:10.1038/nm0901-1021Gautier, M., Zhang, H., & Fearon, I. M. (2008). Peroxynitrite formation mediates LPC-induced augmentation of cardiac late sodium currents. Journal of Molecular and Cellular Cardiology, 44(2), 241-251. doi:10.1016/j.yjmcc.2007.09.007Fearon, I. M., & Brown, S. T. (2004). Acute and chronic hypoxic regulation of recombinant hNav1.5 α subunits. Biochemical and Biophysical Research Communications, 324(4), 1289-1295. doi:10.1016/j.bbrc.2004.09.188Ward, C. A., & Giles, W. R. (1997). Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. The Journal of Physiology, 500(3), 631-642. doi:10.1113/jphysiol.1997.sp022048Saint, D. A. (2009). The cardiac persistent sodium current: an appealing therapeutic target? British Journal of Pharmacology, 153(6), 1133-1142. doi:10.1038/sj.bjp.0707492Zygmunt, A. C., Eddlestone, G. T., Thomas, G. P., Nesterenko, V. V., & Antzelevitch, C. (2001). Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle. American Journal of Physiology-Heart and Circulatory Physiology, 281(2), H689-H697. doi:10.1152/ajpheart.2001.281.2.h689Trenor, 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.0032659Martin, R. L., McDermott, J. S., Salmen, H. J., Palmatier, J., Cox, B. F., & Gintant, G. A. (2004). The Utility of hERG and Repolarization Assays in Evaluating Delayed Cardiac Repolarization: Influence of Multi-Channel Block. Journal of Cardiovascular Pharmacology, 43(3), 369-379. doi:10.1097/00005344-200403000-00007Antzelevitch, C., Belardinelli, L., Zygmunt, A. C., Burashnikov, A., Di Diego, J. M., Fish, J. M., … Thomas, G. (2004). Electrophysiological Effects of Ranolazine, a Novel Antianginal Agent With Antiarrhythmic Properties. Circulation, 110(8), 904-910. doi:10.1161/01.cir.0000139333.83620.5dNoble, D. (2006). Late sodium current in the pathophysiology of cardiovascular disease: consequences of sodium-calcium overload. Heart, 92(suppl_4), iv1-iv5. doi:10.1136/hrt.2005.078782Scirica, B. M., Morrow, D. A., Hod, H., Murphy, S. A., Belardinelli, L., Hedgepeth, C. M., … Braunwald, E. (2007). Effect of Ranolazine, an Antianginal Agent With Novel Electrophysiological Properties, on the Incidence of Arrhythmias in Patients With Non–ST-Segment–Elevation Acute Coronary Syndrome. Circulation, 116(15), 1647-1652. doi:10.1161/circulationaha.107.724880Hoefen, R., Reumann, M., Goldenberg, I., Moss, A. J., O-Uchi, J., Gu, Y., … Lopes, C. M. (2012). In Silico Cardiac Risk Assessment in Patients With Long QT Syndrome. Journal of the American College of Cardiology, 60(21), 2182-2191. doi:10.1016/j.jacc.2012.07.053Silva, J. R., Pan, H., Wu, D., Nekouzadeh, A., Decker, K. F., Cui, J., … Rudy, Y. (2009). A multiscale model linking ion-channel molecular dynamics and electrostatics to the cardiac action potential. Proceedings of the National Academy of Sciences, 106(27), 11102-11106. doi:10.1073/pnas.0904505106STARMER, C. (1991). Lidocaine blockade of continuously and transiently accessible sites in cardiac sodium channels*1. Journal of Molecular and Cellular Cardiology, 23, 73-83. doi:10.1016/0022-2828(91)90026-iRudy, 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/s0033583506004227Clancy, C. E., Zhu, Z. I., & Rudy, Y. (2007). Pharmacogenetics and anti-arrhythmic drug therapy: a theoretical investigation. American Journal of Physiology-Heart and Circulatory Physiology, 292(1), H66-H75. doi:10.1152/ajpheart.00312.2006Zygmunt, A. C., Nesterenko, V. V., Rajamani, S., Hu, D., Barajas-Martinez, H., Belardinelli, L., & Antzelevitch, C. (2011). Mechanisms of atrial-selective block of Na+ channels by ranolazine: I. Experimental analysis of the use-dependent block. American Journal of Physiology-Heart and Circulatory Physiology, 301(4), H1606-H1614. doi:10.1152/ajpheart.00242.2011Nesterenko, V. V., Zygmunt, A. C., Rajamani, S., Belardinelli, L., & Antzelevitch, C. (2011). Mechanisms of atrial-selective block of Na+ channels by ranolazine: II. Insights from a mathematical model. American Journal of Physiology-Heart and Circulatory Physiology, 301(4), H1615-H1624. doi:10.1152/ajpheart.00243.2011Zemzemi, N., Bernabeu, M. O., Saiz, J., Cooper, J., Pathmanathan, P., Mirams, G. R., … Rodriguez, B. (2013). Computational assessment of drug-induced effects on the electrocardiogram: from ion channel to body surface potentials. British Journal of Pharmacology, 168(3), 718-733. doi:10.1111/j.1476-5381.2012.02200.xHille B. Ionic Channels of Excitable Membranes. 2nd ed. Sunderland, MA: Sinauer Associates, 1992.Rajamani, S., Shryock, J. C., & Belardinelli, L. (2008). Rapid Kinetic Interactions of Ranolazine With HERG K+ Current. Journal of Cardiovascular Pharmacology, 51(6), 581-589. doi:10.1097/fjc.0b013e3181799690Rajamani, S., El-Bizri, N., Shryock, J. C., Makielski, J. C., & Belardinelli, L. (2009). Use-dependent block of cardiac late Na+ current by ranolazine. Heart Rhythm, 6(11), 1625-1631. doi:10.1016/j.hrthm.2009.07.042Bassingthwaighte, J., Hunter, P., & Noble, D. (2009). The Cardiac Physiome: perspectives for the future. Experimental Physiology, 94(5), 597-605. doi:10.1113/expphysiol.2008.044099Quinn, T. A., Granite, S., Allessie, M. A., Antzelevitch, C., Bollensdorff, C., Bub, G., … Delmar, M. (2011). Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): Standardised reporting for model reproducibility, interoperability, and data sharing. Progress in Biophysics and Molecular Biology, 107(1), 4-10. doi:10.1016/j.pbiomolbio.2011.07.001Glukhov, A. V., Fedorov, V. V., Lou, Q., Ravikumar, V. K., Kalish, P. W., Schuessler, R. B., … Efimov, I. R. (2010). Transmural Dispersion of Repolarization in Failing and Nonfailing Human Ventricle. Circulation Research, 106(5), 981-991. doi:10.1161/circresaha.109.204891MALTSEV, V., & UNDROVINAS, A. (2006). A multi-modal composition of the late Na+ current in human ventricular cardiomyocytes. Cardiovascular Research, 69(1), 116-127. doi:10.1016/j.cardiores.2005.08.015Drouin, E., Charpentier, F., Gauthier, C., Laurent, K., & Le Marec, H. (1995). Electrophysiologic characteristics of cells spanning the left ventricular wall of human heart: Evidence for presence of M cells. Journal of the American College of Cardiology, 26(1), 185-192. doi:10.1016/0735-1097(95)00167-xBerecki, G., Zegers, J. G., Bhuiyan, Z. A., Verkerk, A. O., Wilders, R., & Van Ginneken, A. C. G. (2006). Long-QT syndrome-related sodium channel mutations probed by the dynamic action potential clamp technique. The Journal of Physiology, 570(2), 237-250. doi:10.1113/jphysiol.2005.096578Valdivia, C. R., Chu, W. W., Pu, J., Foell, J. D., Haworth, R. A., Wolff, M. R., … Makielski, J. C. (2005). Increased late sodium current in myocytes from a canine heart failure model and from failing human heart. Journal of Molecular and Cellular Cardiology, 38(3), 475-483. doi:10.1016/j.yjmcc.2004.12.012Belardinelli, L. (2006). Inhibition of the late sodium current as a potential cardioprotective principle: effects of the late sodium current inhibitor ranolazine. Heart, 92(suppl_4), iv6-iv14. doi:10.1136/hrt.2005.07879