Background: Flecainide and other class-Ic antiarrhythmic drugs (AADs) are widely used in Andersen-Tawil syndrome type 1 (ATS1) patients. However, class-Ic drugs might be proarrhythmic in some cases. We investigated the molecular mechanisms of class-I AADs proarrhythmia and whether they might increase the risk of death in ATS1 patients with structurally normal hearts. Methods and Results: Of 53 ATS1 patients reviewed from the literature, 54% responded partially to flecainide, with ventricular arrhythmia (VA) reduction in only 23%. Of the latter patients, VA persisted in 20-50%. Flecainide was ineffective in 23%, and surprisingly, 13.5% suffered a non-fatal cardiac arrest. In five cardiac-specific ATS1 mouse models (Kir2.1Δ314-315, Kir2.1C122Y, Kir2.1G215D and Kir2.1R67W and Kir2.1S136F), flecainide or propafenone (40 mg/Kg i.p.) differentially prolonged the P wave, and the PR, QRS and QTc intervals compared to Kir2.1WT; Kir2.1S136F had milder effects. Flecainide increased VA inducibility in all mutant mice except Kir2.1S136F, which exhibited significant VA reduction. At baseline, Kir2.1G215D cardiomyocytes had the lowest inward rectifier K+ channel (IK1) reduction, followed by Kir2.1C122Y, Kir2.1R67W and Kir2.1S136F. Kir2.1C122Y cardiomyocytes had a significant decrease in sodium inward current (INa). Flecainide (10 μM) slightly increased IK1 density in Kir2.1WT and Kir2.1S136F, while it decreased both IK1 and INa in Kir2.1C122Y and Kir2.1R67W, despite normal trafficking of mutant channels. Optical mapping in ATS1 patient-specific iPSC-CM monolayers expressing Kir2.1C122Y, Kir2.1G215D and Kir2.1R67W showed an increase in rotor incidence at baseline and under flecainide, confirming the drug's proarrhythmic effect. Lastly, in-silico molecular docking predicts that the Kir2.1-Cys311 pharmacophore-binding site is altered in Kir2.1C122Y heterotetramers, reducing flecainide accessibility and leading to channel closure and arrhythmias. Conclusions: Class-Ic AADs are only partially effective and might be proarrhythmic in some ATS1 patients. Kir2.1 mutations impacting the resting membrane potential and cellular excitability create a substrate for life-threatening arrhythmias, raising significant concern about using these drugs in some ATS1 patients.

The authors have declared no competing interest.

FUNDING: Supported by National heart, Lung and Blood Institute, NIH grant number R01HL163943; La Caixa Banking Foundation project code HR18-00304 (LCF/PR/HR19/52160013); grants PI-FIS-2020 # PI20/01220 and PI-FIS-2023 # PI23/01039 from Instituto de Salud Carlos III (ISCIII) and co-funded by Fondo Europeo de Desarrollo Regional (FEDER), and by The European Union, respectively; grant PID2020-116935RB-I00 and BFU2016-75144-R funded by MCIN/AEI/10.13039/501100011033; Fundación La Marató de TV3 (736/C/2020) ?amb el suport de la Fundació La Marató de TV3?; CIBERCV (CB16/11/00458; CB/11/00222 to CV); European Union's Horizon 2020 grant agreement GA-965286; and Program S2022/BMD7229 -CM ARCADIA-CM funded by Comunidad de Madrid; to JJ; Program S2022/BMD-7223 funded by Comunidad de Madrid, to CV; Grant PID2022-137214OB-C21 (to CV), funded by MCIN/AEI/10.13039/501100011033; The imaging studies were performed in the TRIMA@CNIC node of the ICTS ReDIB Grant ICTS-2018-04-CNIC-16 funded by MCIN/AEI /10.13039/501100011033 and ERDF; project EQC2018-005070-P funded by MCIN/AEI /10.13039/501100011033 and FEDER. AIM-M holds an FPU contract (FPU20/01569) from Ministerio de Universidades. JMRR holds an FPU contract (FPU22/03253) from Ministerio de Universidades. LKG holds an FPI contract (PRE2018-083530), Ministerio de Economía y Competitividad de España co-funded by Fondo Social Europeo, attached to Project SEV-2015-0505-18-2. IMC holds a PFIS contract (FI21/00243) funded by Instituto de Salud Carlos III and Fondo Social Europeo Plus (FSE+), ?co-funded by the European Union?. MLVP held contract PEJD-2019-PRE/BMD-15982 funded by Consejería de Educación e Investigación de la Comunidad de Madrid y Fondo Social Europeo. ACKNOWLEDGEMENTS: We thank the CNIC Viral Vectors Unit for producing the AAV9. Confocal experiments were conducted at the CNIC Microscopy and Dynamic Imaging Unit. We thank the CNIC Bioinformatics Unit for generating the in-silico homology modeling simulations, F-function analysis, and helpful discussions. We also thank the Centro de Supercomputación de Galicia (CESGA) for the use of the Finis Terrae III supercomputer to perform molecular dynamics studies. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MICIU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).

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We obtained skin biopsies from patients carrying the Kir2.1C122Y, Kir2.1G215D and Kir2.1R67W mutation after written informed consent, previously approved by local institutional review (2020-411-1, 2020-582-1), according to the Ethics Committee for Research of CNIC and the Carlos III Institute (CEI PI58_2019-v3), Madrid, Spain.

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