From single-cell to tissue: hiPSC-based modeling uncovers electrophysiological mechanisms and pharmacological modulation in Short QT Syndrome type 5

Z. Xue (Mannheim)¹, F. Liu (Mannheim)¹, Z. Meng (Mannheim)¹, L. Cyganek (Göttingen)², N. Hamdani (Bochum)³, X. Zhou (Mannheim)¹, I. El-Battrawy (Bochum)⁴, I. Akin (Mannheim)^1
1Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ²Universitätsmedizin Göttingen Herzzentrum Göttingen - Stem Cell Unit Göttingen, Deutschland; ³Kath. Klinikum Bochum Cellular Physiology Bochum, Deutschland; ⁴Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland

Background:
Short QT syndrome (SQTS) is a rare inherited channelopathy characterized by abnormal ion channel function and shortened cardiac repolarization, predisposing patients to malignant arrhythmias and sudden cardiac death (SCD). Eight genetic subtypes of SQTS have been identified; however, detailed mechanisms and effective therapies remain limited. SQTS type 5 (SQTS5), caused by CACNB2 mutations, is exceptionally uncommon, with poorly defined electrophysiological features and limited treatment options. Advances in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and gene editing have provided platforms for mechanistic studies and drug screening in inherited arrhythmias. Increasing evidence suggests that anti-arrhythmic drugs exhibit genotype-dependent efficacy, offering new opportunities to understand and manage SQTS5.

Objective:
To establish a patient-specific hiPSC model of SQTS5 to characterize its electrophysiological phenotype, explore pharmacological modulation, identify anti-arrhythmic drugs that prolong action potential duration (APD) and reduce arrhythmic risk, and elucidate underlying mechanisms.

Methods:
hiPSCs carrying a pathogenic CACNB2 variant were generated from a symptomatic SQTS5 patient and differentiated into cardiomyocytes. Isogenic control lines were created through CRISPR/Cas9-mediated correction. Electrophysiological mechanisms were investigated using patch-clamp and calcium transient analyses. Tissue-level conduction, repolarization, and susceptibility to reentrant arrhythmias were assessed in two-dimensional hiPSC-derived cardiac cell sheets (hiPSC-CCSs) by optical mapping. Carbachol and epinephrine were applied to provoke arrhythmia-associated events. Systematic drug screening identified compounds that normalized the electrophysiological phenotype.

Results:
SQTS5-hiPSC-CMs showed markedly shortened APD and reduced L-type calcium current (I_Ca-L) due to delayed activation, slower recovery, and enhanced inactivation compared with corrected cells. At the tissue level, SQTS5-CCSs exhibited shortened APD and wavelength but preserved conduction and increased susceptibility to reentry. Among seven tested anti-arrhythmic drugs, only disopyramide significantly prolonged APD by enhancing I_Ca-L and inhibiting rapid (I_Kr) and slow (I_Ks) delayed rectifier potassium currents, effectively suppressing arrhythmia-like events. In 2D tissues, disopyramide prolonged APD and wavelength, restoring them to control levels and reducing reentrant arrhythmias. It showed minimal effects in corrected lines, indicating genotype-dependent efficacy.

Conclusions:
This patient-specific hiPSC model of SQTS5 recapitulates the phenotype and enables mechanism-based drug testing. Disopyramide restores repolarization and suppresses arrhythmic activity, supporting its potential therapeutic use in SQTS5.

Keywords: Short QT syndrome; CACNB2; hiPSCs; Cardiomyocyte; Cardiac cell sheets; Electrophysiology; Anti-arrhythmic drugs.