SLC4A3-related short QT syndrome assessed in human induced pluripotent stem cell-derived cardiomyocytes: mechanisms of ventricular arrhythmia and sudden cardiac death.

Background And Aims Short QT syndrome (SQTS) is an inherited channelopathy that can cause sudden cardiac death. Recent research has implicated mutations in the SLC4A3 gene as a cause of SQTS, but the mechanisms of shortened action potential duration (APD) and arrhythmia vulnerability have not been described. This study aims to evaluate the underlying pathophysiology causing a shortened APD and ventricular arrhythmia vulnerability in SLC4A3-associated SQTS through mechanistic studies of novel SLC4A3 mutations responsible for familial SQTS.Methods This study evaluated the function and pathophysiology of two novel SLC4A3 variants (p.Arg370Cys and p.Lys531Thr) responsible for SQTS in their respective families. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from each index SQTS patient were developed, as well as an isogenic cell line with variant correction (using CRISPR/Cas9) and HEK 293T cells transfected with SLC4A3 expression constructs expressing either wild type (WT) SLC4A3 or the variants. SLC4A3-SQTS variants were physiologically characterized by patch-clamp analysis, Ca2+ imaging, single cell contraction, intracellular pH measurement, protein structure analyses, immunostaining, and optical mapping studies in a human organoid model.Results SQTS-hiPSC-CMs showed significantly shorter APD and a higher rate of arrhythmia-like events, as recorded by spontaneous action potentials, calcium transient imaging, and rhythmicity of visualized single cell contractions. SQTS-hiPSC-CMs exhibited decreased L-type calcium channel current (ICa-L), and significantly increased Na/Ca exchange current (INCX). Frequent delayed afterdepolarization (DAD) events were recorded from mutant cells but not WT or isogenic cell lines. The intracellular pH value was significantly higher (alkaline) in SQTS-hiPSC-CMs and in transfected heterologous cells expressing mutant SLC4A3, as compared to WT-SLC4A3. Experimental-induced alkalinization of WT-hiPSC-CMs by NH4Cl resulted in shortened APD, enhanced INCX, and reduced ICa-L, similar to observations in cells expressing mutant SLC4A3 proteins. Quinidine and sotalol were found to prolong APD and decrease the occurrence of arrhythmia-like events (DADs) in SQTS-hiPSC-CMs.Conclusions In human cell models, SLC4A3 mutations responsible for SQTS result in loss-of-function leading to intracellular alkalinization, decreased ICa-L, and shortened APD, accounting for the clinical phenotype of short QT. Arrhythmic events in SLC4A3-associated SQTS are provoked by enhanced INCX evoking DADs.