Highlight
• Novel SLC4A3 mutations (p.Arg370Cys and p.Lys531Thr) cause short QT syndrome (SQTS) through loss-of-function effects.
• hiPSC-derived cardiomyocytes from affected patients exhibit shortened action potential duration, intracellular alkalinization, and increased arrhythmogenic delayed afterdepolarizations.
• Decreased L-type calcium current and increased sodium-calcium exchange current underlie electrophysiological alterations.
• Pharmacologic agents quinidine and sotalol can restore action potential duration and reduce arrhythmia-like events in mutant cardiomyocytes.
Study Background
Short QT syndrome (SQTS) is a rare, inherited cardiac channelopathy characterized by abnormally short ventricular repolarization reflected as a shortened QT interval on electrocardiogram. SQTS is clinically important because it predisposes affected individuals to ventricular arrhythmias and sudden cardiac death often occurring at a young age, with limited preventive treatments. While mutations in genes encoding potassium and calcium channels have been implicated, the recent identification of SLC4A3 mutations—a gene encoding a Cl-/HCO3- exchanger involved in intracellular pH regulation—has expanded understanding of SQTS genetic heterogeneity. However, the exact cellular mechanisms by which SLC4A3 mutations shorten action potential duration (APD) and increase arrhythmia vulnerability have remained unclear, limiting targeted therapeutic approaches. The present study addresses this knowledge gap by investigating two novel SLC4A3 variants identified in familial SQTS and elucidating their functional effects using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
Study Design and Methods
This translational study employed a comprehensive approach integrating patient-derived hiPSC-CMs, isogenic CRISPR/Cas9 gene-edited control lines, and heterologous expression in HEK 293T cells to dissect the pathophysiology of SLC4A3-related SQTS. hiPSC lines were generated from two index patients harboring SLC4A3 variants p.Arg370Cys and p.Lys531Thr, accompanied by gene correction to create isogenic wild-type controls. Functional electrophysiologic assays included patch-clamp recordings assessing action potential characteristics and ion currents (L-type calcium current, sodium-calcium exchanger current), and calcium transient measurements to evaluate excitation-contraction coupling. Intracellular pH was quantitatively measured to link SLC4A3 transporter dysfunction to cellular alkalinization. Protein structure modeling and immunostaining helped localize variant effects. Additionally, a human cardiac organoid model enabled optical mapping of electrical behavior in tissue context. The study tested effects of alkalinization induced pharmacologically in control cells and evaluated pharmacologic rescue with quinidine and sotalol.
Key Findings
1. Electrophysiologic Phenotype of SLC4A3-SQTS hiPSC-CMs
hiPSC-CMs carrying SLC4A3 mutations displayed significantly shortened action potential duration (APD) compared to isogenic controls. This was consistent across spontaneous action potentials and calcium transients, with mutant cells exhibiting frequent arrhythmia-like events, particularly delayed afterdepolarizations (DADs), not observed in controls.
2. Ion Current Alterations
Patch-clamp experiments revealed a marked reduction in the L-type calcium channel current (ICa-L) in mutant cardiomyocytes, which irreversibly shortens action potential duration and compromises calcium entry crucial for contraction. Conversely, sodium-calcium exchanger (NCX) current (INCX) was significantly elevated, particularly during diastole, promoting DAD formation and arrhythmogenesis.
3. Intracellular Alkalinization and SLC4A3 Dysfunction
Intracellular pH measurements showed pronounced alkalinization in SLC4A3 mutant hiPSC-CMs and in HEK 293T cells expressing mutant SLC4A3 proteins. Given the role of SLC4A3 as a Cl-/HCO3- exchanger, loss-of-function mutations impair acid extrusion, leading to intracellular alkaline shifts.
The experimental induction of alkalinization with NH4Cl in wild-type hiPSC-CMs reproduced the electrophysiologic phenotype of shortened APD, reduced ICa-L, and enhanced INCX, confirming the causal link between intracellular pH disturbance and electrophysiologic remodeling.
4. Arrhythmia Mechanisms
Enhanced INCX during diastole facilitated cellular calcium overload and triggered DADs, providing a mechanistic basis for ventricular arrhythmia susceptibility in SLC4A3-SQTS. The high frequency of DADs strongly correlates with potential for triggered activity underlying sudden cardiac death risk.
5. Pharmacological Modulation
Quinidine and sotalol, class 1 and III antiarrhythmics respectively, prolonged APD and suppressed DAD frequency in mutant cardiomyocytes. These findings suggest these agents may have therapeutic utility in managing arrhythmias associated with SLC4A3-related SQTS.
Expert Commentary
This study provides compelling evidence that SLC4A3 mutations cause SQTS via an unusual mechanism involving intracellular alkalinization consequent to impaired Cl-/HCO3- exchange, which secondarily alters ionic currents integral to cardiac repolarization and arrhythmia vulnerability. The use of patient-derived hiPSC-CMs combined with gene editing and heterologous systems constructs a robust translational model. By illuminating how disruption of pH homeostasis modulates L-type calcium and NCX currents to cause APD shortening and triggered arrhythmias, this work broadens the conceptual framework beyond classical ion channelopathies in SQTS. Moreover, the responsiveness to quinidine and sotalol supports clinical translation, though their effectiveness and safety in this genetic subgroup require further in vivo validation. Potential limitations include the immaturity of hiPSC-CMs compared to adult cardiomyocytes and the need for confirmation of findings in whole heart models. Nonetheless, this study underscores the need to consider intracellular pH regulation as a modifier of cardiac electrophysiology and arrhythmia risk.
Conclusion
Familial short QT syndrome caused by SLC4A3 loss-of-function mutations represents a novel mechanism of cardiac arrhythmogenesis marked by intracellular alkalinization, reduced L-type calcium channel current, and increased sodium-calcium exchanger activity. These changes shorten ventricular action potentials and predispose to delayed afterdepolarizations and lethal ventricular arrhythmias. Patient-specific hiPSC-CM models provide a powerful platform to dissect pathophysiology and screen pharmacologic interventions. Quinidine and sotalol show promise in normalizing electrophysiological disturbances. Future studies should explore targeted therapies correcting intracellular pH disturbances to reduce sudden cardiac death risk in this rare but deadly channelopathy.
