The pathomechanism of a novel SLC4A3 variant in a family suffering from short-QT syndrome

Z. Meng (Mannheim)¹, B. Kovacs (Zürich)², C. Yan (Mannheim)¹, C. Hoelscher (Mannheim)³, N. L. Rehbehn (Mannheim)³, L. Cyganek (Göttingen)⁴, X. Fan (Mannheim)¹, X. Lei (Mannheim)¹, R. Liu (Mannheim)¹, B. Zhao (Mannheim)¹, G. Yang (Mannheim)¹, F. Duru (Zürich)², A. Hohn (Mannheim)³, A. Aweimer (Bochum)⁵, S. Zhazykbayeva (Bochum)⁶, A. Mügge (Bochum)⁷, M. Jarkas (Bochum)⁸, N. Hamdani (Bochum)⁹, A. M. Saguner (Zürich)², X. Zhou (Mannheim)¹, I. Akin (Mannheim)¹, I. El-Battrawy (Bochum)^10
1Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ²UniversitätsSpital Zürich Klinik für Kardiologie Zürich, Schweiz; ³Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; ⁴Universitätsmedizin Göttingen Herzzentrum Göttingen - Stem Cell Unit Göttingen, Deutschland; ⁵Berufsgenossenschaftlliches Universitätsklinikum Bergmannsheil gGmbH Medizinische Klinik II, Kardiologie und Angiologie Bochum, Deutschland; ⁶Ruhr University Bochum Department of Cellular and Translational Physiology Bochum, Deutschland; ⁷Institut für Physiologie, Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Abteilung für Zelluläre und Translationale Physiologie, Molekulare und Experimentelle Kardiologie Bochum, Deutschland; ⁸Ruhr-Universität Bochum Bochum, Deutschland; ⁹Kath. Klinikum Bochum Cellular Physiology Bochum, Deutschland; ¹⁰Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland

Background: Short QT syndrome (SQTS) is a rare channelopathies causing sudden cardiac death (SCD). Investigations of human cellular phenotypes and effective therapies of SQTS linked to SLC4A3 variants remain sparse.

Aims: The aim of this study was to assess the pathogenic roles of a novel SLC4A3 variant, c.(1108C>T) p.(Arg370His) in a family with several members showing SQTS, to explore underlying pathomechanism by using patient-specific and CRISPR-engineered human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and gene-edited HEK-293 cells. and to search for possible effective drugs for treatment of the disease. 

Methods: hiPSC-CMs from SQTS carrying the variant, from independent healthy donors, and from an isogenic cell line with variant-correction (using CRISPR/Cas9) were employed for this study. In addition, gene-edited HEK-293 cells were generated. Patch-clamp and molecular studies were applied.

Results: The intracellular pH value of SQTS cardiomyocytes was higher compared to healthy donor or isogenic control. CRISPR-insertion of the variant in HEK-293 showed also an increase of intracellular PH value consistent with the SQTS cardiomyocytes. Comparing with hiPSC-CMs from the healthy donors or isogenic controls, cardiomyocytes from the SQTS-patient (SQTS-hiPSC-CMs) displayed abnormal action potentials (APs) with a shortening in the action potential duration (APD) at 50% and 90% of repolarization.  To model the SQTS phenotype we increased the intracellular pH value of healthy donor using NH4Cl, which confirmed the  finding showing a reduction of APD50 and APD90. In SQTS cardiomyocytes and healthy donors treated with NH4Cl a loss of function of ICa-L, but not of IKr or IKs was observed. The occurrence of arrhythmic events (abnormal spontaneous calcium transients) was increased in SQTS compared to healthy donors or isogenic cells. The total expression level of SLC4A3 was reduced in immunostaining and western blot in SQTS compared to healthy donor or isogenic control. Even more, the SLC4A3 was translocated to the nucleus in SQTS compared to healthy donors and isogenic control cells. The oxidation level was increased in SQTS compared to healthy donors and isogenic cells, whereas the phosphorylation level was reduced. Quinidine prolonged APD50 and APD90 and reduced the arrhythmic events.

Conclusions: The novel SLC4A3 c.(1108C>T) p.(Arg370His) is linked to the clinical phenotype of SQTS. The variant increases the intracellular pH value, increases the oxidation of SLC4A3, reduces the phosphorylation of SLC4A3 and translocate SLC4A3 to the nucleus, leading to ion channel dysfunction and APD-shortening as well as arrhythmias.