The D801N Variant in ATP1A3-encoded Na/K-ATPase Alpha 3 causes Cardiac Arrhythmogenesis through Sodium-Calcium Exchanger-mediated Calcium Overload

Abstract

Background: Short QT syndrome (SQTS) is a heritable form of channelopathy that manifests as a shortened QTc on an electrocardiogram and predisposes to arrhythmias and sudden cardiac death. We have recently found that some patients with alternating hemiplegia of childhood, a rare syndrome that manifests with episodic hemiplegia and sudden unexplained death, have shortened QTc and risk of ventricular fibrillation. These patients carry the most common pathogenic missense D801N in the ATP1A3-encoded Na+/K+ ATPase alpha 3 isoform (ATP1A3). The mechanism of how ATP1A3-D801N results in cardiac disease is unknown. We hypothesized that the D801N variant results in reduced ATPase function leading to Ca2+ overload resulting in shortened repolarization time and increased arrhythmogenic risk. Methods: We leverage an in-silico model of human cardiomyocytes, a knock-in Atp1a3D801N mouse model, and induced pluripotent stem cell-derived cardiac myocytes from two AHC patients hosting the ATP1A3-D801N missense variant (iPSC-CMsD801N) as models. We used whole cell patch clamp, live-cell imaging, molecular expression analysis, mouse telemetry, and mouse intracardiac electrophysiology to determine the mechanism that may underlie the cardiac phenotype seen in AHC patients. Results: In comparison to wild type, iPSC-CMsD801N demonstrated reduced action potential duration (APD), and increased delayed after depolarizations, intracellular Ca2+, and SR Ca2+ store. We found increased Ca2+ influx during positive potential through the Na+/Ca2+ exchanger (NCX1) in iPSC-CMsD801N. Suppression of NCX1 Ca2+ influx with the small molecule ORM-10103 rescued both APD and delayed after depolarizations in iPSC-CMsD801N. In silico, we found that the L-type Ca2+ channel had rapid inactivation with lower Na+/K+ ATPase cell function. In vivo, we found that mice do not express Atp1a3, and that Atp1a3D801N do not have shortened QTc but have increased arrhythmic burden in pre-ictal states. We also found that inhibition of Na+/K+ ATPase with high dose ouabain in wild-type mice increases risk of arrhythmias. Conclusions: Our report demonstrates a Ca2+-mediated mechanism of shortened APD and arrhythmic susceptibility that can be rescued by NCX1 modulation, highlighting the emerging role of ATP1A3-D801N in creating a myocardium vulnerable to arrhythmias. Our in vivo findings address a need for better models that can recapitulate human ATP1A3 cardiac expression to determine the neurovascular interplay that contributes to disease.