Abstract 12942: The D801N Variant in ATP1A3 -Encoded Na/K-atpase Alpha 3 Shortens the Cardiac Myocyte Action Potential and Increases Risk for Calcium Mediated Arrhythmias

Background: Alternating hemiplegia of childhood (AHC) is a rare de novo syndrome that manifests with episodic hemiplegia, seizures, dystonia, and, notably, sudden unexplained death. Gene positive patients carry a pathogenic variant in the ATP1A3 -encoded Na/K ATPase alpha 3 isoform (ATP1A3), and the variant D801N is the most common pathogenic variant. Our group recently found that AHC patients with ATP1A3-D801N missense have short QTc and are at risk of ventricular fibrillation.

Hypothesis: We hypothesized that the D801N variant results in reduced ATPase function leading to Ca ²⁺ overload resulting in shortened repolarization time and increased arrhythmogenic risk.

Methods: We differentiated iPSCs into cardiomyocytes from an AHC patient hosting the ATP1A3-D801N variant (iPSC-CM ^D801N ), and an ostensibly healthy individual (iPSC-CM ^WT ). Patch clamp was conducted to determine APD90, and the ratio of cells with delayed after depolarization events (DADs). iPSC-CM ^WT were perfused with 10 ^-7 M ouabain, an ATP1A3 inhibitor, and APD90 and DADs were measured. SR store ([Ca ²⁺ ] _SR ) was measured with Cal-520 dye under 10 mM caffeine. Resting [Ca ²⁺ ] _i was measured with Fura-2 dye, and Na/Ca exchanger (NCX) peak current was measured as Ni ²⁺ sensitive current in a stepwise voltage-clamp protocol.

Results: iPSC-CM ^D801N APD90 was shorter 40% than iPSC-CM ^WT . IPSC-CM ^WT treated with ouabain had 17% shorter APD90 than vehicle. iPSC-CM ^D801N had DADs (3/11 cells) when iPSC-CM ^WT (0/24) did not, and 6/12 ouabain treated iPSC-CM ^WT had DADs vs vehicle treated (0/18). Post caffeine, iPSC-CM ^D801N had higher [Ca ²⁺ ] _SR than iPSC-CM ^WT . Finally, iPSC-CM ^D801N had higher resting [Ca ²⁺ ] _i than iPSC-CM ^WT and iPSC-CM ^D801N had higher peak NCX current density at +80mV but no difference at RMP.

Conclusions: Our results show that both iPSC-CM ^D801N and ouabain-treated iPSC-CM ^WT , have a shorter APD compared to WT controls and exhibit DADs. This is associated with higher NCX-mediated influx of Ca ²⁺ and increased intracellular Ca ²⁺ . Our findings highlight the emerging role of ATP1A3-D801N in creating a myocardium vulnerable to arrhythmias.