Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

Abstract

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pH_i, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pH_i and decrease in [Cl^-]_i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

Fig. 1

Pedigrees and representative ECG recordings in two families with SQTS. a Pedigree of family 1 and 2. Proband marked with an arrow. b ECG recording showing ventricular fibrillation and DC shock conversion to sinus rhythm in the proband in family 1. c ECG-12 from the proband in family 1. d ECG-12 from patient III-2 in family 2

Fig. 2

Immunoblot of AE3 and intracellular pH in HEK-293 cells. a Representative immunoblots of total and membrane localized (biotinylated) AE3 protein in AE3 transfected and AE3_R370H transfected HEK-293 cells. Pan-actin was used as control/reference protein. b Average total and membrane localized AE3 protein relative to pan-actin expression (n = 4) (*p < 0.05 as determined by unpaired Student t-test). c Steady state pH_i of AE3 transfected and AE3_R370H transfected HEK-293 cells in CO₂/HCO₃ ⁻ containing media and CO₂/HCO₃ ⁻ free media. d Representative recording of pH in HEK-293 cells. Numbers indicate parts of the protocols used for calculations of net base transport shown in e. e Average net base uptake for AE3 transfected and AE3_R370H transfected HEK-293 cells. Numbers correspond to the numbers given in c (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by unpaired Student t-test. Error bars throughout represent the s.e.m.

Fig. 3

Loss of slc4a3 in zebrafish embryos replicates the QTc shortening observed in patients. a Systolic interval divided by heart cycle duration is significantly reduced in slc4a3 knockdown embryos 2 days post fertilization (dpf). This phenotype is rescued by overexpression of AE3 in knockdown embryos. n = 19 (Control), n = 14 (Knockdown), and n = 19 (Knockdown + AE3) compiled from three independent experiments, each including all three experimental groups. b Reduced systolic interval in slc4a3 knockdown embryos is not rescued by overexpression of R370H-mutated AE3 in knockdown embryos. n = 13 (Control), n = 16 (Knockdown), and n = 15 (Knockdown + AE3_R370H) compiled from three independent experiments, each including all three experimental groups. c A raw ECG recording of a 2 dpf zebrafish embryo; P,Q,R,S,T waves, and RR and QT intervals indicated. d Corrected QT intervals (QTc) for control and slc4a3 knockdown embryos were calculated with Bazett’s formula. n = 12 (Control) and n = 11 (Knockdown) compiled from three independent experiments, each including both experimental groups. e pH_i is significantly increased in hearts from slc4a3 knockdown embryos 2 days post fertilization (dpf). n = 16 (Control), n = 14 (Knockdown). ^* p < 0.05, **p < 0.01, ***p < 0.001 as determined by one way ANOVA with Tukey’s post-test (a, b) or unpaired student t-test (d, e). Error bars throughout represent the s.e.m.

Fig. 4

Intracellular alkalization and low [Cl⁻]_i shortens the action potential duration and R-T interval. a Action potentials of rabbit hearts measured before and during application of 10 mM NH₄Cl. APD: Action potential duration. b Mean APD during application of NH₄Cl (n = 5). Zero indicates time of intervention. c ECGs in rabbit hearts before and during application of 10 mM NH₄Cl. d Mean RT interval in rabbit hearts (n = 5). In a, c the records are aligned at the pacing stimulus. ***p < 0.001, ****p < 0.0001 as determined by one-way repeated measures ANOVA. Zero indicates time of intervention. e Bar graph showing APD90 of rat ventricular cardiomyocytes patched with 40 mM (open, n = 23) or 10 mM (black, n = 16) chloride in the pipette solution. *p < 0.05 as determined by one-way repeated measures ANOVA. Error bars throughout represent the s.e.m.

Fig. 5

Molecular interaction in direct contact with Arg370. Glu376 is forming a charged hydrogen bond to Arg370 (dashed black lines), while Trp385 is linked through a cation-π interaction (orange dashed line). The figure was prepared with The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC, and the modeling based on an alignment of the AE1 and AE3 sequence in the cytoplasmic region using PDB ID entry 1HYN as template