Po-455617-2 unravelling the arrhythmogenic mechanisms of short qt syndrome type 3 in an animal model of kir2.1m301k gain-of-function mutation

Short QT Syndrome Type 3 (SQTS3) is an extremely rare, highly arrhythmogenic disease caused by gain-of-function mutations in the KCNJ2 gene coding the inward rectifier potassium channel Kir2.1. We investigated arrhythmogenic mechanisms associated with the mutation Kir2.1M301K in a patient presenting an extremely abbreviated QT interval, paroxysmal atrial fibrillation, and ventricular fibrillation inducibility. To study mechanisms of life-threatening arrhythmias produced by Kir2.1M301K. We tested the hypothesis that, in addition to abolishing IK1 rectification, Kir2.1M301K produces functional defects in Kir2.1 partner proteins, predisposing patients to an arrhythmogenic phenotype. We used intravenous cardiac-specific adeno-associated virus-mediated gene transfer to generate mice expressing wild-type (WT) and M301K mutant channel. We characterized the mice using molecular and cellular biology techniques, electrocardiograms (ECGs), intracardiac stimulation and patch-clamping. We confirmed Kir2.1WT or Kir2.1M301K gene expression specifically in the mouse heart. On ECG, the corrected QT (QTc) interval of Kir2.1M301K mice was significantly shorter than Kir2.1WT mice (p<0.0001), and the QRS complex was prolonged (p<0.01). On intracardiac stimulation, 7 out of 8 Kir2.1M301K mice had inducible ventricular tachycardia/fibrillation of >500ms duration, while none of 10 Kir2.1WT mice were inducible (p=0.0009). Compared with Kir2.1WT, the current/voltage relation of Kir2.1M301K cardiomyocytes showed increased outward IK1 at voltages positive to -80 mV (p<0.0001) with no inward-going rectification. However, inward IK1 was reduced at voltages negative to -80 mV (p=0.006). Unexpectedly, Kir2.1M301K cardiomyocytes had a reduced INa density (p<0.0001) with voltage shifted activation and inactivation. Membrane fractionation and western blot analysis revealed that, while 100% of NaV1.5 channels reached the membrane in Kir2.1WT cardiomyocytes, only 77% did in Kir2.1M301K cardiomyocytes. The Kir2.1M301K mouse recapitulates the arrhythmogenic phenotype of the SQTS3 patient. Kir2.1M301K mutation produces both Kir2.1 and NaV1.5 dysfunction, confirming the reciprocal modulation of these channels in macromolecular complexes. This is the first demonstration that a KCNJ2 gain-of-function mutation modifies NaV1.5 current, thus producing severe arrhythmias by both shortening QT interval and reducing ventricular excitability.