B-YIA2-02 CELLULAR AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF TRIADIN KNOCKOUT SYNDROME USING HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES

Abstract

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. TKOS is characterized by QT prolongation, T-wave inversions in the precordial leads, ectopy upon stress testing, and a severe disease expression of cardiac arrest in childhood. To characterize the cellular and electrophysiological phenotype of TKOS using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were generated from two unrelated patients with TKOS and an unrelated control (TKOS Patient 1: p.D18fs*13/D18fs*13; TKOS Patient 2: p.N9fs*5/K147fs*0). CRISPR/Cas9 was used to insert homozygous p.D18fs*13 into a different healthy control line thereby generating a patient-independent TKOS model (TRDN-/-) and isogenic control iPSC-CMs. Protein expression was measured using immunofluorescence. Action potential duration (APD) and L-type calcium channel (LTCC) properties were measured by whole cell patch-clamp. Calcium handling was assessed using Fluo-4 calcium indicator. APD90 was prolonged significantly from 507±20 ms (n=9) in the unrelated control to 662±62 ms (n=9; P<0.05) in TKOS Patient 1 and 653±39 ms (n=9; P<0.05) in TKOS Patient 2. This was confirmed in TRDN-/- iPSC-CMs compared to isogenic control (518±27 ms, n=17 vs. 312±20 ms, n=16; P<0.0001). Additional work in TRDN-/- iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of RyR2 and Casq2 leading to slow and decreased calcium release from the sarcoplasmic reticulum. These abnormal calcium transients lead to slow inactivation of the LTCC which contributes to the observed APD prolongation. Finally, these changes underlie frequent cellular arrhythmias including early- and delayed afterdepolarizations and APD alternans. Here, we characterized the first set of iPSC-CM models of TKOS and provide further evidence for recessive null variants in TRDN as a self-sufficient monogenetic substrate for potentially lethal genetic heart disease. These cells display APD prolongation, calcium handling abnormalities, and arrhythmias which likely underlie the unique clinical and arrhythmogenic phenotype observed in patients with TKOS.