PO-615-05 ACACETIN, A POTENT TRANSIENT OUTWARD CURRENT BLOCKER, MAY BE A NOVEL THERAPEUTIC FOR KCND3-ENCODED KV4.3 GAIN-OF-FUNCTION-ASSOCIATED J-WAVE SYNDROMES

Abstract

The transient outward current (Ito) that mediates early (phase 1) repolarization is conducted by the KCND3-encoded Kv4.3 pore-forming α-subunit. KCND3 gain-of-function mutations have been reported previously as a pathogenic substrate for J wave syndromes (JWS), including the Brugada syndrome (BrS) and early repolarization syndrome (ERS), as well as autopsy-negative sudden unexplained death (SUD). Acacetin, a natural flavone, is a potent Ito current blocker. We hypothesize that Acacetin may be a novel therapeutic for KCND3-mediated JWS. KCND3-V392I was identified in an 18-year-old male with JWS/ERS, and a history of cardiac arrest including ventricular tachycardia/fibrillation and atrial fibrillation/flutter. KCND3-V392I was engineered by site directed mutagenesis and expressed in TSA201 cells. Gene-edited/variant-corrected isogenic control and patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the KCND3-V392I-positive patient were generated. Ito currents and action potentials (APs) were recorded before and after treatment with Acacetin using the whole cell patch-clamp and multielectrode array (MEA) technique. Western blot and immunocytochemistry were performed to investigate KCND3 expression. KCND3-V392I demonstrated a marked gain-of-function phenotype, increasing peak Ito current density by 92% (p<0.05 vs. KCND3-WT) in TSA201 cells and by 61% in Kv4.3-V392I iPSC-CM (p<0.05 vs. isogenic control). KCND3 transcript and translate expression was increased significantly in KCND3-V392I-derived iPSC-CMs (p<0.05 vs. isogenic control). While KCND3-WT revealed an IC50 of 7.2±1.0 μM for Acacetin effect, 30 μM Acacetin dramatically inhibited KCND3-V392I peak Ito current density by 96% (p<0.05 vs. before Acacetin). 10 μM Acacetin, a concentration approaching its IC50 value, inhibited Ito by 54% (P<0.05 vs. before Acacetin) in patient-derived iPSC-CMs and reduced the accentuated AP notch displayed in KCND3-V392I-derived iPSC-CMs. This pre-clinical study provides pharmacological and functional evidence to suggest that Acacetin may be a novel therapeutic for patients with KCND3 gain-of-function-associated JWS by inhibiting Ito and abolishing the accentuated AP notch in patient-derived iPSC-CMs.