PO-615-03 MUTANT ATRIAL NATRIURETIC PEPTIDE INDUCES MITOCHONDRIAL DEFECTS AND ION CHANNEL REMODELING IN A HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED ATRIAL FIBRILLATION MODEL

Abstract

We identified a mutation in NPPA, encoding atrial natriuretic peptide (ANP), as the first non-ion channel gene causing familial atrial fibrillation (AF). However, lack of a mature and comprehensive model prevents a deeper understanding of the underlying cellular mechanisms. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) are ideally suited for modeling heritable AF and personalized pharmacological therapy, but there is limited data on the maturation of iPSC-atrial (a) CMs. We hypothesized that an electro-metabolic maturation (EMM) approach using biochemical cues (T3, IGF-1, dexamethasone), bioenergetic supplement (fatty acids), and electrical stimulation, synergistically promotes metabolic, structural, and electrophysiological maturity of iPSC-aCMs to a level comparable to human atrial cardiomyocytes (haCMs) and will reveal the novel underlying cellular mechanisms of the NPPA mutation. We applied our EMM approach to iPSC-aCMs and compared the metabolic (Seahorse Analyzer, western blots [WB], RT-PCR), structural (immunofluorescence, WB), electrophysiological (patch clamping, optical voltage mapping), and transcriptomic (RNA-seq) maturity with immature iPSC-aCMs and adult haCMs from the same patient. EMM matured iPSC-aCMs from a family carrying the NPPA-S64R mutation, and an isogenic control using CRISPR-Cas9, elucidated the novel underlying mechanism by which the mutation causes AF. EMM iPSC-aCMs displayed improved sarcomeric organization (Fig. 1a) and oxidative capacity (Fig. 1b), as well as more hyperpolarized RMP and increased APA (Fig. 1c,d). Only mature NPPA-S64R iPSC-aCMs demonstrated that the NPPA mutation caused mitochondrial defects resulting in reduced oxidative capacity and electron transport chain dysfunction (Fig. 2a), as well as electrophysiological remodeling causing prolonged APD (Fig. 2b,c) via increased IKs density (Fig. 2d-g). We established a combinatorial EMM approach that promoted comprehensive maturation of iPSC-aCMs. Using this maturation approach, we unmasked the novel electrophysiological remodeling and mitochondrial dysfunction underlying an AF-causing NPPA mutation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)