Abstract Body: Background
Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with ventricular arrhythmias and sudden cardiac death. Multiple variants in the desmosomal protein plakophillin-2 (PKP2) are defined as pathogenic for ACM, but genotyping has identified many variants of uncertain significance (VUS). A patient presented with syncope from sustained ventricular tachycardia (VT) at 250 bpm with a right ventricular origin, and the Arrhythmia and Cardiomyopathic Genetic panel showed the PKP2 L709F variant. To determine the pathogenicity of unknown PKP2 variants and investigate mechanistic underpinnings of ACM, a model enabling characterization of tissue-like electrophysiology is needed. Here, we investigated the electrophysiological phenotype of PKP2 L709F using a novel patient-derived induced pluripotent stem cell (iPSC) cardiac tissue chip (CTC) model.

Hypothesis
The PKP2 L709F variant impairs cardiomyocyte intercellular coupling and electrical propagation, resulting in slowed conduction in the CTC model.

Methods
Patient iPSCs were generated from the proband harboring the PKP2 L709F variant, and isogenic control iPSCs were created by CRISPR/Cas9 gene editing. Differentiated iPSC-derived cardiomyocytes (iPSC-CMs) and line-matched iPSC-derived cardiac fibroblasts (iPSC-CFs) were co-cultured on micropatterned PDMS substrates with physiological stiffness (18 kPa). Cultures were maintained in fatty acid-based maturation medium. Electrical conduction was assessed by optical mapping with FluoVolt™ transmembrane voltage-sensitive dye.

Results
PKP2 L709F CTCs exhibited markedly reduced longitudinal conduction velocity compared with isogenic controls (paced at 1 Hz: 6.03 ± 0.53 cm/s vs. 24.30 ± 1.73 cm/s, p < 0.0001). Immunocytochemical analysis revealed fewer connexin-43 (Cx43) puncta per junction in PKP2 L709F CTCs than in controls (2.10 ± 3.35 vs. 16.53 ± 8.72, p < 0.0001). Optical action potential recordings also showed slower upstroke in PKP2 L709F CTCs (paced at 1 Hz: 352 ± 64 a.u./s vs. 590 ± 99 a.u./s, p = 0.01).

Conclusions
Using the CTC model, PKP2 L709F iPSC-CMs demonstrate slowed conduction and reduced Cx43 expression. These findings support the pathogenicity of the PKP2 L709F variant and demonstrate the utility of a patient-derived CTC model for investigating arrhythmogenesis in ACM and potential therapeutic interventions.