Abstract Body (Do not enter title and authors here): Background: Previously, we discovered that DENND3, a guanine nucleotide exchange factor regulating Rab GTPases, influences cardiac ion channel trafficking. Both electrophysiological dysfunction and arrhythmic events were identified in variant inserted inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) expressing DENND3-p.R534S. This was a novel missense variant identified in a patient with familial idiopathic ventricular fibrillation (IVF). Notably, augmented membrane retention of key cardiac ion channels was observed in both DENND3-p.R534S expressing TSA201 cells and variant inserted iPSC-CMs. To further study the mechanisms underlying arrhythmogenesis associated with IVF, intracellular calcium handling was characterized in these re-engineered heart cells with DENND3-p.R534S.

Methods: Here, iPSC-CMs expressing DENND3-p.R534S and isogenic controls (IC) were studied. Optical action potentials were recorded using the FluoVolt voltage-sensitive dye at a constant pacing rate of 1 Hz. Multi-electrode array (MEA) recordings assessed beat period and conduction dynamics. Intracellular calcium dynamics were analyzed using Fluo-4 calcium imaging.

Results: DENND3-p.R534S iPSC-CMs exhibited frequent arrhythmic activity, including irregular beating patterns (78%, n=28/36), alternans (25%), early afterdepolarizations (EADs, 8%), and delayed afterdepolarizations (DADs, 8%) versus 0% in IC lines (n=0/36, p<0.05). MEA recordings revealed significantly prolonged and irregular beat periods (2.59±0.26s vs. 1.37±0.10s, p<0.05), indicating electrical instability. Calcium imaging further showed significantly reduced calcium transient amplitude in DENND3-p.R534S iPSC-CMs compared to ICs (0.035±0.039ΔF/F₀ vs. 0.302±0.099ΔF/F₀, p<0.05), and slowed upstroke velocity (0.170±0.212 ΔF/F₀●s^-1 vs. 1.340±0.463ΔF/F₀●s^-1) suggesting impaired intracellular calcium release.

Conclusion: The DENND3-p.R534S variant not only disrupts ion channel distribution as shown previously but also markedly perturbs intracellular calcium handling, contributing to electrophysiological instability and arrhythmogenic cellular phenotypes. These findings highlight DENND3’s novel and emerging role in maintaining cardiac cellular homeostasis and rhythm stability.