Abstract Body (Do not enter title and authors here): Background: We identified a novel, ultra-rare missense variant (p.R534S) in DENND3 as a potential disease-susceptibility variant in a multi-generational pedigree of familial idiopathic ventricular fibrillation (IVF). DENND3 is a guanine nucleotide exchange factor and activator for small GTPase Rab proteins involved in endosome and ion channel membrane trafficking. However, the role of DENND3 in the heart and its potential involvement in cardiac arrhythmias are unknown.

Objective: To functionally characterize DENND3-p.R534S and determine its impact on channel trafficking.

Methods: Heterologous co-expression studies in TSA201 cells were performed with DENND3-WT or DENND3-p.R534S and ion channels: KCNQ1 (Kv7.1, IKs), KCNH2 (Kv11.1, IKr), SCN5A (Nav1.5, INa), or CACNA1C (Cav1.2, ICa) as well as Rab proteins (Rab4, Rab5, Rab6, Rab7, Rab11, Rab12). Patient-specific DENND3-p.R534S iPSC-derived cardiomyocytes (iPSC-CMs) and isogenic controls (IC) were created. Localization and expression of ion channel and Rab proteins was assessed using immunofluorescence (IF) by super resolution AiryScan microscopy. GTPase-Glo assays measured Rab protein activity. Electrophysiology studies using patch-clamp and FluoVolt were performed on iPSC-CM models.

Results: IF imaging showed significant increases in membrane expression of KCNQ1 (11714±157to 21929±378, p<0.05), KCNH2 (11378±160 to 31965±544, p<0.05), , SCN5A (10339±8 to 11225±23, p<0.05), and CACNA1C (11879±83 to 13749±138, p<0.05) in DENND3-p.R534S compared to DENND3-WT expressing TSA201 cells. Rab5 distribution was altered significantly by the DENND3-p.R534S variant, with increased dispersion away from the nucleus (0.23±0.01 vs. 0.17±0.01, p<0.05) and increased proximity to the plasma membrane (0.16±0.02 vs. 0.23±0.01, p<0.05). Additionally, Rab5 activity was enhanced significantly (57% increase) by p.R534S versus WT (p<0.05). iPSC-CMs revealed similar increases in channel membrane accumulation and irregular arrhythmic activity including frequent action potential duration alternans and afterdepolarizations in 78% of p.R534S iPSC-CMs versus none in IC cells (p<0.05).

Conclusion: DENND3 is a novel ion channel trafficking protein in the heart. The DENND3-p.R534S variant disrupts cardiac ion channel trafficking. These findings highlight DENND3’s role in cardiac ion channel distribution, cellular function, and potential association with arrhythmogenic risk.