DENND3-p.R534S disrupts dyadic microdomain architecture to drive potentially pro-arrhythmic calcium and electrophysiologic instability

Inherited ventricular arrhythmias (VAs) frequently occur in the absence of pathogenic variants in canonical ion channel genes, suggesting alternative mechanisms of electrical instability. DENND3 is a guanine nucleotide exchange factor that regulates Rab GTPase-mediated trafficking, but its role in cardiac excitation-contraction coupling and membrane microdomain organization remains undefined.

We studied induced pluripotent stem cell-derived cardiomyocytes generated from a CRISPR/CAS9-engineered ultra-rare DENND3-p.R534S variant-inserted line (previously identified in an idiopathic ventricular fibrillation pedigree) and matched isogenic controls. Multielectrode array recordings, live-cell calcium imaging, super-resolution imaging using expansion microscopy, and biochemical analyses were used to assess electrical activity, calcium handling, membrane architecture, and calcium release unit organization. Potentially therapeutic studies were performed using genetic and pharmacologic inhibition of Rab11b. DENND3-p.R534S induced pluripotent stem cell-derived cardiomyocytes exhibited multicellular electrical instability characterized by increased beat-to-beat variability, arrhythmic activity, conduction slowing, and prolonged excitation-contraction delay. These abnormalities were accompanied by heterogeneous and dyssynchronous calcium cycling despite preserved expression of major calcium-handling proteins. Super-resolution imaging revealed disruption of BIN1-dependent membrane architecture and nanoscale uncoupling of Cav1.2 and RyR2. Inhibition of Rab11b restored BIN1 organization, re-established dyadic coupling, normalized calcium cycling, and improved electrical stability.

These findings support a model in which altered trafficking balance contributes to disruption of membrane microdomain organization, leading to dyadic uncoupling, calcium instability, and electrical dysfunction. Modulation of the Rab11b-mediated trafficking pathway restored structural and functional abnormalities, supporting the trafficking-associated pathway as a potential therapeutic target in DENND3-associated ventricular arrhythmia.