Abstract Body: Wolff-Parkinson-White (WPW) syndrome is characterized by an accessory pathway bypassing normal atrioventricular conduction, predisposing patients to ventricular preexcitation, atrioventricular reentrant tachycardia, and atrial fibrillation (AF). While PRKAG2 mutation in familial WPW can drive cardiac glycogen overload and cardiomyocyte hypertrophy, most WPW patients lack PRKAG2 mutations, suggesting a non-monogenic etiology. To investigate origins of arrhythmogenicity, human induced pluripotent stem cells (hiPSCs) were generated from a non-PRKAG2 WPW patient with AF and differentiated into atrial and ventricular cardiomyocytes (CMs). Cardiac microtissues were produced using 95% hiPSC-CMs and 5% primary cardiac fibroblasts and cultured in a metabolic maturation medium for 1 week. Atrial WPW CMs were 88.4% larger than healthy atrial control (WTC11) CMs, and periodic acid-Schiff staining confirmed a 5.4% increase in glycogen storage in atrial WPW vs control microtissues, revealing etiologic similarities to monogenic WPW despite the absence of PRKAG2 mutation. Metabolic assessment via 3D Seahorse assay showed reduced basal and ATP-linked respiration in atrial WPW microtissues vs controls, indicating decreased mitochondrial oxidative phosphorylation potentially driving glycolytic compensatory mechanisms. These differences were not observed between ventricular WPW and control CMs, suggesting chamber-specific pathology. Optical mapping of action potential (AP) and calcium transient (CaT) from 4 batches (35 microtissues/mold, 1-2 molds/batch) revealed reduced AP duration (APD80) by 15.6% (p = 0.061) and 39.6% (p < 0.01) in atrial and ventricular WPW microtissues vs controls, respectively. CaT duration decreased by 35% in atrial and 11.2% in ventricular WPW microtissues vs controls, and CaT rise time and tau were also reduced. These data indicate a shorter effective refractory period and increased proarrhythmic risk in WPW CMs. Ongoing work examines ion channel, calcium handling, and metabolic gene expression to assess pathways linking metabolic remodeling with arrhythmia risk. Altogether, this model recapitulates key clinical features and provides insight into metabolic origins of arrhythmogenicity in non-PRKAG2 WPW.