Omega-3 fatty acids have long been recognized for their cardiovascular benefits, with extensive evidence demonstrating their role in reducing atherosclerotic cardiovascular events and sudden cardiac death. However, recent clinical trials, including JELIS, REDUCE-IT, and RESPECT-EPA, suggest that EPA (eicosapentaenoic acid), but not DHA (docosahexaenoic acid), provides significant cardiovascular protection. While EPA reduces major cardiovascular events, it has also been consistently linked to an increased risk of atrial fibrillation (AFib), as seen in large-scale studies such as the UK Biobank. At the same time, omega-3 fatty acids, particularly EPA, have been shown to reduce ventricular arrhythmias and sudden cardiac death. The reason for this chamber-specific discrepancy remains unclear. This study aims to investigate the mechanistic basis of omega-3 fatty acid-induced arrhythmogenesis using human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues, which provide a physiologically relevant in vitro model for atrial and ventricular electrophysiology. Using these chamber-specific models, we will determine how EPA promotes pro-arrhythmic electrical remodeling in atrial cells while exerting protective effects in ventricular cells and whether DHA exerts distinct or protective effects in AFib susceptibility. We propose three specific aims: Aim 1: Examine the electrophysiological effects of EPA and DHA on hiPSC-derived atrial and ventricular cardiac microtissues using optical mapping and calcium transient imaging to assess changes in action potential duration, conduction velocity, and arrhythmic susceptibility. Aim 2: Investigate mitochondrial dysfunction and oxidative stress as a mechanism of EPA-induced atrial dysregulation using Seahorse XF assays, mitochondrial membrane potential imaging, and reactive oxygen species (ROS) detection. Aim 3: Identify bioactive lipid-mediated signaling pathways involved in EPA-induced atrial remodeling using mass spectrometry-based lipidomics and metabolomics to profile changes in lipid signaling and metabolic pathways. This study will provide critical mechanistic insights into EPA-induced AFib while addressing the paradox of EPA’s protective effects against ventricular arrhythmias. By clarifying the differential electrophysiological effects of EPA and DHA in atrial vs. ventricular cardiomyocytes, this project will establish the foundation for personalized omega-3 supplementation strategies that maximize cardiovascular benefits while minimizing arrhythmic risks.