PROJECT SUMMARY Atrial Fibrillation (AF) is the most common sustained arrhythmia among adults. In AF, dysfunctional atrial cardiomyocytes (aCMs) and fibrosis within the atrial wall result in abnormal impulse generation and disorganized wave front propagation, preventing a coordinated atrial contraction, ultimately increasing the risk of thromboembolic stroke and heart failure in patients. Hypertension predisposes patients to AF due to the increased afterload, or pressure the heart must work against. In addition, the NLRP3 inflammasome has been shown to be consistently activated in AF patients, however, the mechanism of activation has yet to be explained. Despite its growing prevalence, AF treatments remain inadequate. Clinically available anticoagulants and antiarrhythmic drugs have dangerous side effects and fail to address the causal mechanisms of AF, including the dysfunctional aCMs and fibrosis. Preventative strategies are limited to managing underlying conditions. Given that AF is progressive in nature, preventing its onset in susceptible patients may yield better outcomes and significantly improve patient survival. Therefore, we aim to investigate the mechanisms underlying electrical and structural remodeling seen in afterload-induced AF to identify possible upstream targets. The overall hypothesis is that elevated afterload in the cell-in-gel EHT platform will recapitulate pressure overload seen in chronic hypertension and heart failure. The increase in afterload on our EHT will activate the NLRP3 inflammasome, resulting in CF activation, pro-fibrotic signaling cascades, and electrophysiological and structural remodeling seen in AF development. To achieve this, we will utilize a novel physiologically relevant model of AF. Engineered heart tissue, composed of decellularized human atrial tissue recellularized with hiPSC derived aCMs and cardiac fibroblasts, will recapitulate the heterogeneity, complex structure, and functionality of native atrial myocardium. This tissue will be encased within a stiff polyvinyl alcohol hydrogel that will apply multiaxial stress to it. This will mimic the increased afterload seen in hypertension. This novel platform will provide the field with a new and relevant in vitro model of human AF. We will observe AF-like remodeling in loaded control engineered tissue along with an NLRP3-/- tissue. These experiments will determine the critical roles of afterload and the NLRP3 inflammasome in AF development. This research could elucidate the steady rise in AF occurrence and actively work to curtail its prevalence.