Sudden cardiac death (SCD) caused by ventricular tachyarrhythmias (VT/VF) remains a major cause of mortality and morbidity accounting for up to 20% of all deaths in the U.S. Despite numerous advances, available antiarrhythmic drugs failed to reduce mortality. Implantable cardioverter defibrillators (ICDs) remain the main therapy VT/VF. However, ICDs cannot prevent VT/VF, only terminate it. Thus, novel antiarrhythmic targets and novel relevant translational models of SCD are needed for arrhythmia research. This Basic Project will use the Efimov laboratory’s world-recognized capabilities in studying the electrophysiology, metabolism, and contractile properties of the human heart ex vivo acutely. This basic project focuses on testing the hypothesis that RyR2 hyperactivity constitutes a molecular mechanism for triggers and substrates of VT/VF in the human heart. Structural heart disease leads to significant transcriptional, translational and post-translational remodeling, which all converge during progression to HF on metabolic, electrophysiological and mechanical dysfunction. We and others have demonstrated that, despite significant differences in etiologies of HF, Ca signaling is dysregulated in cardiomyopathy. The hypotheses we propose to test here in the ex vivo human heart mirror and complement those being investigated in the Knollmann and Stevenson Projects: Aim 1. To test the hypothesis that RyR2 hyperactivity generates arrhythmogenic triggers in the human donor ventricle. Experiments will utilize coronary perfused left and right ventricular (LV/RV) preparations and organotypic slices from human donor hearts to investigate the effect of pharmacological or genetic RyR2 modulation on delayed and early afterdepolarizations induced by catecholamine application. Aim 2. To test the hypothesis that RyR2 hyperactivity generates an arrhythmogenic substrate in the human donor ventricle. Using the same human preparations, experiments will determine whether increasing RyR2 activity produces conduction slowing and/or shortening of repolarization, generating proarrhythmic wavelength shortening. Aim 3: To test the hypothesis that pharmacological RyR2 suppression inhibits arrhythmogenesis in human ventricles with structural heart disease. Using myopathic human preparations, experiments will investigate the effect of RyR inhibitors dantrolene and ent-verticilide on arrhythmogenic triggers and substrate.