Background. Frequent premature ventricular contractions (PVCs) can cause LV dysfunction (CM), referred to as PVC-cardiomyopathy (PVC-CM). The mechanism responsible and the impact of PVC coupling interval (prematurity) are unclear. Suspected triggers are post-extrasystolic potentiation and LV dyssynchrony. Hypotheses. Our four working hypotheses are: 1) Post-extrasystolic potentiation, associated with PVCs, is a key trigger responsible for PVC-CM. LV dysfunction (Aim 1), impaired EC coupling and dyad remodeling (Aim 2) are greater in early- rather than late-coupled PVCs since this phenomenon is more prominent in early- rather than late-coupled PVCs; 2) Impaired EC coupling in PVC-CM is due to changes in JPH-2 and Cav1.2 pathways (Fig.1); 3) Early changes in dyad, JPH-2 and Cav1.2 pathways precede the development of this CM and the recovery of LV function after PVC cessation, providing evidence that these changes are a primary cause of CM (Aim 2); and 4) Baseline clinical and structural / molecular phenotype can identify those animals that will develop PVC-CM when exposed to 33% PVC burden. Aim 1. Evaluate the impact of post-extrasystolic potentiation and PVC coupling interval in the development of LV dysfunction associated with chronic exposure to frequent PVCs (PVC-CM). Aim 2. Determine the temporal structural and molecular changes responsible for decrease in Ca2+ release and Junctophylin-2, and their role on the pathophysiology of PVC-CM and recovery upon PVC cessation. Aim 3. Identify baseline echocardiographic, hemodynamic and molecular features that can predict the development of, or resilience to PVC-CM despite identical ventricular ectopy. Methods. 56 animals will undergo pacemaker implant to reproduce frequent ventricular ectopy (PVCs). They will be randomized to one of 4 groups: 1) early-coupled 50% PVCs (n=13), 2) late-coupled 50% PVCs (n=13), 3) early-coupled PVCs 33% PVCs (n=18), or 4) control (n=12) groups. At the end of a 12-week PVC period, a recovery phase (disabling PVCs) will be allowed in 6 animals of each group exposed to 50% burden and sham group (Fig. 6). Serial cardiac evaluation and biopsies will allow us to assess LV function, dyad structure, Ca2+ transients (EC coupling), changes in JPH-2 and Cav1.2 expression, function and distribution and their mediators at baseline and different time points of PVC-CM in all groups. Significance. This study will: 1) provide an understanding of the role of post-extrasystolic potentiation and LV dyssynchrony in the mechanism of PVC-CM; 2) assess the impact of PVC coupling interval in the development or severity of PVC-CM, 3) identify molecular mechanisms behind impaired EC coupling in PVC-CM and 4) identify baseline clinical and molecular phenotypes that distinguish patients at risk to develop PVC-CM. Understanding the mechanism of PVC-CM will help us to identify high-risk patients for development of PVC- CM, but most importantly find future targets to prevent and treat subjects with PVC-...