Mechanisms and time course of cell death in pulsed field ablation

Pulsed Field Ablation (PFA) is increasingly used for atrial fibrillation, but the mechanisms of PFA-induced cell death remain incompletely understood. This study aimed to characterize the early temporal changes in structural and molecular markers after cardiac PFA. Epicardial left ventricular ablations were performed using the AblaView System, with lesions created at predefined locations. Hearts were harvested for histopathological and immunohistochemical analysis at 10, 40, and 100 min, while a separate survival cohort was also examined at 1 month. Structural damage and lesion borders were assessed using routine histology and Desmin staining, and regulated cell death pathways, inflammation, and metabolic stress were evaluated via immunohistochemistry. PFA produced lesions with comparable biophysical parameters across all time points. Within minutes, structural markers revealed loss of cardiomyocyte striation, increased intercellular spacing, and disappearance of myocardial anisotropy. RIPK3 and GSDMD increased significantly over time (both p < 0.05), indicating potential activation of necroptotic and pyroptotic pathways, while Caspase-8 showed transient elevation consistent with extrinsic apoptotic signaling. Myeloperoxidase staining increased over time ( p < 0.05), reflecting neutrophil-driven inflammation, and GLUT1 inhibition at 100 min suggested hypoxia and metabolic stress. At 1-month, ablated regions were replaced by fibrotic scar tissue. Overall, PFA was associated with early structural disruption and temporal changes in markers linked to apoptotic, necroptotic, pyroptotic, and inflammatory signaling. These findings provide a descriptive framework for future mechanistic and lesion-validation studies.