Impact of Pulse Duration on Cardiac Electroporation: Nanosecond Pulses Enhance Cardiomyocyte Selectivity and Promote a Raman-Detected Shift Toward Apoptotic Cell Death

Abstract Background Pulsed Field Ablation (PFA), a cardiac ablation technique using microsecond pulsed electric fields (µsPEF), is widely used in clinical settings, while nanosecond pulsed electric fields (nsPEF) have recently entered clinical trials. Selective ablation of cardiomyocytes over endothelial cells is critical to prevent adverse remodeling, arrhythmias, and thrombosis, yet comparative data on nsPEF vs. µsPEF remain limited. This study investigates the cytotoxic effects and cell death mechanisms induced by nsPEF and µsPEF in cardiac and endothelial cells. Methods Human cardiomyocytes and endothelial cells were exposed to varying electric field intensities with nsPEF and µsPEF using custom-built automated setup to assess permeabilization and cell death. Raman spectroscopy evaluated biochemical changes in cardiomyocytes following electroporation. Ex vivo epicardial ablation was performed on murine hearts using customized electrodes. Results Maximal cardiomyocyte death occurred 24 hours after both pulse types in vitro. Ex vivo, both pulse types produced visible myocardial lesions as early as 1 hour post-exposure, with lesion size progressively increasing up to 4 hours. µsPEF induced significantly greater endothelial damage (ED50: 1.18 kV/cm) than damage to cardiomyocytes (ED50: 1.28 kV/cm), whereas nsPEF affected both cell types equally (ED50: 7.27 kV/cm vs. 7.24 kV/cm). Raman spectroscopy analysis of exposed cells indicated that µsPEF predominantly triggered necrotic or unregulated cell death, while nsPEF exposure was associated with regulated, apoptotic cell death. Conclusions Pulse duration critically determines electroporation selectivity and downstream death pathways. nsPEF favored regulated cell death and cardiomyocyte selectivity, highlighting its potential to improve the safety and durability of PFA.