Nanosecond Pulse Lengths Enhance the Selectivity of Pulsed Field Ablation for Cardiomyocytes While Minimizing Plasma Membrane Damage

Pulsed Field Ablation (PFA) is a cardiac ablation technique based on electroporation, primarily using bipolar microsecond pulses (µsPFA) in clinical settings. Recently, Nanosecond Pulsed Field Ablation (nsPFA) has entered early clinical trials, but the differences between nsPFA and µsPFA in cardiomyocytes remain poorly understood. Selective ablation of cardiomyocytes is crucial to prevent endothelial cell damage, which could lead to tissue remodeling and arrhythmogenesis. Cardiomyocytes are generally believed to be more sensitive to electroporation than endothelial cells, but the evidence for this sensitivity is limited. This study aims to compare and determine the cytotoxic mechanisms and efficacy of nanosecond and microsecond pulsed electric fields (nsPEF and µsPEF) in cardiac and endothelial cells.

Objective:

The primary objective was to investigate and compare the effects of nsPEF and µsPEF on cardiomyocytes and endothelial cells, focusing on cell-killing efficiency, membrane permeabilization, and the kinetics of ablation lesion formation.

Methods:

In vitro experiments were performed using human and murine cardiomyocytes and endothelial cells. Cells were exposed to varying electric field intensities under nano- and microsecond pulse protocols to assess permeabilization and cell death. The study employed Ca²⁺ transient imaging to evaluate membrane permeabilization. For ex vivo experiments, epicardial ablation was conducted on murine hearts using customized electrodes.

Results:

In vitro, both nsPEF and µsPEF took 24 hours to achieve maximal cell death (p < 0.05). Ex vivo ablation lesions formed 3-3.5 hours earlier with nsPEF than with µsPEF (p < 0.05). While nsPEF demonstrated no significant difference in cell-killing efficiency between cardiomyocytes and endothelial cells (p = 0.7204), µsPEF exhibited a significantly higher vulnerability in endothelial cells (ED50: 1.1 kV/cm) compared to cardiomyocytes (ED50: 1.35 kV/cm, p < 0.001). Both nsPEF and µsPEF permeabilized intracellular compartments, increasing cytoplasmic Ca²⁺ concentrations, although µsPEF was more effective at permeabilizing the plasma membrane in cardiomyocytes. Contrary to common belief, nsPEF was not more efficient in permeabilizing intracellular structures than µsPEF.

Conclusions:

Pulse duration significantly impacts the selectivity of pulsed electric fields. nsPEF showed greater selectivity in targeting cardiac cells, whereas both nsPEF and µsPEF permeabilized intracellular compartments. These findings highlight the importance of optimizing pulse parameters to achieve effective and safe ablation, suggesting that pulse duration influences tissue selectivity during ablation procedures.

Key Words: Nanosecond Pulsed Field Ablation, Microsecond Pulsed Field Ablation, Electroporation, Cardiomyocyte, Permeabilisation, Ca²⁺