Does The Fractionation of Exposure to Nanosecond Pulsed Electric Fields Matter?

Pamela Weronika Sowa (Tübingen)1, V. Novickij (Vilnius)2, D. Heinzmann (Tübingen)1, J. Schreieck (Tübingen)1, O. Borst (Tübingen)1, M. Gawaz (Tübingen)1

1Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Kreislauferkrankungen Tübingen, Deutschland; 2Vilnius Gediminas Technical University Institute of High Magnetic Fields Vilnius, Lettland


Pulsed field ablation (PFA) is an emerging cardiac ablation method based on the biophysical phenomenon of electroporation. Recent advances in understanding the bioeffects of nanosecond pulsed electric fields (nsPEF) suggest that they could be safer than already introduced into the clinic longer microsecond electric pulses, and more efficient in causing a cytotoxic effect in cardiac cells. Nevertheless, the optimized protocols, including irreversibility thresholds, and cell recovery time-constants are still lacking and the metabolic changes leading to its cytotoxic and anti-arrhythmic effects remain unknown. Here we studied approaches increasing cardiomyocytes' susceptibility to PFA without changing the total delivered energy, focusing on the electrosensitization and the impact of cell orientation in the electric field. 

We applied monolayers of HL-1 murine cardiomyocytes, MHEC 5-T murine endothelial cells, and H9C2 rat cardiomyocytes and constructed a robotic system precisely positioning stimulation electrodes orthogonal to the substrate. Contact electrodes produced the electric field gradually decaying with distance from them, allowing the comparison of cell killing by a range of electric field strengths in a single sample. Moreover, the biochemical reaction resulting from the current flow have been considered and minimized with the adjusted design of electrode array. Rectangular monophasic pulses were delivered by nsPEF and μsPEF generator. We used wide-field fluorescence microscopy for measuring cell viability (by propidium staining at 2- to 24 h after exposure). We determined the electric field distribution generated between different electrodes using the Maxwell equation solver and fit them with stained areas to obtain dose-response curves. 

After a train of 200, 10 kV/cm, 300-ns pulses at 10 Hz, the murine cardiomyocytes death increased from 70% after 2 h to 95% after 6h. Achieving electrosensitization by switching the electroporation protocol to fractionated nsPEF (4x 50, 10kV/cm, 300-ns pulses at 50 sec interval) accelerated time killing peak to 2 hours after ablation. Unexpectedly, endothelial cells showed higher susceptibility to nsPEF with abrupt cell death already 2 h after exposure to pulses. Ablation field of endothelial cells achieved with the same electric field parameters was about 1.7 times greater compared to cardiomyocytes. Finally, evaluating the cell orientation in the electric field we found that H9C2 cells positioned perpendicular to the electric field were more susceptible to PEF, enabling efficient cell killing at a lower electric field.

This new model for the study of cardiac PFA provides novel insights into its electrophysiological characteristics, and facilitates protocol optimization. The results of our study may support future clinical applications of nsPEF for cardiac ablation. 

Support: This research has been made possible by the Junior Clinician Scientist Program (University of Tübingen). 

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