Pulsed Field Ablation (PFA) is a new non-thermal ablation method that works by irreversible electroporation. This method has advantages such as no thermal damage, selective ablation of tissue gradients, and fast speed. Recent studies have shown that PFA has potential advantages in the treatment of atrial fibrillation, but the ablation parameters and underlying mechanisms are not clear. This research is still in the initial stage, and the dose-effect relationship of the major cells (cardiomyocytes and smooth muscle cells) in the ablation process by PFA is not clear. The ablation dose and surgical methods in animal experiments, as well as evaluation indicators, need to be studied. This article conducts preliminary experimental research on the killing effect from two aspects: cytology and large animal experiments. It clarifies the dose-effect relationship and evaluation system of pulsed field ablation, and explores the ablation effect and mechanism of PFA on myocardial tissue through in vivo and in vitro experiments, providing experiment evidence for further clinical trials.

Two ablation modes in vitro of single-cell system (ablation in electrode cup) and monolayer cell system (ablation in inserts with electrode tips) were established to perform PFA for myocardial cell H9C2 and smooth muscle cell A7r5. MTT and live/dead cell staining methods were used to evaluate the ablation effects of PFA (field strength range: 250-1250 V/cm, pulse modes: bidirectional short pulse, unidirectional short pulse, and unidirectional long pulse). Calcium staining was used to observe the immediate ablation effect of PFA on PFA, immunofluorescence was used to observe the effect of PFA ablation on the expression of CX45 protein in two types of cells, and scanning electron microscopy was used to observe the microscopic manifestations of cell damage caused by PFA. Three Bama miniature pigs (80 kg) were used to verify the in vivo ablation effect of PFA on myocardial cells, and pacing conduction detection was used to determine the immediate ablation effect. Imaging was used to determine whether there was pulmonary vein stenosis, and HE, Masson staining, and TUNEL staining were used to observe the pathological changes of myocardial cells after PFA ablation.

In the single-cell PFA system, H9C2(2-1) and A7R5 cells showed no significant difference in their response to PFA, and their survival rate depended on the field strength and pulse mode. In contrast, in the monolayer cell system, H9C2(2-1) was significantly more sensitive to PFA than A7R5, and showed a central contraction of the entire cell layer. After ablation, the expression of CX45 protein and intercellular connections in H9C2(2-1) increased. Bidirectional pulses had weaker ablation effects than unidirectional pulses, but there was no difference in cell contraction and intercellular connections among the three pulse modes. Scanning electron microscopy confirmed that irreversible electroporation occurred in both types of cells after PFA ablation. Animal experiments showed that bidirectional pulse PFA could effectively block the electrical activity between the pulmonary vein and the atrium, and there was no significant muscle contraction or pulmonary vein stenosis. Apoptosis of ablated tissue was evident, and connective tissue was undamaged.

Bidirectional pulse PFA can significantly ablate myocardial cells, maintain intercellular connections intact, and reduce muscle contraction. It is an optimized strategy for PFA ablation in atrial fibrillation.