Study On Effect And Mechanism Of Pulsed Electric Field Ablation For Atrial Fibrillation

Objective:Pulsed field ablation（PFA）is a novel non-thermal ablation mode,and its mechanism is irreversible electroporation.Recent studies have shown that PFA has potential advantages in atrial fibrillation ablation,but the parameters and underlying mechanisms of ablation remain unclear.This study explored the effect and mechanism of PFA on myocardial tissue ablation through in vitro and in vivo experiments,and provided experimental evidence for the clinical application of PFA.Methods:In the cell experiment,two kinds of cell ablation systems were established,including the classical single-cell suspension ablation system（electrode cup system）and the self-designed monolayer adherent cell ablation system（culture cell insert system）.Rat embryonic cardiomyocyte H9C2（2-1）was used as the main object of study,rat thoracic aorta smooth muscle cell A7r5 and human neuroblastoma cell SH-SY5Y were used as the comparative object of study.The effects of different ablation parameters including field intensity（250-1250 V/cm）,pulse modes（bidirectional short pulse,unidirectional short pulse,unidirectional long pulse）and ablation energy on PFA injury of cardiomyocytes were evaluated by MTT assay and live/dead cells staining.Ca²⁺flash and intercellular junction protein Cx45 were observed by LSM,and surface scanning electron microscopy was used to observe irreversible electroporation of target cells after PFA treatment.Eighteen Bama pigs（about 50KG）were used to study the safety and effectiveness of PFA ablation of myocardial tissue in vivo.PFA ablation was performed at the left upper pulmonary vein of the left atrium（1000V/cm,1000 pulses,effective time 8000μs）.A4-pole catheter pacing was performed to observe the electrical conduction of pulmonary veins.The end points were 3,7 and 30 days after surgery,respectively.Blood indexes,adjacent tissues and animal survival conditions were observed to evaluate safety.Pathological changes of myocardial tissue were observed by HE,Masson and Tunel staining.Six pigs underwent renal artery ablation at the same time as myocardial ablation to evaluate the effects of different field intensities（1000-2000V/cm）on ablation range and depth.Results:Cell experiments showed that PFA had selective killing effect on cardiomyocytes.MTT results showed that the survival rate of cardiomyocytes was negatively correlated with field intensity.In the monolayer adherent cells ablation system,monolayer cell membrane shrinks and curls from high field intensity to low field intensity.Live/dead cells staining showed that PFA had field-dependent damage to all three kinds of cells.PFA ablation showed obvious selectivity to cardiomyocytes.Compared with the three pulse modes,the ablation effect of bidirectional short pulse was lower than that of unidirectional short and long pulse,which was related to the polarity of monolayer cells.Surface electron microscopy showed that electroporation on the surface of cardiomyocytes was irreversible and the severity was the highest.Ca²⁺scintillation was intense during the ablation of cardiomyocytes,and intercellular interstitial protein Cx45 fluoresced under the condition of monolayer membrane shrinkage.Animal experiments show that PFA is safe and effective in myocardial ablation.All 18experimental pigs survived after PFA ablation without adverse reactions.Intraoperative pacing experiments showed that PFA ablation could block the electrical activity of pulmonary veins.Pathological results 3 days after PFA showed clear boundary of ablation area,deep penetration of ablation wall,ablation myocardial atrophy,increased cell space,infiltration of inflammatory cells,proliferation of fibrous connective tissue,and large-scale apoptosis of ablation myocardium.7 days after PFA,the inflammatory reaction was still significant,and the proliferation of fibrous connective tissue was obvious.Thirty days after surgery,the inflammatory response disappeared,and the ablation area was replaced by a large number of fibrous connective tissue hyperplasia.The results of renal artery ablation showed that the field intensity was positively correlated with the ablation range and intensity of PFA.Conclusion:PFA is highly selective for the ablation of myocardial cells,and PFA is safe and effective in vivo ablation of myocardium.The preclinical study of this study shows that PFA is a safe and effective method for atrial fibrillation ablation,and provides experimental basis and data support for its clinical transformation.