Testing a Novel Pulsed Field Ablation Catheter in Mouse Hearts: Lesion Analysis and Cell-Type Identification

Introduction:
Atrial Fibrillation (AF) is one of the most common cardiac arrhythmias worldwide. Current evidence indicates that catheter ablation is superior to antiarrhythmic drugs for restoring and maintaining sinus rhythm in AF patients. Unlike traditional thermal ablation techniques, pulsed field ablation (PFA) uses short, high-voltage electrical pulses to cause irreversible electroporation. This technique offers thus a non-thermal approach that is comparable in procedural efficiency and safety to thermal ablation, and has the advantage of being faster. However, studying the fundamental mechanisms of PFA requires the use of translational models, for which standard catheters (designed for use in patients) are not suitable. The aim of this work was to test a specially designed catheter for PFA in small-scale models – such as mouse hearts and cardiac tissue slices – and to develop an appropriate method for analysing the lesions generated by this catheter.

Methods:
Pulsed electric fields were applied in a Langendorff-perfused mouse heart. Biphasic electric currents were delivered using a PFA generator system (AQUILA, Stockert GmbH) and a 1.25 mm monopolar linear-tip catheter (Stockert GmbH). The heart received two lesions on the left ventricle, followed by a 30 minutes maturation period. A 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution was then perfused for 15 minutes to stain the lesions. Subsequently, the tissue was cryo-embedded and sectioned at a thickness of 30 µm. A similar protocol was performed in 400 µm thick mouse ventricular tissue slices (n=4). Lesion size was measured manually using ImageJ, and with a semi-automatic thresholding method implemented in a self-designed Python script. Immunohistofluorescence imaging was used to identify cell types in the TTC-stained tissue.

Results:
In a perfused mouse heart, local application of pulsed electric fields generated two PFA lesions in the left ventricular tissue. The lesions appeared as faint discolorations, visible within minutes, and clearly identifiable after TTC perfusion, enabling precise lesion measurement. For the two lesions, manual vs. script-based analyses showed maximal depth/lesion area of 1.54 mm/1.81 mm2 vs. 1.39 mm/ 1.69 mm2, and 1.43 mm/ 2.06 mm2 vs. 1.33 mm/1.97 mm2, respectively. The semi-automated method consistently produced slightly lower values, with average differences of approximately 9% for depth and 5% for area. The algorithm appeared to better track fine lesion boundaries, though further analysis is needed to clarify these differences. Processing speed improved by a factor of three, with potential for further optimization. In mouse cardiac slices, the application of pulsed electric fields also resulted in lesions detectable by TTC staining. Confocal imaging of slices labelled with anti-α actinin (for cardiomyocytes) and anti-vimentin antibodies (for nonmyocytes, predominantly fibroblasts) enabled distinction between major cell types. These findings confirm the compatibility of TTC staining with immunofluorescence-based cell type identification in PFA-treated tissue.

Conclusion:
This study demonstrates the feasibility of using custom catheters to induce PFA lesions in small-scale cardiac models and highlights the value of semi-automated quantification for lesion size characterization, allowing faster and standardized assessment.