Enhanced Lesion Depth and Preserved Deep Thermal Safety of Sequential Radiofrequency and Pulsed Field Ablation: An Ex Vivo Porcine Heart Study

Background Catheter ablation has become a standard rhythm-control strategy for atrial fibrillation, but creating lesions deep enough to be effective—without risking thermal damage to nearby structures, especially the esophagus and left atrial posterior wall—remains a difficult clinical problem. Radiofrequency ablation (RFA) reliably produces thermal coagulative necrosis, yet the same thermal mechanism puts periesophageal tissue at risk for deep injury. Pulsed field ablation (PFA) works by irreversible electroporation and creates myocardial-selective, non-thermal injury, though the lesions it produces tend to be relatively shallow. Sequential dual-energy ablation, in which RFA and PFA are delivered in the same procedure, could combine the greater depth of thermal coagulation with the tissue selectivity of electroporation. How the order of energy delivery affects lesion formation, deep tissue temperature dynamics, and the cell death mechanisms involved, however, has not been systematically examined. Methods We used an ex vivo porcine heart model to compare lesion formation and deep thermal safety across four ablation strategies: RFA alone (ablation index [AI] 350, 400, 450, and 500), PFA alone (1 or 2 discharges), RFA followed by PFA (RFA + PFA), and PFA followed by RFA (PFA + RFA), with six repetitions per parameter combination (n = 6). Lesion depth and width were measured with triphenyltetrazolium chloride (TTC) staining. Tissue injury patterns and cell death modes were assessed by hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Deep tissue temperature was recorded continuously at 3 mm and 7 mm below the ablation site using PT100 platinum resistance temperature probes (1.5 mm diameter, 2 cm length, accuracy ± 0.1°C, 0.1 s sampling interval); peak temperature (Tmax), temperature rise (dT), maximum heating rate (dT/dt), and the 7 mm-to-3 mm attenuation ratio were calculated as thermal safety indicators. Results In the RFA-only group, lesion size grew as AI increased, and AI 500 produced the deepest and widest lesions. PFA-only lesions were comparatively small but showed the typical injury pattern of electroporation. Sequential dual-energy ablation caused more myocardial damage than either energy alone; the RFA + PFA group (RFA AI 500 + PFA 2 discharges) reached the greatest lesion depth (approximately 6.9 mm), which was significantly deeper than RFA AI 500 alone. Temperature recordings showed a 4–11°C rise at 3 mm across groups, whereas at 7 mm the rise was much smaller (dT < 1°C in all groups), with 7 mm-to-3 mm attenuation ratios well below 1. The maximum heating rate at 3 mm was highest with RFA (approximately 17.90°C/s) and lowest with PFA (approximately 8.58°C/s). On histology, RFA mainly produced thermal coagulative necrosis with minimal TUNEL positivity (around 2%), whereas PFA led to apoptosis-like changes and the highest proportion of TUNEL-positive cells. The RFA + PFA group showed primarily necrotic injury, while the PFA + RFA group had a mixture of necrosis and apoptosis-like changes. Conclusion Sequential RFA and PFA produces larger ex vivo myocardial lesions than either modality alone, mainly by increasing depth, while heat spread to 7 mm remains limited—pointing to an acceptable thermal safety margin for nearby structures. The order of energy delivery matters: it shapes lesion morphology, temperature response, and cell death patterns, with RFA + PFA producing mainly necrosis and PFA + RFA retaining some apoptosis-like features. These preclinical data support further development of dual-energy ablation and suggest that optimizing the energy sequence is key to balancing efficacy and safety.