Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No.860974. Background The success-rate of pulmonary vein isolation (PVI) is modest in persistent atrial fibrillation (AF), but additional empirical ablation, such as posterior wall isolation (PWI), has not been shown to reduce the rate of AF recurrence. The "seatbelt", an anterior linear ablation lesion connecting the right superior pulmonary vein to the mitral annulus, is a novel substrate modification approach that is yet to be tested in a large-scale randomised clinical trial. Purpose To use in-silico trials with human-based modelling and simulation to compare the efficacy of (i) PVI alone, (ii) PVI and PWI (PVI+PWI), or (iii) PVI and seatbelt (PVI+SB) for the prevention of atrial arrhythmia in large populations of virtual patient models. Methods AF was simulated in a cohort of 400 virtual atrial models with anatomical and electrophysiological variability, with AF persisting for >7 seconds defined as sustained. Models which sustained AF were each independently subjected to all three ablation approaches before repeating the AF induction protocol. Results Three hundred forty-four (86%) atrial models developed sustained AF at baseline. After applying the ablation lesions, substantially fewer models sustained atrial arrhythmias in the PVI+SB group (50 [14%] vs. 134 [39%] with PVI+PWI vs. 172 [50%] with PVI). PVI+SB was more effective than PVI and PVI+PWI in preventing both AF and atrial flutter (i.e., presence of a single rotor), since PVI and PVI+PWI led to the formation of anatomical re-entries around the rings of the pulmonary veins and the posterior wall of the left atrium, respectively (Figure). Therefore, PVI+SB restricted atrial flutter mainly to the right atrium (either anatomical, around the inferior cava vein, or functional, meandering throughout the venous portion of the right atrium, Figure). Importantly, in all three ablation strategies, the likelihood of AF recurrence was directly proportional to the surface area of the right atrium (AF probability increased 0.95%/cm², 0.88%/cm² and 0.56%/cm² after PVI+SB, PVI+PWI and PVI, respectively). The role of the right atrial size was less accentuated for PVI alone, since after the ablation the entire left atrial body was still available for rotor anchoring. Moreover, an enlarged left atrium enabled the appearance of micro-re-entries in the anterior and inferior wall of the left atrium, even after applying floor and roof lines. One atrial model additionally presented a rotor in the posterior wall of the left atrium after completely isolating it from the left atrial body. Conclusion(s) In a cohort of 400 virtual atrial models, PVI+SB reduced sustained atrial arrhythmias by 72% compared to PVI alone and by 64% to PVI+PWI, but AF could still originate in an enlarged right atrium.