## 4 Discussion
This method has the capability to distinguish dense collagenous fibrotic patches, as demonstrated in a previous study performed ex vivo in chronically infarcted porcine hearts [27], and has already been translated to preclinical in vivo free-breathing 3D imaging of infarct scars, using a 1.4 mm isotropic resolution [16]. Furthermore, in the imaging protocol, we plan to include T2-based methods to evaluate edema resorption, a reversible side-effect of DOX which we recently observed in [24] (in three animals). Lastly, with respect to cardiac functional assessment, the gradual decline in ejection fraction observed in the current work within the first 9 weeks post-COX indicated an early sub-acute and sub-chronic occurrence of biomechanical dysfunction, in agreement with the swine study of cardiotoxicity that used intracoronary DOX injections [15]. We suggest that the collagen deposition has substantially contributed to the overall EF decline, and will likely have a critical role in the further evolvement towards heart failure. Regarding the third novel aspect, to the best of our knowledge, this is the first preclinical study to report X-ray guided electro-anatomical mapping of endocardial bipolar voltages in a large animal model post-chemotherapy, as most studies have focused only on longitudinal assessment of cardiac mechanical function. The recorded intracardiac iEGM signals, acquired with a catheter-based clinical systems, revealed small patches of fibrotic tissue with reduced bipolar voltage amplitude, which is typically seen clinically in scarred myocardium post-infarction. Our findings also suggest that early electrical remodeling takes place within the first couple of months post-COX, with prolonged QRS-T intervals accompanying the low voltages in fibrotic areas. The small patches of dense collagenous fibrosis located within areas of abnormal iEGM waves' morphology likely created unexcitable obstacles in the electrical wave pathway, generating reentrant waves, spiral wave break, and chaotic electrical propagation. This eventually led to lethal ventricular fibrillation during the rapid pacing procedure, similarly to the scar-related ventricular arrhythmias induced in post-infarction. Future work will include the monitoring of spontaneous arrhythmia episodes via MR-compatible implantable cardiioverter defibrillators, in order to study the risk of lethal arrhythmia development post-chemotherapy. We acknowledge that one study limitation is the small number of animals. However, in this work, we aimed to describe in detail the experimental protocol and qualitative results, using a minimum number of animals for tests and respecting the '3 Rs principle' in animal research (i.e., replacement, reduction, and refinement). Nonetheless, we plan to expand the cohort in the future by including more animals, which will enable us to give statistical power to the study. We envision that preclinical large animal models of cardiotoxicity will substantially help researchers better understand the mechanistic effects of cardiotoxicity. Such models can also provide a robust translational platform for testing new individual cardio-protective strategies or a synergistic combination of those [28,29]. These could slow the irreversible myocardial injury and associated dysfunction post-chemotherapy, and restrict further progression towards the heart failure stage. Our future work will also focus on testing 3D virtual models in order to predict the electro-mechanical function post-cardiotoxicity, integrating MRI-defined fibrosis areas and electrical remodeling information. Such in silico computer models can be exploited to virtually predict risk of arrhythmia and impaired mechanical contraction (including the EF index) [30] either for screening of drugs' cardiotoxicity [31] or to design more efficient therapeutic strategies, in conjunction with information provided by early imaging biomarkers and knowledge gained from animal models of cardiotoxicity [32].