## 2 Methods
### In Vivo MR Imaging Protocol and Associated Analysis  
MR imaging studies were conducted on a 3T whole body scanner (MR 750, General Electric Healthcare, Waukesha, WI, USA). Prior to imaging (Figure 2a), each animal was sedated using an anesthetic mixture of atropine (0.05 mg/kg) and ketamine (30 mg/kg), and was supported through mechanical ventilation. Anesthesia was maintained with isoflurane/O\({}_{2}\) (1-5%). For image acquisition, an 8-channel cardiac anterior array coil was placed on each pig. The heart rate and associated physiological signals were continuously monitored. The proposed MR imaging protocol presented in this paper included: a short-axis 2D CINE sequence for heart function evaluation, as well as a high-resolution 3D late gadolinium enhancement (LGE) for the identification and quantification of fibrosis. Amiodarone was injected in order to avoid arrhythmic events during the MR imaging study, which kept the heart rate stable and below 100 bpm. The heart rate stabilization improved image acquisition process and, consequently, the quality of reconstructed images.  
As per the diagram previously presented in Figure 1, the MR images were acquired at the following time points: (a) at baseline healthy state (pre-DOX injections) in both groups; (b) at one week after the completion of DOX injections in all four animals; (c) at five weeks post-DOX injections in Group 1 (i.e., in pig #1 and pig #2, respectively), and (d) at nine weeks post-DOX injections in Group 2 (i.e., in pig #3 and pig #4, respectively).  
For the assessment of cardiac function, we utilized a steady-state free precession (SSFP) sequence in Cine mode, with the following MR parameters: 16-20 short-axis slices  
Figure 1: Pipeline of the experimental study to evaluate DOX cardiotoxicity pre- and post-treatment, as indicated by the arrows (the arrowheads point to the end of longitudinal studies).  
Figure 2: Example of a preclinical MR imaging study: (**a**) experimental set-up with the pig on the table of the 3T MR scanner (prior to placing the animal inside the bore); and (**b**) long-axis image serving for the prescription of the short-axis Cine images.  
to cover the entirety of the heart (prescribed on longitudinal-axis images, as in Figure 2b), 8 views/segment, 20 cardiac phases/slice, repetition time TR= 4.2 ms, echo time TE = 1.8 ms, flip angle = 45\({}^{\circ}\), matrix size = 224 \(\times\) 160, in-plane resolution of 1 mm \(\times\) 1 mm, and slice thickness = 5 mm (with no gap between slices). All Cinematic images were acquired using ECG- gating and breath holds.  
Contrast-enhanced imaging was performed by employing a free-breathing 3D late gadolinium enhancement (LGE) method, approximately 5-6 min after injecting a bolus of gadolinium-based contrast agent Gd-DTPA (0.2 mmol/kg, [PERSON], Bayer Healthcare Pharmaceuticals, Berlin, Germany). The 3D LGE method with isotropic voxels size was based on a 3D inversion recovery fast gradient echo (IR-FGRE) sequence with fat suppression and respiratory navigation (initial inversion time TI = 300 ms, repetition and echo times TR/TE = 3.5/1.5 ms, bandwidth BW = 100 kHz, flip angle = 15\({}^{\circ}\), and an isotropic spatial resolution of 1.4 mm \(\times\) 1.4 mm \(\times\) 1.4 mm), similarly to the method we previously used in preclinical MR imaging studies to evaluate chronic infarct scars [16].