## 2 Materials and Methods
### Extracellular Matrix Characterization  
After decellularization, several areas from the heart, evenly spaced between the base and the apex (\(n=6\)-8 tissue samples per heart), were isolated, fixed in 4% buffered formaldehyde solution (Roti-Histofix, Carl Roth GmbH, Karlsruhe, Germany), embedded in paraffin and cut with a microtome into histological sections of 5 \(\upmu\)m. Before staining, the specimens were deparaffinized with xylene (Sigma-Aldrich, St. Louis, MO, USA; Merck & Co., Kenilworth, NJ, USA), treated with graded ethanol solutions and rehydrated according to the manufacturer's protocols. The hematoxylin and eosin (H&E) staining method (Sigma-Aldrich, St. Louis, MO, USA; Merck & Co., Kenilworth, NJ, USA) was first used for characterization of the ECM. The staining was performed by incubating the tissue in hematoxylin solution for 5 min, followed by immersion in eosin for 30 s, and the washing steps were performed with tap water. H&E staining was used for the visualization of residual cellular remnants (basophilic nuclei appear in blue/black and eosinophilic cytoplasm  
Figure 2: System set-up of the modified Langendorf experimental device. (**a**) General view of the setup. (**b**) 4-h and (**c**) 8-h retrograde perfusion of the rat heart with 1% SDS in the control group (modified Langendorf experimental device in the absence of an electric field). (**d**) A complete decellularized rat heart from the control group after 14 h of perfusion with 1% SDS. (**e**) 4-h and (**f**) 8-h retrograde perfusion of the rat heart with 1% SDS in the presence of an alternating rectangular electric field. The electric contacts with the power supply are marked by black arrows. (**g**) A complete decellularized rat heart from the electric field group after 8 h of perfusion with 1% SDS (the heart becomes translucent when all the cellular material is washed out, while preserving the anatomy, vascular network and ECM characteristics of the native organ). and collagen in pink). For a better characterization of the ECM, sections were also stained via [PERSON]'s trichrome staining protocol (Bio-Optica, Milano, Italy) for visualization of collagenous connective tissue fibers. In [PERSON]'s trichrome staining, hematoxylin (5 min incubation) stains the nuclei of the cells black, acid fuchsin (3 min immersion) dyes the cytoplasm red, and the connective tissue is stained with aniline blue (10 min). Argrentic impregnation (Sigma-Aldrich, St. Louis, MO, USA; Merck & Co., Kenilworth, NJ, USA) lends itself especially well to the demonstration of reticular connective tissue. The reticulum is sharply impregnated, appearing brownish-black. If the processes of oxidation and reduction are accomplished, the background is almost colorless, with elements other than fibers (nuclei or cytoplasm) being silver-negative. Characterization of ECM content was also conducted, utilizing the orcein staining technique, according to the manufacturer's protocol (Orcein for elastic fibers; Bio-Optica, Milano, Italy) for the visualization of elastic fibers, which appeared in dark brown. All imaging was performed under a light microscope Leica DM750 Clinical Microscope (Leica Microsystems, Wetzlar, Germany) and a Panthera L Microscope (Motic, Richmond, CA, USA). Before the characterization of the extracellular matrix, the hearts from the control group and the electric field group were kept for 16 h in the decellularization solution. The process was considered finished when there were no traces of myocardial tissue remaining from the macroscopic point of view. Scanning electron microscopy (SEM) was used to characterize the morphological changes following decellularization. The protocol involved 2.5% (_v_/_v_) buffered glutaraldehyde (in PBS) fixation for 1 h at room temperature, followed by washing with PBS several times (three times, 15 min each), 1% osmium tetroxide (OsO\({}_{4}\); Sigma-Aldrich, St. Louis, MO, USA; Merck & Co., Kenilworth, NJ, USA) post-fixation for 1 h, followed again by fresh buffer.