Pathology

All animals were anesthetized using an intraperitoneal injection of 30 mg/kg sodium pentobarbital and then euthanized by thoracotomy and heart removal 3 weeks after surgery. Cardiac tissues were excised and placed in glass receptacles containing phosphate-buffered saline (PBS). Residual blood in the heart was extruded with gentle pressure. After the cardiac tissues were washed with PBS, the residual tissues of the aortic arch and pericardium were carefully removed. With ophthalmology scissors, the atria were removed along the atrioventricular groove, and the right ventricular free wall was cut from the lower right side of the interventricular septum. Thus, the left ventricle and interventricular septum remained. The tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at 4 µm. Hematoxylin/eosin staining was then used to visualize global morphology. Masson staining was used to assess the fibrosis level. To examine cell size, wheat germ agglutinin (WGA) staining was performed. Slides were stained for 30 min with WGA-FITC-labeled antibody (1:40, L4895, Sigma Aldrich) at room temperature. Staining with Hoechst (1:10,000. 33342, Thermo Scientific) for 5 min was done to visualize the nuclei in blue. Images of tissue sections were captured with a BX53 microscope (Olympus), and analysis was performed in a blinded fashion using ImageJ.