Immunofluorescence and Morphometric Examination

Cells attached to the PDMS construct were fixed with 10% formalin, permeabilized, blocked in 10% normal horse serum for 1 hour, and incubated for 1 hour at room temperature with primary antibodies raised against Cx43 (6.4 µg/ml; Sigma‐Aldrich), sarcomeric α‐actinin (11.5 µg/ml ascites fluid; Sigma‐Aldrich), GATA‐4 (4 µg/m.; R&D Systems Inc., Minneapolis, MN, https://www.rndsystems.com), MEF2 (4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), and SERCA2 (4 µg/ml; Santa Cruz Biotechnology). Secondary antibodies were conjugated with Cy2 and Cy3 (7.5 µg/ml; Jackson ImmunoResearch, West Grove, PA, https://www.jacksonimmuno.com), and actin fibers (actinF) were stained with Phalloidin Alexa 568 (0.161 µM; Thermo Fisher Scientific Life Sciences). Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (0.1 µg/ml; Sigma‐Aldrich). Images were acquired with the Axio Observer Z1 inverted microscope (Zeiss, Stuttgart, Germany, http://www.zeiss.com).

Hearts were cross‐sectioned from apex to base (10‐μm‐thick sections spaced every 300 μm). Eight serial cryosections per animal were stained with Masson’s trichrome (collagen, green; myocardium, red; nuclei, black or brown; cytoplasm, pink) for morphometry. All sections were blindly examined and photographed using a SMZ 800 stereoscope (Nikon, Tokyo, Japan, http://www.nikon.com). Infarct size volume, expressed as a percentage of the total left ventricle (LV) wall volume, was calculated by addition of partial scar volumes of each section. Scar thickness was calculated as the mean of at least three measurements made in three different sections for each animal. Additionally, Gallego’s modified trichromic (collagen, blue; myocardium, yellow‐pink; elastic fibers, purple; nuclei, fuchsia) and Movat’s pentachromic (nuclei, black; collagen, yellow; ground substance, blue; muscle, purple; elastic fibers, brownish gray) stainings were carried out on heart cross‐sections from all groups.

Further immunoanalyses were performed on cryosections by using specific monoclonal antibodies against Cx43 (Sigma‐Aldrich), sarcomeric α‐actinin (Sigma‐Aldrich), GATA‐4 (R&D Systems), MEF2 (Santa Cruz Biotechnology), SERCA2 (Santa Cruz Biotechnology), biotinylated GSLI B4 isolectin (10 µg/ml; Vector Laboratories, Burlingame, CA, http://vectorlabs.com), cTnI (10 µg/ml; Abcam, Cambridge, MA, http://www.abcam.com), phospho‐histone H3 (PH3) (1 µg/ml; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), CD31 (4 µg/ml; Abcam), smooth muscle actin (SMA) (1:50 ascites fluid; Sigma‐Aldrich), and vimentin (10 µg/ml; Abcam). Secondary antibodies were conjugated with Cy2 and Cy3 (Jackson ImmunoResearch). Streptavidin was conjugated with Alexa 488 (Thermo Fisher Scientific Life Sciences). Nuclei were counterstained with DAPI (Sigma‐Aldrich). Images were captured under a laser confocal microscope (Axio‐Observer Z1; Zeiss). Quantitative histological measurements were made by using ImageJ analysis software (NIH, Bethesda, MD, https://imagej.nih.gov).