Actc1

Cells were fixed in 3.7% (v/v) paraformaldehyde for 10 min, then permeabilized and blocked in PBS with 4% (v/v) bovine growth serum (Thermo Scientific) and 0.2% (v/v) Triton X-100 for 1 h, and subsequently incubated with primary antibodies for AcH3 (Millipore), Actc1 (Sigma), sarcomeric MyHC (R&D), Nppa (Millipore), Ryr2 (Sigma), Pln (Abcam) in blocking buffer overnight on a rocker at 4 °C. Secondary antibody incubation (Alexa 647, Life Technologies) was performed in blocking buffer for 1 h at room temperature. The fixed PDMS membranes were then mounted on slides in Vectashield^® mounting medium and analysed with confocal microcospy (Zeiss LSM-780 inverted) or conventional epifluoresence microscopy with a Zeiss Axio-Observer Z1 widefield fluorescent microscope.
High-throughput image analysis was performed with a custom made Matlab^® script. Briefly, cell nuclei were detected using contrast enhancement methods, then segmented with an edge detection algorithm (Canny; Matlab^®), following which elliptical fitting was performed to compute the aspect ratio (short axis/long axis) and the angle between the long axis and the groove direction.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was performed, as previously described.2, 3 Briefly, hearts were rapidly extracted and fixed in 10% formalin (Sigma, St. Louis, MO, USA). Ventricular tissue was paraffin embedded and sectioned (5 μm), and was performed by the Department of Pathology at the Cincinnati Children Hospital Medical Center. Apoptotic nuclei were determined using the DeadEnd™ Fluorometric TUNEL system (Promega, Madison, WI, USA), according to the manufacturer's instruction. Sections were stained with Actc1 (Sigma‐Aldrich; 1:50) for cardiomyocyte visualization and mounted with VECTASHIELD^® Antifade Mounting Medium Plus DAPI to stain nuclei. TUNEL‐positive nuclei were examined with a fluorescence microscope.2, 3 Caspase‐3 activity was determined in NTG and S10F cardiac homogenates, using the Caspase‐3 Colorimetric Assay Kit (BioVision Incorporated, K105), in accordance with the manufacturer's instruction.