Measurement of Cardiomyocyte Surface Area

Cell surface area of NRCMs was measured as described elsewhere (Xu et al., 2014). Briefly, after treatment with different agents, the cells were fixed with 4% paraformaldehyde for 1 h at 4°C, then permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. After blocking with 5% BSA for 2 h, the cells were incubated with anti-α-actinin antibody (abcam, ab18061) overnight at 4°C, followed by incubation with donkey anti-mouse AlexaFluor 555-conjugated secondary antibody (Thermo Fisher Scientific, A-21422) for 2 h at room temperature. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Life Technologies, D1306). The cells were visualized under a FV1000 laser scanning confocal microscope. The areas of 100 randomly selected cells were determined using ImageJ software.