Di 4 anepps

Optical mapping was performed with a high-speed CMOS camera system (MiCAM Ultima, Brainvision, Tokyo, Japan) as reported previously⁵² . The heart organoids were stained with 15 μM di-4-ANEPPS (Wako, Tokyo, Japan) for 15 min, followed by washout with PBS. Next, the samples were incubated with 30 μM of blebbistatin (Sigma-Aldrich, St. Louis, MO) for 15 min to eliminate the motion artifact. After washout, the heart organoids were placed in a glass-bottom dish filled with Tyrode’s solution (135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 0.33 mM NaH₂PO₄, 5.5 mM d-glucose, and 5.0 mM HEPES at pH = 7.40 adjusted with NaOH and aerated with 100% O₂). E-4031 was used in the final concentration of 100 nM or 1 μM. The temperature was maintained at 37 °C throughout the procedure. All optical mapping was recorded using a ×5 objective lens, resulting in a spatial resolution of 20 μm × 20 μm/pixel, and data sampling was performed at 500 frames per second. The obtained data were analyzed using BV analysis software (Brainvision).

For confocal fluorescence imaging of the structural proteins for myocyte morphology, the hearts were fixed using Langendorff perfusion with 2% paraformaldehyde for 30 min (n = 29 hearts). We used Alexa Fluor 633 phalloidin (dilution 1:400, Life Technologies) for actin histochemistry. For sarcomeric α-actinin immunohistochemistry, incubation with a primary antibody (dilution 1:500, mouse monoclonal IgG, Sigma‒Aldrich, USA) for 48 h at 4 °C was followed by incubation with a secondary antibody for 48 h (dilution 1:500, Alexa Fluor 488, anti-mouse, Thermo Fisher Scientific, USA) prior to visualisation at room temperature. Confocal fluorescence images of both phalloidin and α-actinin were obtained using samples from the subepicardial surface of the heart using an FV-1000 confocal microscope (Olympus, Japan). The excitation wavelengths for phalloidin and sarcomeric α-actinin were 633 nm and 488 nm, respectively, and the emission wavelengths were > 650 nm and between 500 and 600 nm, respectively. To confirm membrane permeabilisation by saponin, we perfused in advance the membrane dye di-4-ANEPPS at 5 μM (Wako Pure Chemicals, Japan) and the membrane-impermeable DNA dye propidium iodide (PI) at 7.5 μM (Dojindo, Japan) for 5 min and subsequent washout for another 5 min for confocal imaging (n = 3 hearts).