Tyrode s solution

A concentrated solution of Rhod-2 (Thermo Fisher Scientific) was prepared by dissolving 50 μg of the calcium indicator in 100 μl of DMSO. The solution was then diluted 1:250 in Tyrode’s solution (Alfa Aesar). Blebbistatin (Sigma-Aldrich) was added at a final concentration of 5 μM (from a 5 mM stock solution). Cardiac OBB tissues were first rinsed with Tyrode’s solution (Alfa Aesar) and incubated with the calcium indicator solution for 30 min. Next, the tissues were rinsed once with a Blebbistatin-containing Tyrode’s solution. The samples were subjected to a 555-nm light illumination, and the wide-field signal was captured by a scientific complementary metal-oxide semiconductor camera (sCMOS, Andor technology). Data were acquired using Micro-Manager open source software. During imaging, the plate containing the cardiac tissues was placed on top of a heated pad (Stoelting).

iPSC-CMs cultured on 35-mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) at 37°C, 5% CO₂ were loaded with 2 μM Fluo-4 AM (Thermo Fisher Scientific, Waltham, MA, USA) with 0.02% F-127 (Thermo Fisher Scientific, Waltham, MA, USA) in Tyrode’s Solution (Alfa Aesar, Tewksbury, MA, USA) for 30 min. Following washout, Tyrode’s Solution was added, and cells were imaged. During imaging, cells were kept in a heated 37°C stage-top environment chamber supplied with 5% CO₂. Imaging of Ca²⁺ transients was taken under a 40× water objective using a Nikon Eclipse Ti (Melville, NY, USA) light microscope. iPSC-CMs were paced at 0.5 Hz using an IonOptix MyoPacer Field Stimulator (Westwood, MA, USA). Time-lapse videos were taken at a speed of 20 ms/frame for 20 s. For recordings with ISO, 1 μM ISO (I2760; MilliporeSigma) was added to the Tyrode’s Solution, and video recordings were taken between 6 and 10 min of incubation. The raw data were exported to Excel software (Microsoft, Redmond, WA, USA) and analyzed with a custom Excel-based script to correct for photo bleaching.