Indo 1 am

For calcium transient, the cells were loaded with 5 μM Indo-1 AM (1006; Dojindo) dissolved in FluoroBrite Dulbecco’s modified Eagle’s medium (DMEM) (A1896701; Gibco) containing 0.1% pluronic F-127 (P3000MP; Thermo Fisher Scientific) and incubated at 37°C for 1 h. After washing twice with PBS, the cells were incubated in FluoroBrite DMEM for 30 min at 37°C. For measuring membrane potential, the prepared cells were treated with the FluoVolt membrane potential kit (F10488; Thermo Fisher Scientific) for 30 min at 37°C. The medium was replaced with FluoroBrite DMEM containing 2% FBS and incubated for another 30 min. The calcium transient and membrane potential were measured under pacing at 1 Hz. The imaging data were acquired and analyzed using the FDSS/uCELL system (C13299; Hamamatsu Photonics, Hamamatsu, Japan).

The medium was replaced with ECM supplemented with 10 μM Indo-1 AM (I006, Dojindo, Japan) and 0.02% Pluronic F-127 (59005, Biotium, USA) for 50 min to incorporate the dye and ECM for 20 min to de-esterize the intracellular dye. We obtained time-lapse images using a two-photon laser microscope (LSM880 NLO, Carl Zeiss, Germany) by perfusing HBSS without calcium (17461-05, Nacalai Tesque, Japan) under a 20-cm hydraulic head distance to obtain a non-pulsatile flow of 11 mL/min. In the suramin group, we used these media supplemented with 0.25 μg/mL suramin sodium.

Intracellular Ca²⁺ transients were measured using Indo-1 AM. Isolated cardiomyocytes were loaded with 10 μmol/L Indo-1 AM (Dojindo) for 10 min at 37°C and electrically stimulated at 1 Hz. Ca²⁺ transients were measured as the ratio of fluorescence emitted at 405/480 nm after excitation at 340 nm. Ca²⁺ transients were recorded under an inverted IX71 microscope with a 20× water immersion objective lens (UApo N340) and a High-performance EvolveTM EMCCD camera and analysed using MetaMorph software.