Assessment of Ca2+ transients in 2D hiPSC‐CM

PLNic and PLN p. Arg14del hiPSC‐CM were seeded onto gelatin‐coated glass coverslips (24x24 mm, VWR) in a 6‐well culture dish at a density of 2 × 10⁵ hiPSC‐CM per dish (10 cm²). The hiPSC‐CMs were cultured for 7 ± 2 days (RPMI, B‐27) until analysis. 2.5 µM Fura 2‐AM (5 mM stock in DMSO) was dissolved in 2 ml Tyrode’s solution for 2D Ca²⁺ transient analysis. The cardiomyocytes were washed with 2 ml Tyrode’s solution and loaded with Fura 2‐AM for 15 min protected from light. The Fura 2‐AM‐containing buffer was replaced by 3–4 ml Tyrode’s solution for a 15‐min washing step. The coverslips with the hiPSC‐CM were removed from the culture wells for analysis with the IonOptix system (IonOptix, Milton, USA). The cells were continuously superfused with 37°C pre‐warmed Tyrode’s solution and paced at 0.5 Hz. Cells were included into the analysis, when they showed a regular beating pattern and followed the electrical pacing signal. Furthermore, cells should be either in a single or in a small cell cluster format (3–5 cells), but not part of larger cell aggregates. Initially, a stable baseline was recorded. Electrical stimulation was switched off, and 20 mM caffeine was applied to the cells. After 10–15 s, the perfusion was switched back to normal Tyrode’s solution and cells were electrically stimulated again. The recording was continued for approximately 30 s. A maximum of three cells per coverslip was recorded.