Calcium Transient Measurement of iPSC-CMs

Both Ctrl- and RYR2^–/–-iPSC-CMs around day 80 were dissociated and replated on coverslips at a density of 200,000 cells/well. Cells were allowed to recover for at least 10 days post-replating. For measurement, cells were loaded with Fura-2 (Thermo Fisher Scientific) at a final concentration of 5 μM in cardio culture medium for 30 min at 37°C and washed twice with the medium. Prior to measurement, cells were incubated for 10 min to enable complete de-esterification of intracellular Fura-2. Intracellular Ca²⁺ events were recorded using a 40 × objective on an Olympus IX70 microscope fitted with an IonOptix system (Ionoptix, Milton, MA) at 35°C. Samples were excited at 340 and 380 nm with a switching frequency of 200 Hz and the emitted fluorescence was collected at 510 nm. The cytosolic Ca²⁺ level was measured as the ratio of fluorescence at 340 and 380 nm (340/380 nm).

Spontaneous whole-cell Ca²⁺ transients were recorded in normal Tyrode’s solution containing (in mM): NaCl 138, KCl 4, CaCl₂ 1.8, MgCl₂ 1, NaH₂PO₄ 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). To normalize the Ca²⁺ transient frequency, Ctrl- and RYR2^–/–-iPSC-CMs were field-stimulated using a MyoPacer (Ionoptix, Milton, MA) at a pacing frequency of 0.5 Hz (6 V, 10 ms). Monotonic transient analysis was performed using the LabChart Pro software (ADInstrument) and the following parameters were determined: peak amplitude of Ca²⁺ transients (the Fura-2 ratio at systole subtracted by the Fura-2 ratio at diastole), decay rate (tau), as well as duration and frequency of Ca²⁺ transients.

Calcium sensitivity analysis was performed as described previously (Jiang et al., 2007). Briefly, Ctrl- and RYR2^–/–-iPSC-CMs loaded with Fura-2 were first exposed to 0 mM Ca²⁺ Tyrode’s solution until no spontaneous Ca²⁺ transient was detected and then continuously perfused with Tyrode’s solution containing Ca²⁺ of increasing concentrations (0, 0.1, 0.2, 0.3, 0.5, 1.0, and 2.0 mM). In the end, caffeine (10 mM; Sigma-Aldrich) was applied to confirm the activity of RYR2.

To examine the caffeine-induced Ca²⁺ release in iPSC-CMs, cells loaded with Fura-2 were washed with depolarization solution containing (in mM): NaCl 112, KCl 30, CaCl₂ 1.8, MgCl₂ 1, NaH₂PO₄ 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). Cytosolic Ca²⁺ level was measured before and after continuous addition of caffeine with increasing concentrations (from 0.025 to 5.0 mM).

To investigate the role of IP3R-mediated Ca²⁺ release in Ca²⁺ handling of iPSC-CMs, spontaneous Ca²⁺ transients were recorded before and after the application of the IP3R antagonists 2-aminoethoxydiphenyl borate (2-APB, 20 μM; Tocris) and Xestospongin C (XeC, 1 μM; abcam). To determine the contribution of SERCA-mediated SR Ca²⁺ uptake to Ca²⁺ cycling in iPSC-CMs, spontaneous Ca²⁺ transients were recorded before and after the application of the SERCA inhibitor thapsigargin (5 μM; Millipore). The changes of Ca²⁺ transient amplitude and frequency caused by the addition of these inhibitors were determined.

For these experiments, we used an imaging chamber (RC-47FSLP, Warner instruments), which is furnished with a field stimulation and a built-in aspiration port. For fluid control, a 12-valve superfusion system (DAD-VM, ALA Scientific Instruments) combined with a multi-tube pre-heater (MPRE8, Cell MicroControls) was used, which together allows a gentle, rapid, and direct solution change around the measured cells. The overflow solution was removed via the aspiration port by suction with a peristaltic pump (minipuls 3, Gilson).