Measurement of the Ca²⁺ binding ratio of cardiomyocytes. (A) Representative traces of [Ca²⁺]_i observed in the preparatory protocols before the measurement of the Ca²⁺ buffering ratio in the whole-cell voltage clamp of the fura-2 pentapotassium salt loaded cardiomyocytes. After confirming the Ca²⁺ transient evoked by step pulses (from −70 to 0 mV, 200 ms, 0.5 Hz), SERCA was inhibited by thapsigargin while continuing the stimulation pulses. Then, 20 mM caffeine was applied to confirm the depleted state of the SR. Finally, the bath was changed from NT to 0 Na/0 Ca solution to inhibit NCX. (B) After reaching the steady-state condition, a higher rate of depolarizing pulses (from −40 mV to 0 mV, 100 ms, 1 Hz, 9–10 times) was applied to induce inward Ca²⁺ current (middle panel) and associated increase of [Ca²⁺]_i (upper panel). The inward Ca²⁺ current was integrated and plotted as the quantity of electric charge (lower panel) for the calculation of Ca²⁺ binding ratio (see Materials and methods).