Ell [21]. Due to the lack of T-tubule, Ca2+ sparks are restricted to the cell periphery in neonatal cells and rabbit Purkinje cells whereby the SR membrane is associated directly with the plasma membrane which is similar to the dyadic cleft in ventricular myocytes [17?9,22]. Studies with mouse and human ESC-derived CMs have revealed that Ttubules are either absent [23,24], or less developed with poorly organized T-tubule system [25,26]. Our present study of hiPSCCMs show a U-shaped Ca2+ wavefront with rise of Ca2+ occurs initially at the cell periphery and then diffuses to the centre of the cell with an obvious delay. This indicates spatial separation between L-type Ca2+ channels and RyRs in ultrastructural organization which is likely due to the lack of t-tubules [7]. Similar findings of such segregation were reported previously [7,18,27,28]. Spontaneous Ca2+ spark could be activated either by the cytosolic Ca2+ affecting the entire population of RyRs, or by Ca2+ entry into myocyte through the L-type channels. It is reported that about 50 of spontaneous sparks are attributable to spontaneous and infrequent openings of L-type Ca2+ channels at resting membrane potential [12]. The present results showed that blocking the L-type Ca2+ channels by nifedipine reduced over 60 of the rate of spark occurrence suggesting that Ca2+ sparks in hiPSC-CMs were triggered predominately by the L-type Ca2+ channel dependent triggering mechanisms.Nifedipine did not inhibit caffeine-induced Ca2+ transients. Similar phenomena were also demonstrated after the application of nifedipine or Ca2+-free extracellular solution in cat ventricular myocytes or hiPSC-CMs [8,29]. Accordingly, the main Ca2+ source for the caffeine-induced Ca2+ transients is not dominated by Ca2+ influx via L-type Ca2+ channels [8]. To study the effect of Ca2+ concentration on Ca2+ sparks, 5 mM CaCl2 was applied to extracellular solution. Under high extracellular Ca2+ condition, spontaneous Ca2+ sparks synchronously activated nearby Ca2+ release units and produced multiple sparks, called a “compound spark” or “macrospark”, from neighboring Ca2+ release units [30]. Similar observations were seen in Fig. 6, our findings were consistent with those reported in adult ventricular myocytes [31]. Therefore, Ca2+ sensitivity of RyRs in hiPSC-CMs was similar to those in adult ventricular myocytes. The decrease in the 15857111 amplitude of sparks might be related to a decrease in the amount of Ca2+ release per RyR opening. In the present study, ryanodine elevated the rate of spark occurrence and the temporal and spatial properties of Ca2+ sparks, without affecting the amplitude of Ca2+ sparks. RyR2 is the predominant RyR isoform in cardiac muscle, this RyR isoform is essential for E-C coupling and 15857111 amplitude of sparks might be related to a decrease in the amount of Ca2+ release per RyR opening. In the present study, ryanodine elevated the rate of spark occurrence and the temporal and spatial properties of Ca2+ sparks, without affecting the amplitude of Ca2+ sparks. RyR2 is the predominant RyR isoform in cardiac muscle, this RyR isoform is essential for E-C coupling and 24786787 Ca2+ sparks in cardiac myocytes [4]. It was reported that Ca2+ sparks activity was absent after genetic ablation of RyR2 in stem cell-derived cardiomyocytes [4]. The expression of RyR2 gene in hiPSC-CMs has been confirmed previously [7,8]. Therefore, our results indicated that a functional RyR2-mediated SR Ca2+ release is present in hiPSC-CMs. In hiPSC/hESC-CMs, the mechanism of E-C coupling remains contentious. Some reports supported classical model of E-C coupling [8,32]. Alternatively, it was suggested that Ca2+ used byCalcium Sparks in iPSC-Derived CardiomyocytesFigure 4. The characteristics of spontaneous Ca2+ sparks in hiPSC-CMs. (A) Two representative Ca2+ sparks (a and b) and an overlay of 160 original.