Spontaneous inward currents reflecting oscillatory activation of Na^sup +^/Ca^sup 2+^ exchangers in human embryonic stem cell-derived cardiomyocytes

Na^sup +^/Ca^sup 2+^ exchanger current (I^sub NCX^) triggered by spontaneous Ca^sup 2+^ release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca^sup 2+^ clock model"). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between -40 and 40 mV (-80±10.6 pA, at -20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca^sup 2+^]^sub i^ synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca^sup 2+^]^sub e^, L-type Ca^sup 2+^ channel (VOCC^sub L^) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K^sup +^ channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCC^sub L^, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca^sup 2+^ release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCC^sub L^, oscillatory Ca^sup 2+^ releases via RyR induce sharp increases in subsarcolemmal [Ca^sup 2+^]^sub i^ and inward I^sub NCX^ (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca^sup 2+^ clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.