Spontaneous inward currents reflecting oscillatory activation of Na⁺/Ca²⁺ exchangers in human embryonic stem cell-derived cardiomyocytes

Abstract

Na⁺/Ca²⁺ exchanger current (I_NCX) triggered by spontaneous Ca²⁺ release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms (“Ca²⁺ 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 [Ca²⁺]_i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca²⁺]_e, L-type Ca²⁺ channel (VOCC_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⁺ 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_L, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca²⁺ release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCC_L, oscillatory Ca²⁺ releases via RyR induce sharp increases in subsarcolemmal [Ca²⁺]_i and inward I_NCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative “Ca²⁺ clock” would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.