title: Spontaneous inward currents reflecting oscillatory activation of Na⁺/Ca²⁺ exchangers in human embryonic stem cell-derived cardiomyocytes.
abstract: Na(+)/Ca(2+) exchanger current (INCX) triggered by spontaneous Ca(2+) release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca(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(2+)]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca(2+)]e, L-type Ca(2+) channel (VOCCL) 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, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca(2+) release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca(2+) releases via RyR induce sharp increases in subsarcolemmal [Ca(2+)]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca(2+) clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.
authors.forename: Seong Woo
authors.initials: SW
authors.lastname: Choi
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.|Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Daejeon, Republic of Korea.
authors.forename: Hyang-Ae
authors.initials: HA
authors.lastname: Lee
authors.affiliation: Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Republic of Korea.
authors.forename: Sung-Hwan
authors.initials: SH
authors.lastname: Moon
authors.affiliation: Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Republic of Korea.
authors.forename: Soon-Jung
authors.initials: SJ
authors.lastname: Park
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.|Ischemic/Hypoxic Disease Institute, College of Medicine, Seoul National University, Seoul, Republic of Korea.
authors.forename: Hae Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.|Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Daejeon, Republic of Korea.
authors.forename: Ki-Suk
authors.initials: KS
authors.lastname: Kim
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.|Ischemic/Hypoxic Disease Institute, College of Medicine, Seoul National University, Seoul, Republic of Korea.
authors.forename: Yin Hua
authors.initials: YH
authors.lastname: Zhang
authors.affiliation: Department of Physiology, College of Medicine, Inje University, Busan, Republic of Korea. jaeboum@gmail.com.
authors.forename: Jae Boum
authors.initials: JB
authors.lastname: Youm
authors.affiliation: Department of Physiology, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea. sjoonkim@snu.ac.kr.|Ischemic/Hypoxic Disease Institute, College of Medicine, Seoul National University, Seoul, Republic of Korea. sjoonkim@snu.ac.kr.
authors.forename: Sung Joon
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D000200:Action Potentials; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D024661:Potassium Channels, Inwardly Rectifying; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger
keywords: Ca2+ release; Embryonic stem cell; Heart; NCX; Pacemaker current