ABSTRACT
Introduction: Dysfunction of the sinoatrial node (SAN) cells causes arrhythmias, and many patients require artificial cardiac pacemaker implantation. However, the mechanism of impaired SAN automaticity remains unknown, and the generation of human SAN cells in vitro may provide a platform for understanding the pathogenesis of SAN dysfunction. The short stature homeobox 2 (SHOX2) and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) genes are specifically expressed in SAN cells and are important for SAN development and automaticity. In this study, we aimed to purify and characterize human SAN-like cells in vitro, using HCN4 and SHOX2 as SAN markers. Methods: We developed an HCN4-EGFP/SHOX2-mCherry dual reporter cell line derived from human induced pluripotent stem cells (hiPSCs), and HCN4 and SHOX2 gene expressions were visualized using the fluorescent proteins EGFP and mCherry, respectively. The dual reporter cell line was established using an HCN4-EGFP bacterial artificial chromosome-based semi-knock-in system and a CRISPR-Cas9-dependent knock-in system with a SHOX2-mCherry targeting vector. Flow cytometry, RT-PCR, and whole-cell patch-clamp analyses were performed to identify SAN-like cells. Results: Flow cytometry analysis and cell sorting isolated HCN4-EGFP single-positive (HCN4+/SHOX2-) and HCN4-EGFP/SHOX2-mCherry double-positive (HCN4+/SHOX2+) cells. RT-PCR analyses showed that SAN-related genes were enriched within the HCN4+/SHOX2+ cells. Further, electrophysiological analyses showed that approximately 70% of the HCN4+/SHOX2+ cells exhibited SAN-like electrophysiological characteristics, as defined by the action potential parameters of the maximum upstroke velocity and action potential duration. Conclusions: The HCN4-EGFP/SHOX2-mCherry dual reporter hiPSC system developed in this study enabled the enrichment of SAN-like cells within a mixed HCN4+/SHOX2+ population of differentiating cardiac cells. This novel cell line is useful for the further enrichment of human SAN-like cells. It may contribute to regenerative medicine, for example, biological pacemakers, as well as testing for cardiotoxic and chronotropic actions of novel drug candidates.
ABSTRACT
INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called "iCell cardiomyocytes" under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents. METHODS: iCell cardiomyocytes were cultured for 8-14 days (38-44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp. RESULTS: iCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were -62.7 ± 13.7, -8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 µM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 µM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 µM, respectively. CONCLUSIONS: In iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.
