The ability to generate patient-specific human induced pluripotent stem cells (iPSCs) offers a new paradigm for modelling human disease and for individualizing drug testing. Congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific human iPSC line from a patient with type-2 LQTS (which is due to the A614V missense mutation in the KCNH2 gene). The generated iPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp and extracellular multielectrode recordings revealed significant prolongation of the action-potential duration in LQTS human iPSC-derived cardiomyocytes (the characteristic LQTS phenotype) when compared to healthy control cells. Voltage-clamp studies confirmed that this action-potential-duration prolongation stems from a significant reduction of the cardiac potassium current I(Kr). Importantly, LQTS-derived cells also showed marked arrhythmogenicity, characterized by early-after depolarizations and triggered arrhythmias. We then used the LQTS human iPSC-derived cardiac-tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium-channel blockers) or ameliorate (calcium-channel blockers, K(ATP)-channel openers and late sodium-channel blockers) the disease phenotype. Our study illustrates the ability of human iPSC technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.

Human embryonic stem cell-derived cardiomyocytes (hES-CMs) are thought to recapitulate the embryonic development of heart cells. Given the exciting potential of hES-CMs as replacement tissue in diseased hearts, we investigated the pharmacological sensitivity and ionic current of mid-stage hES-CMs (20-35 days post plating). A high-resolution microelectrode array was used to assess conduction in multicellular preparations of hES-CMs in spontaneously contracting embryoid bodies (EBs). TTX (10 µM) dramatically slowed conduction velocity from 5.1 to 3.2 cm s −1 while 100 µM TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca 2+ channel blockers nifedipine or diltiazem (1 µM) had a negligible effect on conduction. These results suggested a prominent Na + channel current, and therefore we patch-clamped isolated cells to record Na + current and action potentials (APs). We found for isolated hES-CMs a prominent Na + current (244 ± 42 pA pF −1 at 0 mV; n = 19), and a hyperpolarization-activated current (HCN), but no inward rectifier K + current. In cell clusters, 3 µM TTX induced longer AP interpulse intervals and 10 µM TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca 2+ channel block) and 2 mM Cs + (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum upstroke velocity (dV /dt max ) of 118 ± 14 V s −1 in control conditions; in contrast, partial block of Na + current significantly reduced stimulated dV /dt max (38 ± 15 V s −1 ). RT-PCR revealed Na V 1.5, Ca V 1.2, and HCN-2 expression but we could not detect Kir2.1. We conclude that hES-CMs at mid-range development express prominent Na + current. The absence of background K + current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of Na V 1.5; thus, the Na V 1.5 Na + channel is important for initiating spontaneous excitability in hES-derived heart cells.

BackgroundThe ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/Principal FindingsRT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122.Conclusions/SignificanceOur study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.