Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Background The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. Results BCs exhibited spontaneous beating (60.5 ± 2.6 bpm) and maximum-diastolic-potential (MDP) of 67.8 ± 0.8 mV (n = 155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7 ± 1.9 pA/pF at +40 mV; n = 14). Recovery of Ito from inactivation (at −80 mV) showed slow kinetics (τ1 = 200 ± 110 ms (12%) and τ2 = 2380 ± 240 ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype.

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair

“…In general, human PSC-derived cardiomyocytes are distinguished by their relatively low levels of the inward rectifier potassium current (I K1 ) [62, 82] and high levels of the pacemaker current (I f ) [62, 83]. The functional consequence is a relatively depolarized MDP and greater electrical instability.…”

“…The functional consequence is a relatively depolarized MDP and greater electrical instability. On the other hand, current densities reported for a number of other major ionic currents including the fast sodium (I Na ) [84–86], the L-type calcium(I CaL ) [85, 87], transient outward potassium (I to ) [82] and delayed rectified potassium (I Kr and I Ks ) currents [85] are not greatly dissimilar between human PSC-derived and adult cardiomyocytes; although in some instances there are more subtle differences in isoform expression and/or kinetics. Indeed, modeling experiments suggest that the single-cell electrophysiological properties of human PSC-derived cardiomyocytes could be largely "normalized" if they can be induced to have more adult-like expression levels of I K1 and pacemaking currents [88, 89].…”

Novel therapeutic strategies for the management of ventricular arrhythmias associated with the Brugada syndrome

“…An obstacle that we and others have encountered is the fact that these cells are immature and are unable to recapitulate the BrS phenotype because I to is very slow to recover from inactivation. 183 The development of wedge preparations or whole hearts by reseeding collagen scaffolds using progenitor cells or iPSC generated from fibroblasts isolated from patients with BrS is another futuristic approach to creation of human models of BrS that we and others are pursuing.…”

Myocardial Reprogramming Medicine: The Development, Application, and Challenge of Induced Pluripotent Stem Cells

“…Immature differentiated cardiomyocytes lack the consistent properties of adult mature ventricular cardiomyocytes such as gene expression profile, morphology, and electrophysiological function [158,259,260]. For instance, the majority of hiPSC-derived cardiomyocytes lack the action potential notch characteristic which is central to the disease phenotype of inherited cardiac arrhythmia syndromes [261]. The diversity of various induced cardiomyocyte phenotypes is dependent on their derivation, age, and culture conditions.…”