Characterization of a Transient Outward K+ Current in Hips-Derived Cardiomyocytes

Abstract

Background: Human induced pluripotent stem cell (hiPS)-cardiomyocytes can be used to create in vitro models of genetic disease such as Brugada Syndrome (BrS). Central to the development of BrS is the Ca2+-independent transient outward K+ current (Ito). In this study, we characterized Ito in single hiPS -cardiomyocytes and determined its functional role in beating clusters.Methods: Embryoid bodies (EBs) were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Whole cell patch clamp was used to record Ito in single hiPS cardiomyocytes. Action potential (AP) recordings from spontaneously beating clusters (BCs) were made using sharp microelectrodes. All recordings were done at 36°C.Results: BCs exhibited spontaneous APs with an average rate of 54.9±30.1 bpm and maximum diastolic potential (MDP) of −65.6±9.3 mV (n=122). A small phase 1 repolarization which could be blocked by 4-AP (1 mM) was observed in 6/149 hiPS BCs suggesting the presence of Ito. Interestingly, in single dissociated hiPS cardiomyocytes, patch clamp analysis revealed a robust Ito (13.4±1.79 pA/pF at +40 mV, n=14) in the majority of cells studied. Recovery of Ito (at −80 mV) showed a fast and slow phase as follows: i) 1=271±93 ms and 2= 2697±103 (n=8 cells). These observations demonstrate that Ito is present but the slow recovery suggests minimal contribution during the course of an action potential. Mathematical modeling of APs from hiPSC-CMs confirmed these observations.Conclusion: There is a disconnect between the presence of Ito in cells and the absence of phase 1 repolarization in BCs. In BCs the depolarized MDP and fast spontaneous AP rate suggests negligible contribution of Ito to phase 1 repolarization. Our results point to an important deficiency of hiPSC-CMs in recapitulating the phenotype of adult native myocytes.