Journal of Molecular and Cellular Cardiology. 2013 Jul;60:36-46.

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

Jonathan M. Cordeiro, Vladislav V. Nesterenko, Serge Sicouri, Robert J. Goodrow Jr., Jacqueline A. Treat, Mayurika Desai, Yuesheng Wu, Michael Xavier Doss, Charles Antzelevitch, José M. Di Diego.

Department of Experimental Cardiology, Masonic Medical Research Laboratory, Utica, NY 13501, USA.

 

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.

 

Supplements:

The cardiac action potential (AP) is an important physiological parameter: 1) it is responsible for electrical conduction, 2) it modulates the refractory period, and 3) associated with each action potential is a corresponding contraction.  The AP is a coordinated change in membrane potential brought about by changes in different ionic currents.  In the human heart, the AP is divided into five phases, 0 through 4 (Figure 1, left panel).  The initial depolarization (upstroke) to approximately +40 mV (Phase 0) is generated by Na+ influx into the cell resulting in a measurable current (INa). The brief repolarization (Phase 1), or “notch,” following the upstroke is due to activation of a Ca2+-independent transient outward K+ current (Ito).  The plateau (Phase 2) of the AP is the result of a balance of K+ efflux through voltage-gated potassium channels and an influx of Ca2+ through voltage-gated calcium channels (ICa). In the repolarization phases (Phase 3 and 4), activation of several potassium channels brings about final repolarization.

The physiological and functional role of an Ito gradient centers around repolarization and excitation-contraction (EC) coupling.  Underlying this Phase 1 or “notch” in the adult ventricular AP is the presence of Ito. A gradient of this current has been observed in ventricular tissue with the outer epicardium showing a greater amount compared to the endocardium.  In contrast to adult tissue, APs recorded from human induced pluripotent stem cell (hiPSC) derived beating clusters showed a depolarized resting potential, slow upstroke velocity and no phase 1 repolarization (or notch), suggesting the complete absence of Ito Figure 1, right panel). Given that Ito is absent in neonates but develops with age, we anticipated that Ito would also be absent in hiPSC cardiomyocytes (CM) in that they are developmentally immature.  To test this hypothesis, we used voltage clamp techniques to record Ito from isolated hiPSC-CM. Spontaneously active single dissociated cells were studied. After a brief step to −50 mV to inactivate sodium channels, voltage steps from −40 to +50 mV elicited a rapidly activating and inactivating Ito. Surprisingly, the vast majority of these cells exhibited a relatively large Ito (peak current 13.7±1.91 pA/ pF at +40 mV), an amplitude comparable to that of human adult ventricular myocytes.

The basis for the apparent disconnect between the absence of phase 1 repolarization in beating clusters and the presence of a robust Ito in single hiPSC cells was further investigated.  We suspected that alterations in the gating parameters of Ito in these cells may be responsible for the absence of phase 1 repolarization.  We measured recovery from inactivation of Ito and found it was very slow in hiPSC-CM.  Our results suggest that Ito is present in hiPSC-CM cells but (at −80 mV) suggests that its contribution to the action potential is minimal.

Our study demonstrates that Ito is present in hiPSC-CM but that its functional role at normal heart rates is small due to the slow recovery kinetics of this current and depolarized resting potential of hiPSC-CM.  Future studies should be directed at improving the resting potential by enhancing IK1 in an effort to generate hiPSC-CM that display more mature ventricular electrophysiological phenotypes. Once this goal is achieved, the potential of hiPSC-CM for safety pharmacology or for cell replacement therapy and heart regeneration may be expanded. The ability to generate a mature adult phenotype is also critical to development of human models of inherited cardiac arrhythmias syndromes, such as Brugada Syndrome, in which Ito is believed to be central in generating the disease phenotype.

 Charles Antzelevitch-fig1Figure 1: Left panel.  Schematic showing action potential and ionic currents recorded from mature ventricular myocyte from the epicardial region.   Right panel. Action potentials recorded from a hiPCS-CM.  Note the absence of phase 1 repolarization.

 

Corresponding authors:

Charles Antzelevitch, PhD,           ca@mmrl.edu

José M. Di Diego, MD,                    didiego@mmrl.edu

Jonathan Cordeiro, PhD                                Cordeiroj@mmrl.edu

Department of Experimental Cardiology

Masonic Medical Research Laboratory

2150 Bleecker Street

Utica, NY 13501 USA

Phone: 1 315 735 2217

Fax: 1 315 735 5648

 

Funding: This study was supported by a Grant-in-Aid (10GRNT4210016) from the American Heart Association (Dr. Di Diego), a New York Stem Cell Science (NYSTEM) grant C026424 (Dr. Antzelevitch), a grant HL47678 from NHLBI (Dr. Antzelevitch), an American Heart Association (AHA) grant 11SDG5410034 (Dr. Doss), and by New York State, Florida, Massachusetts and Connecticut Masons.

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