Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs—and consequently the profile of individual membrane currents active during that action potential—differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to “normalize” the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4–6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near −80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen–Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.

Abstract-One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na ϩ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (I pst ) at Ϫ20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized I pst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na ϩ currents (I Na ) in HEK-293 cells expressing either wild-type or 1795insD channels. I pst for 1795insD channels varied between 0.8Ϯ0.2% and 1.9Ϯ0.8% of peak I Na . Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, I pst and the negative shift were implemented into SA node AP models. The Ϫ10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas I pst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate I pst (1% to 2%) and the shift reduced sinus rate by Ϸ10%. An additional increase in I pst could result in plateau oscillations and failure to repolarize completely. Thus, Na ϩ channel mutations displaying an I pst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current. Key Words: long-QT syndrome Ⅲ ion channels Ⅲ sinoatrial node Ⅲ electrophysiology Ⅲ sudden death T he inherited long-QT syndrome is a familial rhythm disorder typically characterized by prolongation of the QT-interval on the ECG and life-threatening cardiac arrhythmias. 1,2 One form of the familial long-QT syndrome (LQT3) is caused by mutations in the gene encoding the cardiac sodium channel (SCN5A). 3 To date, the biophysical properties of the mutant sodium channels have been assessed for at least 21 of the distinct genetic mutations in SCN5A that have been linked to LQT3 (see online Table 1, available in the online data supplement at http://www.circresaha.org). In most of these mutations, incomplete or slowed channel inactivation induces a small persistent sodium inward current (I pst ) during prolonged depolarization (see online Table 1). This small I pst at plateau potentials is sufficient to delay repolarization of the action potential and underlies the QT-prolongation on the ECG.Previous research has focused on the correlation between altered Na ϩ channel kinetics and ventricular action potential prolongation, because the latter renders the heart vulnerable to tachyarrhythmias, especially Torsade de Pointes. In addition to the characteristic QT-prolongation, however, bradycardia and sinus pauses have also been associated with the LQT3 phenotype, indicating a ro...

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.