Jan G. Zegers

Contribution of Sodium Channel Mutations to Bradycardia and Sinus Node Dysfunction in LQT3 Families

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+ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (Ipst) at −20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8±0.2% and 1.9±0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The −10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by ≈10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst 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.

Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: a dynamic clamp study with virtual IK1.

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.

Ca2+‐activated Cl− current in rabbit sinoatrial node cells

The Ca2+‐activated Cl− current (ICl(Ca)) has been identified in atrial, Purkinje and ventricular cells, where it plays a substantial role in phase‐1 repolarization and delayed after‐depolarizations. In sinoatrial (SA) node cells, however, the presence and functional role of ICl(Ca) is unknown. In the present study we address this issue using perforated patch‐clamp methodology and computer simulations. Single SA node cells were enzymatically isolated from rabbit hearts. ICl(Ca) was measured, using the perforated patch‐clamp technique, as the current sensitive to the anion blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS). Voltage clamp experiments demonstrate the presence of ICl(Ca) in one third of the spontaneously active SA node cells. The current was transient outward with a bell‐shaped current‐voltage relationship. Adrenoceptor stimulation with 1 μm noradrenaline doubled the ICl(Ca) density. Action potential clamp measurements demonstrate that ICl(Ca) is activate late during the action potential upstroke. Current clamp experiments show, both in the absence and presence of 1 μm noradrenaline, that blockade of ICl(Ca) increases the action potential overshoot and duration, measured at 20 % repolarization. However, intrinsic interbeat interval, upstroke velocity, diastolic depolarization rate and the action potential duration measured at 50 and 90 % repolarization were not affected. Our experimental data are supported by computer simulations, which additionally demonstrate that ICl(Ca) has a limited role in pacemaker synchronization or action potential conduction. In conclusion, ICl(Ca) is present in one third of SA node cells and is activated during the pacemaker cycle. However, ICl(Ca) does not modulate intrinsic interbeat interval, pacemaker synchronization or action potential conduction.

Dynamic action potential clamp as a powerful tool in the development of a gene-based bio-pacemaker

The development of a genetically engineered ‘biological pacemaker’, or ‘bio-pacemaker’, is a rapidly emerging field of research. One of the approaches in this field is to turn intrinsically quiescent myocardial cells, i.e., atrial or ventricular cells, into pacemaker cells by making them express the cardiac hyperpolarization-activated ‘pacemaker current’ If (known in neurophysiology as Ih), which is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) gene family. We carried out ‘dynamic action potential clamp’ (dAPC) experiments in which we record current from a HEK-293 cell transfected with HCN4, which is the dominant HCN isoform in the sinoatrial (SA) node. This HCN4-transfected HEK-293 cell is voltage-clamped by the action potential generated in a real-time simulation of a human atrial cell (Courtemanche-Ramirez-Nattel model). In a continuous feedback loop, this current is injected into the atrial cell, so that this cell effectively expresses an HCN4-based pacemaker current. With sufficiently high ‘expression levels’ of HCN4 current the atrial cell is turned into a pacemaker cell with an SA nodal like action potential. Lower expression levels are sufficient if the inward rectifier potassium current (IK1), which is largely responsible for the stable resting potential of atrial cells, is ‘down-regulated’ by 50%, thus mimicking the gene therapy strategy to create a bio-pacemaker by down-regulation of IK1 and (over-)expression of If. Our dAPC experiments provide direct insights into the effects of introducing HCN4 current into an atrial cell, illustrating that dynamic action potential clamp can be a powerful tool in the process of developing a gene-based bio-pacemaker.

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

Development of a Genetically Engineered Cardiac Pacemaker: Insights from Dynamic Action Potential Clamp Experiments

In this chapter, we briefly review the use of dynamic clamp in cardiac cellular electrophysiology and present novel results obtained with the ‘dynamic action potential clamp’ (dAPC) technique. This is a technique that we recently developed to study the effects of long-QT syndrome-related ion channel mutations by effectively replacing the associated native ionic current of a cardiac myocyte with wild-type or mutant current recorded from a HEK-293 cell that is voltage clamped by the free-running action potential of the myocyte. Here we demonstrate that the dAPC technique can also be used as a powerful tool in the development of a ‘biological pacemaker’ or ‘bio-pacemaker,’ i.e. a genetically engineered cardiac pacemaker. We record ‘pacemaker current’ from an HCN4-transfected HEK-293 cell and inject this current into a model of a human atrial cell of which the free-running membrane potential is used to voltage clamp the HEK-293 cell. Thus we explore the conditions under which this HCN4-based pacemaker current turns the atrial cell into a pacemaker cell.

Bradycardia in LQT3 patients: insights from 0D models

Type 3 of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene (SCN5A). LQT3 carriers regularly present with bradycardia and sinus pauses. This has been reported for carriers of the /spl Delta/K1500, /spl Delta/KPQ, E1784K, D1790G, and 1795insD mutations, which all show a persistent inward sodium current and/or a negative shift in voltage dependence of inactivation. The 1795insD channel had previously been characterized by a -10 mV. We hypothesized that the relative bradycardia that has been reported for carriers of the 1795insD mutation is, at least in part, due to intrinsic slowing of the sinus node. Therefore, we carried out computer simulations to test the effects of the 1795insD mutation on the intrinsic pacemaker activity of sinus node cells. We found that the negative shift in inactivation reduces sinus rate by slowing diastolic depolarization, whereas the persistent inward current reduces sinus rate by an increase in action potential duration, and that these effects are almost additive. Also, sinus node cells may fail to repolarize. We conclude that sodium channel mutations may account for the relative bradycardia and sinus node dysfunction seen in LQT3 patients.