Jamie I. Vandenberg

Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult Scn5a+/− mice demonstrate a ≈50% reduction in sodium conductance. Scn5a+/− hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.

HERG K+ channels: friend and foe

The K+ channel encoded by the human ether-à-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.

Cell swelling and ion transport pathways in cardiac myocytes

Cell swelling causes stretch and/or deformation of cell membranes and the underlying cytoskeletal network as well as dilution of intracellular contents [l]. It is therefore not surprising that most mammalian cells, including cardiac myocytes, respond to swelling by modulating transporters and or ion channels that permit efflux of intracellular osmolytes (and osmotically obliged water) which will tend to restore cell volume to its original value [2-51. This is of particular significance in the heart as many of the transport pathways modulated by cell swelling are electrogenie, so their modulation will alter excitability of the heart. This is likely to be most important in the context of myocardial ischaemia and reperfusion as this is when cell swelling is most significant [6] and when arrhythmias are most common [7]. Recent work, using a range of techniques including patch-clamp analysis of isolated cardiac myocytes, is beginning to unravel the mechanisms underlying osmotic modulation of electrical activity in the heart and it is this work that will be the primary focus of this review.

Domain Reorientation and Rotation of an Intracellular Assembly Regulate Conduction in Kir Potassium Channels.

Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell–derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.

The sodium channel β‐subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart

1 Cardiac sodium channels are composed of a pore‐forming α‐subunit, SCN5a, and one or more auxiliary β‐subunits. The aim of this study was to investigate the role of the recently discovered member of the β‐subunit family, SCN3b, in the heart. 2 Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3 Co‐expression of SCN3b with the cardiac‐specific α‐subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co‐expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4 When measured in cell‐attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half‐voltage of steady‐state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half‐voltage of steady‐state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5 The rates of inactivation for SCN5a co‐expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at −90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6 Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Abstract. Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field.

Effects of premature stimulation on HERG K+ channels

1 The unusual kinetics of human ether‐à‐go‐go‐related gene (HERG) K+ channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K+ channels, transfected in Chinese hamster ovary cells, at 37 °C. 2 HERG K+ channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3 HERG K+ channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90% repolarization (APD90) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD90– 20 ms) and atrial (APD90– 30 ms) action potentials. 4 The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5 A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre‐open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6 These properties of HERG K+ channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

Human ether-a-go-go related gene (hERG) K+ channels: Function and dysfunction

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.

Drug Binding to the Inactivated State Is Necessary but Not Sufficient for High-Affinity Binding to Human Ether-à-go-go-Related Gene Channels

Drug block of the human ether-à-go-go-related gene K+ channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.