sigma(2)-receptor ligand-mediated inhibition of inwardly rectifying K(+) channels in the heart.

The sigma(2)-receptor agonist, ifenprodil, was suggested as an inhibitor of G protein-coupled inwardly rectifying potassium channels. Nevertheless, an analysis of the role of sigma(2) receptors in cardiac electrophysiology has never been done. This work aims i) to identify the roles of cardiac sigma(2) receptors in the regulation of cardiac K(+) channel conductances and ii) to check whether sigma(2)-receptor agonists exhibit class III antiarrhythmic properties. The sigma(2)-receptor agonists ifenprodil, threo-ifenprodil, LNP250A [threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one] (a derivative of ifenprodil devoid of alpha(1)-adrenergic and N-methyl-d-aspartate glutamate receptor-blocking properties), and 1,3-di(2-tolyl)guanidine were used to discriminate the effects linked to sigma(2) receptors from those of the sigma(1) subtype, induced by (+/-)-N-allylnormetazocine (SKF-10,047). The sigma(2)-receptor antagonist 3-alpha-tropanyl-2(pCl-phenoxy)butyrate (SM-21) was employed to characterize sigma(2)-mediated effects in patch-clamp experiments. In rabbits, all sigma(2)-receptor agonists reduced phenylephrine-induced cardiac arrhythmias. They prolonged action potential duration in rabbit Purkinje fibers and reduced human ether-a-go-go-related gene (HERG) K(+) currents. (+)-SKF-10,047 was completely inactive in the last two tests. The effects of threo-ifenprodil were not antagonized by SM-21. In HERG-transfected COS-7 cells, SM-21 potentiated the ifenprodil-induced blockade of the HERG current. These data suggest that sigma(2)-receptor ligands block I(Kr) and that this effect could explain part of the antiarrhythmic properties of this ligands family. Nevertheless, an interaction with HERG channels not involving sigma(2) receptors seems to share this pharmacological property. This work shows for the first time that particular caution has to be taken toward ligands with affinity for sigma(2) receptors. The repolarization prolongation and the early-afterdepolarization can be responsible for "torsades de pointe" and sudden cardiac death.

Torsades de pointes complicating atrioventricular block: evidence for a genetic predisposition.

BACKGROUND: The prevalence of genetic risk factors has not been systematically evaluated in the setting of complete atriventricular (AV) block complicated by long QT syndrome (LQTS). OBJECTIVE: This study was performed to determine to what extent acquired LQTS in the context of AV block has a genetic substrate. METHODS: Among 420 recipients of pacemakers implanted over a 3-year period, we identified retrospectively 29 patients with complete AV block and a QT interval >600 ms in duration. A second study group included 22 randomly selected patients who had AV block and a QT interval <600 ms. Normal controls were 100 consecutive individuals without medical history. Genetic studies screening for HERG, KCNQ1 KCNE1, KCNE2, and SCN5A mutations were performed. RESULTS: We identified four mutations on genes encoding potassium channels in five patients with AV block and acquired LQTS. These mutations were not found among patients with AV block and a QT interval <600 ms in duration or in healthy volunteers. Functional expression of three HERG mutations (R328C, R696C, and R1047L) had a dominant negative effect on wild-type I(Kr). One KCNE2 mutation (R77W) identified in a patient treated with flecainide did not alter I(Kr). CONCLUSIONS: This study showed that complete AV block complicated by LQTS was associated with HERG mutations in 17% of cases. Further studies are needed to identify factors, genetic or environmental, which may be implicated in bradycardia-related abnormalities of ventricular repolarization.

Expression of human ERG K+ channels in the mouse heart exerts anti-arrhythmic activity.

OBJECTIVE: The K(+) channel encoded by the human ether-a-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (I(Kr)) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart. METHODS AND RESULTS: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the T wave in transgenic mice, but not in WT mice. Injection of BaCl(2) (25 mg/kg) induced short runs of ventricular tachycardia in 9/10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of I(Kr) subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential. CONCLUSION: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

Long-term amiodarone administration remodels expression of ion channel transcripts in the mouse heart.

BACKGROUND: The basis for the unique effectiveness of long-term amiodarone treatment on cardiac arrhythmias is incompletely understood. The present study investigated the pharmacogenomic profile of amiodarone on genes encoding ion-channel subunits. METHODS AND RESULTS: Adult male mice were treated for 6 weeks with vehicle or oral amiodarone at 30, 90, or 180 mg x kg(-1) x d(-1). Plasma and myocardial levels of amiodarone and N-desethylamiodarone increased dose-dependently, reaching therapeutic ranges observed in human. Plasma triiodothyronine levels decreased, whereas reverse triiodothyronine levels increased in amiodarone-treated animals. In ECG recordings, amiodarone dose-dependently prolonged the RR, PR, QRS, and corrected QT intervals. Specific microarrays containing probes for the complete ion-channel repertoire (IonChips) and real-time reverse transcription-polymerase chain reaction experiments demonstrated that amiodarone induced a dose-dependent remodeling in multiple ion-channel subunits. Genes encoding Na+ (SCN4A, SCN5A, SCN1B), connexin (GJA1), Ca2+ (CaCNA1C), and K+ channels (KCNA5, KCNB1, KCND2) were downregulated. In patch-clamp experiments, lower expression of K+ and Na+ channel genes was associated with decreased I(to,f), I(K,slow), and I(Na) currents. Inversely, other K+ channel alpha- and beta-subunits, such as KCNA4, KCNK1, KCNAB1, and KCNE3, were upregulated. CONCLUSIONS: Long-term amiodarone treatment induces a dose-dependent remodeling of ion-channel expression that is correlated with the cardiac electrophysiologic effects of the drug. This profile cannot be attributed solely to the amiodarone-induced cardiac hypothyroidism syndrome. Thus, in addition to the direct effect of the drug on membrane proteins, part of the therapeutic action of long-term amiodarone treatment is likely related to its effect on ion-channel transcripts.

A common antitussive drug, clobutinol, precipitates the long QT syndrome 2.

QT prolongation, a classic risk factor for arrhythmias, can result from a mutation in one of the genes governing cardiac repolarization and also can result from the intake of a medication acting as blocker of the cardiac K(+) channel human ether-a-go-go-related gene (HERG). Here, we identified the arrhythmogenic potential of a nonopioid antitussive drug, clobutinol. The deleterious effects of clobutinol were suspected when a young boy, with a diagnosis of congenital long QT syndrome, experienced arrhythmias while being treated with this drug. Using the patch-clamp technique, we showed that clobutinol dose-dependently inhibited the HERG K(+) current with a half-maximum block concentration of 2.9 microM. In the proband, we identified a novel A561P HERG mutation. Two others long QT mutations (A561V and A561T) had been reported previously at the same position. None of the three mutants led to a sizeable current in heterologous expression system. When coexpressed with wild-type (WT) HERG channels, the three Ala561 mutants reduced the trafficking of WT and mutant heteromeric channels, resulting in decreased K(+) current amplitude (dominant-negative effects). In addition, A561P but not A561V and A561T mutants induced a approximately -11 mV shift of the current activation curve and accelerated deactivation, thereby partially counteracting the dominant-negative effects. A561P mutation and clobutinol effects on the human ventricular action potential characteristics were simulated using the Priebe-Beuckelmann model. Our work shows that clobutinol has limited effects on WT action potential but should be classified as a "drug to be avoided by congenital long QT patients" rather than as a "drug with risk of torsades de pointes".

New KCNQ1 mutations leading to haploinsufficiency in a general population; Defective trafficking of a KvLQT1 mutant.

OBJECTIVE: KCNQ1 mutations lead to the long QT syndrome (LQTS), characterized by a prolonged QT interval, syncopes and sudden death. However, some mutations are associated with non-penetrant phenotype (no symptoms, QTc normal or borderline). The objective of this study was to determine whether KCNQ1 variants are associated with borderline QTc prolongation in a general population and to evaluate the frequency of carriers. METHODS: We selected 2008 unrelated and untreated healthy individuals from a non-patient population. The KCNQ1 gene was screened by denaturing high-performance liquid chromatography (dHPLC) in 50 men and 50 women presenting the longest QTc intervals (403 to 443 ms). RESULTS: We identified a nonsense mutation, Y148X, and an in-frame deletion of the serine residue 276 (DeltaS276), in S2 and S5 transmembrane domains, respectively. DeltaS276 KvLQT1 channels expressed in COS-7 cells failed to conduct any K+ current in the homozygous state. Besides, a slight reduction in channel activity was observed when coexpressed with WT KvLQT1 and IsK. Confocal microscopy performed on transfected COS-7 cells revealed that DeltaS276 KvLQT1 was retained in the endoplasmic reticulum, whereas WT KvLQT1 was localized in the cell membrane. The two mutation carriers presented borderline QTc interval prolongation at slow heart rate but a 24-h ECG recording revealed a marked QTc prolongation at higher heart rate for the Y148X carrier. CONCLUSIONS: In this population, two subjects with borderline QTc prolongations (438 and 443 ms) were carriers of KCNQ1 mutations leading to haploinsufficiency and are potentially at risk of developing drug-induced arrhythmia. The study provides the first demonstration of a defective cell surface localization of a KvLQT1 mutant missing one amino acid in a transmembrane domain.

Mutation in the KCNQ1 gene leading to the short QT-interval syndrome.

BACKGROUND: The electrocardiographic short QT-interval syndrome forms a distinct clinical entity presenting with a high rate of sudden death and exceptionally short QT intervals. The disorder has recently been linked to gain-of-function mutation in KCNH2. The present study demonstrates that this disorder is genetically heterogeneous and can also be caused by mutation in the KCNQ1 gene. METHODS AND RESULTS: A 70-year man presented with idiopathic ventricular fibrillation. Both immediately after the episode and much later, his QT interval was abnormally short without any other physical or electrophysiological anomalies. Analysis of candidate genes identified a g919c substitution in KCNQ1 encoding the K+ channel KvLQT1. Functional studies of the KvLQT1 V307L mutant (alone or coexpressed with the wild-type channel, in the presence of IsK) revealed a pronounced shift of the half-activation potential and an acceleration of the activation kinetics leading to a gain of function in I(Ks). When introduced in a human action potential computer model, the modified biophysical parameters predicted repolarization shortening. CONCLUSIONS: We present an alternative molecular mechanism for the short QT-interval syndrome. Functional and computational studies of the KCNQ1 V307L mutation identified in a patient with this disorder favor the association of short QT with mutation in KCNQ1.

Microarray analysis reveals complex remodeling of cardiac ion channel expression with altered thyroid status: relation to cellular and integrated electrophysiology.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I(to,f) and I(K,slow) densities, and increased I(Ks) density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the alpha1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl-, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel alpha- and beta-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.