Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K(+) channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells.

BACKGROUND AND PURPOSE: Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I(Kr) currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K(+) channels. EXPERIMENTAL APPROACH: Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. KEY RESULTS: Zolpidem caused acute hERG channel blockade in oocytes (IC(50) = 61.5 µM) and in HEK 293 cells (IC(50) = 65.5 µM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I-V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: Zolpidem inhibits cardiac hERG K(+) channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.

Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes.

To evaluate the roles of the C-termini of K + channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K + channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when co-expressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K + currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.

Implication of the C-terminal region of the alpha-subunit of voltage-gated sodium channels in fast inactivation.

The alpha-subunit of both the human heart (hH1) and human skeletal muscle (hSkM1) sodium channels were expressed in a mammalian expression system. The channels displayed slow (hH1) and fast (hSkM1) current decay kinetics similar to those seen in native tissues. Hence, the aim of this study was to identify the region on the alpha-subunit involved in the differences of these current-decay kinetics. A series of hH1/hSkM1 chimeric sodium channels were constructed with the focus on the C-terminal region. Sodium currents of chimeric channels were recorded using the patch-clamp technique in whole-cell configuration. Chimeras where the C-terminal region had been exchanged between hH1 and hSkM1 revealed that this region contains the elements that cause differences in current decay kinetics between these sodium channel isoforms. Other biophysical characteristics (steady-state activation and inactivation and recovery from inactivation) were similar to the phenotype of the parent channel. This indicates that the C-terminus is exclusively implicated in the differences of current decay kinetics. Several other chimeras were constructed to identify a specific region of the C-terminus causing this difference. Our results showed that the first 100-amino-acid stretch of the C-terminal region contains constituents that could cause the differences in current decay between the heart and skeletal muscle sodium channels. This study has uncovered a direct relationship between the C-terminal region and the current-decay of sodium channels. These findings support the premise that a novel regulatory component exists for fast inactivation of voltage-gated sodium channels.

Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes.

UNLABELLED: Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death. OBJECTIVE: In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene). METHOD: The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique. RESULTS: The R1512W mutation found in our first patient diagnosed with Brugada syndrome produced a slowing of both inactivation and recovery from inactivation. The R4132G mutation found in our second patient who also presented Brugada syndrome, resulted in no measurable sodium currents. Both Brugada syndrome patients showed ST segment elevation and right bundle-branch block, and had experienced syncopes. The E1784K mutation found in the LQTS showed a persistent inward sodium current, a hyperpolarized shift of the steady-sate inactivation and a faster recovery from inactivation. CONCLUSION: The different clinical manifestations of these three mutations most probably originate from the distinct electrophysiological abnormalities of the mutant cardiac sodium channels reported in this study.

Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel.

UNLABELLED: The conserved isoleucine-phenylalanine-methionine (IFM) hydrophobic cluster located in the III-IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate in these channels. OBJECTIVES: The aim of our study was to probe the contribution of each amino acids of the IFM cluster to the inactivation. METHODS: A combination of site-directed mutagenesis, cysteine covalent modification and electrophysiological recording techniques were used to elucidate the role of isoleucine1485 and methionine1487 on hH1 sodium channels expressed in tsA201 cells. RESULTS: Mutant I1485C behaves like mutant F1486C studied earlier: producing an incomplete inactivation (residual current), a slowing and change in the voltage-dependence of the time constants of current decay, a shift of the steady-state inactivation to more depolarized voltages, and a faster recovery from inactivation than the wild-type hH1. The electrophysiological parameters of mutant M1487C are similar to those of wild-type hH1 except for the presence of a residual current. Exposure of the cytoplasmic surface of the mutants to MTS reagents MTSES, MTSET and MTSBn further disrupted inactivation. In order to explain differences in the amplitude of the sustained currents recorded in the presence of MTSES or MTSET, we studied the effects of exposure of mutants 11485C, F1486C and M1487C to acidic and basic pH in the absence and presence of MTSES and MTSET. The effects of MTSES [negatively charged (-)] and MTSET (+) on the amplitude of the residual current of mutant F1486C were modulated by changes in intracellular pH. CONCLUSION: Isoleucine1487 and methionine1485, which surround phenylalanine1487 contribute to stabilizing the inactivation particle for fast inactivation.

Effective use of BCH-2763, a new potent injectable direct thrombin inhibitor, in combination with tissue plasminogen activator (tPA) in a rat arterial thrombolysis model.

Current therapeutic use of heparin as an adjunct to thrombolytic therapy for myocardial infarction is suboptimal with respect to efficacy and bleeding risk. In a rat carotid arterial thrombolysis model (FeCl3-induced injury) we evaluated the combined effect of tPA (2.0 mg/kg/30 min) with our potent injectable direct thrombin inhibitor, BCH-2763 (Ki 0.11 nM; MW 1.5 kDa), which, unlike heparin, inhibits bound and free thrombin; comparisons were with standard heparin (SH), other direct thrombin inhibitors, r-hirudin (MW 6.5 kDa) and hirulog (MW 2.3 kDa), or tPA alone. Time to lysis (TL), patency time (PT), aPTT (fold increase) and bleeding time (BT) were determined. ED100 (100% of rats reperfused) for BCH-2763, hirulog or r-hirudin was 1, 3 or 2 mg/kg/60 min, respectively; 67% of rats reperfused with SH at the highest dose tested (220 U/kg/60 min) and 43% with tPA alone. At these doses, TL (min) was shorter (p < 0.01) with BCH-2763 (0.5 +/- 0.1), hirulog (3.3 +/- 2.3) or r-hirudin (2.3 +/- 1.0) than SH (66.3 +/- 30.8) or tPA alone (93.4 +/- 21.4). The aPTT fold increase after 15 min infusion was markedly greater (p < 0.001) for SH (32.0 +/- 0.8) than BCH-2763 (3.7 +/- 0.5), hirulog (5.2 +/- 0.3) or r-hirudin (4.5 +/- 0.8) in combination with tPA or tPA alone (1.1 +/- 0.1). In addition, the BT (min) for BCH-2763 (3.0 +/- 0.4) was similar to tPA alone (1.6 +/- 0.3), but prolonged (p < 0.05) for hirulog (7.5 +/- 2.7), r-hirudin (6.6 +/- 0.8) or SH (7.3 +/- 1.8). Comparisons at same aPTT fold increase revealed that in combination with tPA, BCH-2763 required a lower anticoagulant level to shorten the TL and prolong the PT than hirulog, r-hirudin or SH. Thus, in this rat arterial thrombolysis model direct thrombin inhibitors are more effective than SH as antithrombotic adjuncts to tPA. BCH-2763 is effective at a lower gravimetric dose and more modest aPTT fold increase than hirulog or r-hirudin with less alteration in haemostasis, which may confer an improved safety index.

Electrophysiological study of chimeric sodium channels from heart and skeletal muscle.

The alpha-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the alpha-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 alpha-subunit framework confirmed the following conclusions. (i) The D4 and or/C-ter. are responsible for the slow inactivation of hH1 sodium channels. (ii) Concerning the other differences between rSkM1 and hH1: steady-state activation and inactivation, kinetics of recovery from inactivation, the phenotypes are determined probably by more than one domain of the alpha-subunit.

BCH-2763, a novel potent parenteral thrombin inhibitor, is an effective antithrombotic agent in rodent models of arterial and venous thrombosis--comparisons with heparin, r-hirudin, hirulog, inogatran and argatroban.

Current clinical use of heparin as an antithrombotic agent is limited by suboptimal efficacy and safety considerations. Thrombin's central role in thrombosis makes it an attractive target to develop more effective and safer antithrombotic agents. BCH-2763 is a novel, potent (Ki: 0.11 nM), low molecular weight (1.51 kDa), bivalent direct thrombin inhibitor. The antithrombotic efficacy of BCH-2763 in vivo following i.v. bolus plus infusion in rats was compared in arterial and venous thrombosis models with two other bivalent direct thrombin inhibitors, r-hirudin and hirulog, with two catalytic site-directed thrombin inhibitors, inogatran and argatroban, and with heparin. In vivo efficacy was related to inhibition in vitro of fibrin clot formation, thrombin-induced aggregation of rat or human washed platelets and activity of free and plasma clot-bound thrombin. All the direct thrombin inhibitors were effective on both arterial and venous thrombosis at markedly lower fold aPTT increases than heparin. The antithrombotic doses of all inhibitors against venous thrombosis were less than against arterial thrombosis. The rank order of potency based on doses (mg/kg/h) required for full efficacy against arterial thrombosis was BCH-2763 (1.2) > inogatran (1.5) > r-hirudin (1.8) > hirulog (3.3) > argatroban (> 3.0); heparin required a markedly higher dose (5.7). In venous thrombosis the doses required for full efficacy were substantially lower for the bivalent (BCH-2763: 0.12; r-hirudin: 0.12; hirulog: 0.18) than for the catalytic site-directed (inogatran: 0.48; argatroban: 0.90) thrombin inhibitors; the dose required for heparin was 0.19. All the direct thrombin inhibitors caused similar shifts in aPTT at doses required to inhibit arterial thrombosis, but BCH-2763 inhibited venous thrombosis at lower aPTT fold increases. In vivo antithrombotic efficacy of direct thrombin inhibitors correlated with their inhibitory activity in vitro against fibrin clot formation and platelet aggregation. In contrast to heparin, all the direct thrombin inhibitors inhibited plasma clot-bound thrombin, but the relative IC50s did not correlate with their antithrombotic efficacy. In summary, direct thrombin inhibitors are more effective than heparin in inhibiting arterial and venous thrombosis in rats with less aPTT increases. BCH-2763 is effective at lower doses than the other direct thrombin inhibitors and for venous thrombosis at a smaller aPTT increase. BCH-2763 may offer an improved therapeutic index in the treatment of thromboembolic complications over heparin and other direct thrombin inhibitors.

Restoration of fast inactivation in an inactivation-defective human heart sodium channel by the cysteine modifying reagent benzyl-MTS: analysis of IFM-ICM mutation.

It has been suggested that the region linking domain III and IV of voltage-gated sodium channels forms the inactivation gate. A combination of site-directed mutagenesis, cysteine covalent modification, and electrophysiological recording techniques was used to identify the role of the Phe1486, a conserved phenylalanine residue located in the III-IV linker of Na+ channels. This Phe1486 is part of a hydrophobic amino acid cluster (IFM) that was proposed to play an essential role in the fast inactivation of voltage-gated sodium channels. Expression in tsA201 cells of an altered human heart 1 Na+ channel (hH1/F1486C) in which Phe1486 was replaced by a cysteine is associated with the appearance of a residual current, a loss of voltage-dependence of the time constants of inactivation, a shift of the steady-state inactivation to more depolarized voltages, and a recovery from inactivation that is faster than the wild-type hH1. Exposure of the cytoplasmic surface of mutant F1486C to the methanthiosulfonate reagents, MTSEA, MTSET, and MTSES, further disrupted macroscopic inactivation, but exposure to MTSBN completely restores fast inactivation and the voltage-dependence of fast inactivation. These findings support the formulation that the IFM motif of the III-IV-linker of voltage-gated sodium channels serves as an essential component of the inactivation particle and that the phenyl group of Phe1486 may play a crucial role in inactivation gate closure.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control.

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells.