Steven N. Ebert

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

QTc interval prolongation associated with intravenous methadone

&NA; Numerous medications prolong the rate‐corrected QT (QTc) interval and induce arrhythmias by blocking ionic current through cardiac potassium channels composed of subunits expressed by the human ether‐a‐go‐go‐related gene (HERG). Recent reports suggest that high doses of methadone cause torsades de pointes. To date, no controlled study has described an association between methadone and QTc prolongation. The only commercial formulation of parenteral methadone available in the United States contains the preservative chlorobutanol. The objectives of this study are to determine: (1) whether the administration of intravenous (i.v.) methadone causes QTc prolongation in humans; (2) whether methadone and/or chlorobutanol block cardiac HERG potassium currents (IHERG) in vitro. Over 20 months, we identified every inpatient with at least one electrocardiogram (ECG) performed on i.v. methadone. For each patient, we measured QTc intervals for every available ECG performed on and off i.v. methadone. Concurrent methadone doses were also recorded. Similar data were collected for a separate group of inpatients treated with i.v. morphine. In a separate set of experiments IHERG was evaluated in transfected human embryonic kidney cells exposed to increasing concentrations of methadone, chlorobutanol, and the two in combination. Mean difference (±standard error) per patient in QTc intervals on and off methadone was 41.7 (±7.8) ms, p<0.0001. Mean difference in QTc intervals on and off morphine was 9.0 (±6.1) ms, p=0.15. The approximately linear relationship between QTc measurements and log‐dose of methadone was significant (p<0.0001). Methadone and chlorobutanol independently block IHERG in a concentration‐dependent manner with IC50 values of 20±2 &mgr;M and 4.4±0.3 mM, respectively. Chlorobutanol potentiates methadones ability to block IHERG. Methadone in combination with chlorobutanol is associated with QTc interval prolongation. Our data strongly suggest that methadone in combination with chlorobutanol is associated with QTc interval prolongation.

In vivo androgen treatment shortens the QT interval and increases the densities of inward and delayed rectifier potassium currents in orchiectomized male rabbits

OBJECTIVESnWomen have longer rate-corrected QT intervals (QTc) and are at higher risk for developing life-threatening torsades de pointes ventricular arrhythmias than men, especially after taking medications that block cardiac human ether-a-go-go-related gene (HERG)-encoded K(+) channels. The purpose of the present study was to determine if the male sex steroid hormone, dihydrotestosterone (DHT), influences QT intervals in orchiectomized (Orch) male rabbits.nnnMETHODSnECG and whole-cell patch-clamp analyses were employed to evaluate cardiac repolarization and K(+) currents in hearts isolated from orchiectomized (Orch) male New Zealand White rabbits receiving subcutaneous sustained release pellets for either dihydrotestosterone (DHT) or placebo. The efficacy of the treatment paradigm was monitored by measuring plasma DHT concentrations before and after the treatment period (10-14 days).nnnRESULTSnThe results show that rate- and drug-induced QT-lengthening is attenuated in hearts from DHT-treated rabbits relative to placebo-treated controls. No significant changes in QRS were observed in response to DHT, thereby indicating that DHT influences QT primarily through an effect on ventricular repolarization. In addition, hearts from DHT-treated rabbits displayed significantly less QT lengthening in response to quinidine challenge compared to placebo controls. Current densities for two important cardiac repolarizing K(+) currents, I(K1) and I(Kr), were found to be significantly increased in ventricular myocardium of DHT-treated rabbits. Further, the half-maximal voltage of activation (V(1/2)) for I(Kr) was significantly shifted to more negative potentials in myocytes from DHT vs. placebo hearts (21.2+/-1.2 vs. 30.2+/-1.4 mV, respectively, n=12, P<0.001). Corresponding changes in rabbit ether-a-go-go-related gene (RERG) mRNA were not found when examined by Northern blot hybridization.nnnCONCLUSIONSnThese results suggest that the presence of male sex steroid hormones in male rabbits helps to suppress rate- and drug-induced delays in cardiac repolarization. DHT action produces increased current densities for I(K1) and I(Kr) and a left-shift in the V(1/2) for I(Kr) that could account, at least in part, for the observed QTc differences between males and females. Since little change was seen in ventricular RERG gene expression, DHT action in the heart may influence I(Kr) via post-transcriptional and/or post-translational mechanisms.

Female gender is a risk factor for torsades de pointes in an in vitro animal model.

Recent clinical observations indicate that female gender is associated with a higher risk of developing torsades de pointes (TdP) cardiac arrhythmia. In this study, we used the Langendorff technique in isolated perfused rabbit hearts and the whole-cell patch-clamp technique in ventricular myocytes to examine the gender difference in TdP incidence and gain insight into the underlying mechanisms. Isolated rabbit hearts were perfused by using the Langendorff technique. TdP was induced by abrupt changes of cycle length (deltaCL) in the presence of Tyrodes solution containing 1 mM 4-aminopyridine (4AP) and 50% reduced Mg2+ and K+ (low K/Mg). The effects of 1 mM 4AP on cardiac potassium currents were characterized by using the patch-clamp technique. Results demonstrated that (a) no significant gender difference was observed in the absolute QT interval before or after 4AP perfusion in the presence of low K/Mg; (b) 4AP caused marked QT prolongation in the ECG; (c) a significantly higher TdP incidence (nine of 12) was found in female hearts compared with male hearts (three of 12; p < 0.05); (d) 1 mM 4AP primarily inhibited Ito, although a slight inhibition of IKr also occurred in low-K/Mg Tyrodes solution. (e) No inhibition of IK1 was observed. (f) No gender difference was found in the potassium current block produced by 4AP. Female gender is associated with a higher incidence of TdP in an experimental isolated heart model and mechanisms subsequent to QT prolongation may contribute to such a gender difference.

Comparison of tegaserod (HTF 919) and its main human metabolite with cisapride and erythromycin on cardiac repolarization in the isolated rabbit heart.

Tegaserod (HTF 919) is a new drug being developed for gastrointestinal motility disorders. Because other gastrointestinal prokinetic agents, such as cisapride and erythromycin, cause slowing of cardiac repolarization and have been implicated in the development of the potentially fatal ventricular arrhythmia, torsades de pointes, a study was initiated to determine whether tegaserod and its main human metabolite adversely influence cardiac repolarization. By using isolated Langendorff-perfused rabbit hearts, we show that QT intervals remain unchanged at concentrations of tegaserod from 0.5 to 10 microM. It was not until the tegaserod concentration was increased to 50 microM (roughly 500-5,000 times more concentrated than those typically found in human plasma after administration of recommended clinical dosages), that a small, but significant increase in the QT interval (12+/-4%; p < 0.05; n = 4) was observed. No significant changes in QT occurred in the presence of the tegaserod metabolite at any of the concentrations tested (0.5-50 microM). In contrast, cisapride caused QT lengthening at concentrations as low as 0.1 microM, with significant QT increases occurring when 5-50 microM cisapride was used (22+/-4% to >70%, respectively; p < 0.01; n = 4). Erythromycin also caused significant lengthening of QT intervals (11+/-2%; p < 0.001; n = 4), although 100 microM concentrations of this drug were required to achieve this effect. These results demonstrate that both cisapride and erythromycin can slow cardiac repolarization at therapeutic doses and that tegaserods lack of QT prolongation at therapeutic doses suggests that it has the potential to be a safer alternative to cisapride as a gastrointestinal prokinetic agent.

Targeted Insertion of the Cre-Recombinase Gene at the Phenylethanolamine n-Methyltransferase Locus: A New Model for Studying the Developmental Distribution of Adrenergic Cells

To evaluate the developmental distribution of adrenergic cells in vivo, we inserted the Cre‐recombinase gene into the locus encoding for the epinephrine biosynthetic enzyme phenylethanolamine n‐methyltransferase (Pnmt) and crossed these Pnmt‐Cre mice with ROSA26 reporter (R26R) mice to activate LacZ (encoding β‐galactosidase) expression in cells that were selectively derived from the adrenergic lineage. Our data show the following: (1) Insertion of Cre‐recombinase into the Pnmt locus created a functional knockout of Pnmt expression with concomitant loss of epinephrine in homozygous PnmtCre/Cre mice; (2) Despite the reduction in Pnmt expression and epinephrine production in PnmtCre/Cre mice, these mice were viable and fertile, with no apparent developmental defects; (3) When crossed with R26R mice, Pnmt‐Cre activation of LacZ expression faithfully recapitulated Pnmt expression in vivo; and (4) LacZ expression was activated in substantial numbers of pacemaking, conduction, and working cardiomyocytes. Developmental Dynamics 231:849–858, 2004.

conventional Antihistamines Slow Cardiac Repolarization in Isolated Perfused (langendorff) Feline Hearts

We examined the effects of conventional antihistamines on cardiac repolarization by using the isolated perfused feline heart model. Representative drugs from the major classes of antihistamines were tested. Each of the antihistamines evaluated in this study elicited a dose-dependent slowing of cardiac repolarization, as indicated by the QT prolongations observed from electrocardiogram (ECG) tracings recorded during these experiments. The concentrations of drugs tested ranged from 1 to 30 microM. Of the drugs analyzed, clemastine and hydroxyzine appeared to be the most potent (relative EC50 values, 5.2 and 6.6 microM, respectively), causing the QT to lengthen by as much as 40-50% at a concentration of 10 microM. Brompheniramine, chlorpheniramine, and diphenhydramine displayed intermediate potencies with respect to QT prolongation (relative EC50 values, 11-13 microM), whereas cyproheptadine, chlorcyclizine, and promethazine were the least potent of the antihistamines tested (relative EC50 values, 16-20 microM). It is concluded that the antihistamines evaluated in this study act directly on the heart to slow cardiac repolarization. These findings could have important clinical relevance for patients taking excessive dosages of conventional antihistamines and those at risk of developing cardiac arrhythmias.

Generating Mouse Models for Studying the Function and Fate of Intrinsic Cardiac Adrenergic Cells

Abstract: Embryos lacking the ability to synthesize epinephrine and norepinephrine die (probably due to cardiac failure) without exogenous supplementation while mutant neonates can grow into fertile adults without supplementation. These experiments define a critical period during embryogenesis, when norepinephrine and/or epinephrine are essential for mouse development. The critical period is prior to sympathetic innervation of the heart and prior to synthesis of catecholamines by the adrenal medullae. Recent work indicates that the developing heart is likely to be a major source of catecholamines in the developing mammalian embryo. The spatial pattern of biosynthetic enzymes suggests an association of the intrinsic cardiac adrenergic cells with the developing pacemaker and cardiac conduction cells. To address the functional characteristics and the fate of these cardiac adrenergic cells, we have developed two mouse models that allow us to identify and to characterize the adrenergic cells and their descendants.