Androgenic effects on ventricular repolarization: A translational study from the international pharmacovigilance database to iPSC-cardiomyocytes.

BACKGROUND: Male hypogonadism, arising from a range of etiologies including androgen-deprivation therapies (ADTs), has been reported as a risk factor for acquired long-QT syndrome (aLQTS) and torsades de pointes (TdP). A full description of the clinical features of aLQTS associated with ADT and of underlying mechanisms is lacking. METHODS: We searched the international pharmacovigilance database VigiBase for men (n=6 560 565 individual case safety reports) presenting with aLQTS, TdP, or sudden death associated with ADT. In cardiomyocytes derived from induced pluripotent stem cells from men, we studied electrophysiological effects of ADT and dihydrotestosterone. RESULTS: Among subjects receiving ADT in VigiBase, we identified 184 cases of aLQTS (n=168) and/or TdP (n=68; 11% fatal), and 99 with sudden death. Of the 10 ADT drugs examined, 7 had a disproportional association (reporting odds ratio=1.4-4.7; P<0.05) with aLQTS, TdP, or sudden death. The minimum and median times to sudden death were 0.25 and 92 days, respectively. The androgen receptor antagonist enzalutamide was associated with more deaths (5430/31 896 [17%]; P<0.0001) than other ADT used for prostate cancer (4208/52 089 [8.1%]). In induced pluripotent stem cells, acute and chronic enzalutamide (25µM) significantly prolonged action potential durations (action potential duration at 90% when paced at 0.5Hz; 429.7±27.1 (control) versus 982.4±33.2 (acute, P<0.001) and 1062.3±28.9ms (chronic; P<0.001), and generated afterdepolarizations and/or triggered activity in drug-treated cells (11/20 acutely and 8/15 chronically). Enzalutamide acutely and chronically inhibited delayed rectifier potassium current, and chronically enhanced late sodium current. Dihydrotestosterone (30nM) reversed enzalutamide electrophysiological effects on induced pluripotent stem cells. CONCLUSION: QT prolongation and TdP are a risk in men receiving enzalutamide and other ADTs. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03193138.

Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy.

Mutations in cardiac Troponin T (TnT) are responsible for approximately 15% of all cases of familial hypertrophic cardiomyopathy (FHC). This review summarizes recent data from in vitro assays, transgenic models and clinical studies on how TnT mutations alter the regulation of cardiac muscle contraction. Each TnT mutation has somewhat different effects on myofilament properties (increased myofilament Ca(2)+ sensitivity, decreased maximal force, decreased binding affinity to the thin filament, impaired pH-regulation). But when the in vitro data are correlated with the results from the transgenic models, essentially all mutations can be predicted to result in: (1) impaired relaxation, (2) reduced diastolic compliance, (3) reduced contractile reserve, (4) preserved systolic function under baseline conditions, and (5) cardiac dysfunction under inotropic stimulation. Thus, the alterations of myofilament function caused by TnT mutations likely play an important role in the pathogenesis of FHC.

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, I(Ks). 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.

Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness.

INTRODUCTION: The monophasic action potential (MAP) technique has been validated in humans and larger animals, but, in mice, MAP recordings available to date show little resemblance to the murine ventricular transmembrane action potential (TAP) measured by conventional microelectrodes. We developed a miniaturized MAP contact electrode technique to establish in isolated mouse hearts: (1) optimal electrode size; (2) validation against TAP; (3) relationship between repolarization and refractoriness; and (4) regional repolarization differences. METHODS AND RESULTS: In 30 Langendorff-perfused mouse hearts, MAP electrodes of tip diameter 1.5, 1.0, and 0.25 mm were tested by comparing MAPs and TAPs from epicardial and endocardial surfaces of both ventricles. Only the MAP contact electrode of 0.25-mm tip diameter consistently produced MAP recordings that had wave shapes nearly identical to TAP recordings. MAP durations measured at 30%, 50%, 70%, and 90% repolarization (APD30, APD50, APD70, APD90) highly correlated with TAP measurements (r = 0.97, P < 0.00001). APD50 was significantly longer in endocardial than in epicardial recordings (right ventricle: 9.3+/-1.1 msec vs 3.9+/-1.1 msec; left ventricle: 9.9+/-2.1 msec vs 6.2+/-1.9 msec; both P < 0.001), demonstrating transmural repolarization differences. Effective refractory period (ERP) determined at basic cycle lengths from 70 to 200 msec correlated with 80%+/-6% of total repolarization, with an ERP/APD90 ratio of 0.85+/-0.14. CONCLUSION: Murine myocardial repolarization, regional repolarization heterogeneity, and relation to refractoriness can be assessed reliably by this miniaturized MAP contact electrode technique, which renders action potential wave shapes similar to that of intracellular microelectrodes. This technique may be useful for exploring repolarization abnormalities in genetically altered mice.

Inotropic stimulation induces cardiac dysfunction in transgenic mice expressing a troponin T (I79N) mutation linked to familial hypertrophic cardiomyopathy.

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and a high incidence of sudden death, despite causing little or no cardiac hypertrophy. In skinned fibers, I79N increased myofilamental calcium sensitivity (Miller, T., Szczesna, D., Housmans, P. R., Zhao, J., deFreitas, F., Gomes, A. V., Culbreath, L., McCue, J., Wang, Y., Xu, Y., Kerrick, W. G., and Potter, J. D. (2001) J. Biol. Chem. 276, 3743-3755). To further study the functional consequences of this mutation, we compared the cardiac performance of transgenic mice expressing either human TnT-I79N or human wild-type TnT. In isolated hearts, cardiac function was different depending on the Ca(2+) concentration of the perfusate; systolic function was significantly increased in Tg-I79N hearts at 0.5 and 1 mmol/liter. At higher Ca(2+) concentrations, systolic function was not different, but diastolic dysfunction became manifest as increased end-diastolic pressure and time to 90% relaxation. In vivo measurements by echocardiography and Doppler confirmed that base-line systolic function was significantly higher in Tg-I79N mice without evidence for diastolic dysfunction. Inotropic stimulation with isoproterenol resulted only in a modest contractile response but caused significant mortality in Tg-I79N mice. Doppler studies ruled out aortic outflow obstruction and were consistent with increased chamber stiffness. We conclude that in vivo, the increased myofilament Ca(2+) sensitivity due to the I79N mutation enhances base-line contractility but leads to cardiac dysfunction during inotropic stimulation.

Remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin.

Overexpression of cardiac calsequestrin (CSQ) impairs Ca2+ signalling in murine myocytes, leading to marked cardiac hypertrophy. Here we report on contractile, histological and electrophysiological changes accompanying the development of cardiac hypertrophy and failure in CSQ-overexpressing mice. CSQ mice developed contractile dysfunction after 60 days of age, with only 40% survival at 6 months. Four- to 6-month-old CSQ mice revealed biventricular dilatation, cardiomyocyte hypertrophy, patchy interstitial fibrosis and tissue calcifications. Cardiac hypertrophy of CSQ mice was accompanied by progressive P-R and Q-T interval prolongation, conduction blocks, 2-fold prolongation of the ventricular action potential and increased cellular membrane capacitance. Remodelling of ionic currents included marked reduction of both density and absolute magnitude of transient outward (Ito) and inward rectifying (IK1) K+ currents. The density, but not the absolute magnitude, of basal and isoproterenol (isoprenaline)-stimulated Ca2+ current (ICa) was decreased by 42% and the inactivation kinetics of ICa were significantly slowed. Na+ current density was suppressed by 50%, but its steady-state activation and inactivation were shifted to more positive potentials. The density of Na+-Ca2+ exchange current was increased by 35%. In CSQ but not in control myocytes dialysed with cAMP, isoproterenol continued to enhance ICa. This apparent lower responsiveness of ICa to cAMP could be reversed by the non-hydrolysable cAMP analogue 8-Br-cAMP or the phosphodiesterase inhibitor IBMX, suggesting high phosphodiesterase activity of CSQ myocytes. In young CSQ mice (< 60 days) with compensated cardiac hypertrophy, only Ito was significantly suppressed. All other currents remained relatively intact. An increase in cardiac Ca2+-storage capability by overexpression of CSQ results in a dilated cardiomyopathy with tissue fibrosis, calcifications, impaired beta-adrenergic signalling and progressive remodelling of ionic currents. The extent of the changes in ionic currents was age dependent.

Cardiac actions of erythromycin: influence of female sex.

CONTEXT: Erythromycin is a widely used antibiotic that infrequently causes QT-prolongation and torsades de pointes cardiac arrhythmias. For antiarrhythmic drugs, women are at a higher risk for these cardiac arrhythmias, but few other classes of drugs have been studied. OBJECTIVES: To determine whether female sex is a risk factor for cardiac arrhythmias associated with erythromycin, and if this can be correlated with in vitro measurements of the QT-response to erythromycin in male and female rabbit hearts. DESIGN: Food and Drug Administration (FDA) MEDWATCH database analysis and in vitro experiment. MAIN OUTCOME MEASURES: Cardiac arrhythmia reports associated with erythromycin from 1970 until 1996 classified by patient sex and age, and effect of female sex on erythromycin-induced QT-prolongation in isolated perfused rabbit hearts. RESULTS: We observed a sex difference in cardiac arrhythmias associated with administration of erythromycin. A total of 346 cases were found in the FDA database: 201 females (58%), 110 males (32%), and 35 unspecified (10%). Forty-nine were life-threatening ventricular arrhythmias and deaths directly related to intravenous erythromycin lactobionate: 33 women (67%) and 16 men (33%) (P=.03). During the same period, no sex imbalance was present in the prescription pattern for intravenous erythromycin lacobionate (men 47%, women 49%, unspecified 4%). Perfusion with erythromycin caused significantly greater QT-prolongation in female rabbit hearts (mean [SD], 11.8% [2.3%]) than in male hearts (6.9% [2.1%]; P = .03). CONCLUSIONS: As has been shown in reports of antiarrhythmic drugs, we found a female predominance in the FDA reports of erythromycin-associated cardiac arrhythmias. Based on in vitro experiments, a sex difference in cardiac repolarization response to erythromycin is a potential contributing factor.

Dispersion of ventricular repolarization in the voltage domain.

Dispersion of ventricular repolarization, assessed as QT dispersion in the ECG or by multiple monophasic action potential (MAP) recordings, is defined as the difference between the earliest and latest repolarization. It is thus measured in the time domain. However, myocardial refractoriness is primarily a function of the membrane potential during phase 3 repolarization. The purpose of this study, therefore, was to measure dispersion of ventricular repolarization in the voltage domain and to study its relation to VF inducibility. To further validate this concept, the effect of chronic amiodarone treatment on the voltage dispersion were assessed. MAPs were recorded simultaneously at 10 epicardial and endocardial sites in isolated rabbit hearts, both under baseline conditions (n = 8) and after chronic amiodarone treatment (n = 8). Repolarization dispersion in the voltage domain was calculated as the difference between the highest and lowest repolarization level of all 10 MAPs at 10-ms steps, starting from the MAP plateau level to complete repolarization. Plotting these voltage differences along the time axis resulted in a dispersion curve, which rose during early repolarization, reached a peak during phase 3 repolarization, and thereafter declined toward zero. There was a close correlation between VF vulnerability in response to electrical field stimuli and the time during which voltage dispersion was maximal (r = 0.828, P < 0.0001). Amiodarone caused a right-ward shift of both the dispersion curve (P = 0.007) and VF vulnerability (P = 0.025), but did not change the magnitude nor the shape of the voltage dispersion curve and its relation to VF vulnerability. Repolarization dispersion in the voltage domain describes an alternate approach for evaluating the heterogeneity of ventricular repolarization and may help to characterize arrhythmia susceptibility under experimental conditions.

Assessment of joint review of radiologic studies by a primary care physician and a radiologist.

OBJECTIVES: To assess whether the joint review of radiologic studies by the primary care physician and the radiologist affects patient care and health care costs. DESIGN: Prospective study. SETTING: Student health clinic at a university hospital. PATIENTS: University students seen during acute care visits. INTERVENTION: Joint weekly review of all radiologic studies ordered at the student health clinic between July 1992 and June 1993 by a staff radiologist and internist. MEASUREMENTS AND MAIN RESULTS: The outcome measures were: (1) change of radiologic diagnosis after review process and its effect on patient management; (2) expenses saved or incurred by the review process. Of 323 films ordered, 305 were reviewed, resulting in revisions of 23 (8%) of the initial readings. Sixteen revisions (5%) led to a change in patient management; the remainder were clinically insignificant. In these 16 cases, cancellation or simplification of further workup resulted in savings of $1,967. The cost for extra physician time was $5,499. Thus, the review process incurred a net cost of $3,532. Except for the reduction in diagnostic studies, no therapeutic benefit for the patients could be identified. Film readings in our radiology department were conservative, with a positive predictive value of 85% and a negative predictive value of 99.7%. CONCLUSIONS: Routinely reviewing every radiologic study did not affect patient outcome in an outpatient clinic with low prevalence of disease. Given our radiologists' conservative film-reading practice, a review of only abnormal studies may prove more cost-effective in a healthy population. This type of assessment has implications for improving the efficiency of a changing health care system.