Cardiovascular event rates and trajectories of LDL-cholesterol levels and lipid-lowering therapy in patients with atherosclerotic cardiovascular disease: A population-based cohort study.

BACKGROUND: An understanding of cardiovascular event rates and low-density lipoprotein cholesterol (LDL-C) levels and trajectories in patients with atherosclerotic cardiovascular disease is needed to evaluate treatment goals and adherence to guidelines. METHODS: We conducted a population-based cohort study in the North and Central Denmark Regions. Patients with prevalent atherosclerotic cardiovascular disease (myocardial infarction, non-hemorrhagic stroke, or peripheral artery disease) during 2006-2009 were identified. All patients received lipid-lowering therapy (statins or ezetimibe) and had LDL-C levels ≥1.8 mmol/L at baseline (January 1, 2010). We followed patients for 6 years until a primary composite outcome of cardiovascular death, myocardial infarction, non-hemorrhagic stroke, hospitalization for unstable angina, or coronary revascularization. Additionally, we characterized changes in LDL-C levels and use of statins during follow-up. RESULTS: The study included 10,772 patients (median age 69.2 years, 60.4% male). The overall event rate for the primary outcome was 62.7 (95% confidence interval: 59.2-66.2) per 1000 person-years. This event rate was higher among men than among women and increased with age and baseline LDL-C levels. Approximately 25% of patients with LDL-C measurements during follow-up achieved LDL-C levels below 1.8 mmol/L. Of the approximately two-thirds of patients using statins at the end of follow-up, nearly all patients (97%) received high-intensity therapy. CONCLUSIONS: In this population of patients with atherosclerotic cardiovascular disease, we found high cardiovascular event rates, which increased with baseline LDL-C levels. Although most patients were on high-intensity statin therapy at end of follow-up, only one-quarter reached the guideline-recommended target LDL-C level ≤ 1.8 mmol/L.

Circadian rhythm in QT interval is preserved in mice deficient of potassium channel interacting protein 2.

Potassium Channel Interacting Protein 2 (KChIP2) is suggested to be responsible for the circadian rhythm in repolarization duration, ventricular arrhythmias, and sudden cardiac death. We investigated the hypothesis that there is no circadian rhythm in QT interval in the absence of KChIP2. Implanted telemetric devices recorded electrocardiogram continuously for 5 days in conscious wild-type mice (WT, n = 9) and KChIP2-/- mice (n = 9) in light:dark periods and in complete darkness. QT intervals were determined from all RR intervals and corrected for heart rate (QT100 = QT/(RR/100)1/2). Moreover, QT intervals were determined from complexes within the RR range of mean-RR ± 1% in the individual mouse (QTmean-RR). We find that RR intervals are 125 ± 5 ms in WT and 123 ± 4 ms in KChIP2-/- (p = 0.81), and QT intervals are 52 ± 1 and 52 ± 1 ms, respectively(p = 0.89). No ventricular arrhythmias or sudden cardiac deaths were observed. We find similar diurnal (light:dark) and circadian (darkness) rhythms of RR intervals in WT and KChIP2-/- mice. Circadian rhythms in QT100 intervals are present in both groups, but at physiological small amplitudes: 1.6 ± 0.2 and 1.0 ± 0.3 ms in WT and KChIP2-/-, respectively (p = 0.15). A diurnal rhythm in QT100 intervals was only found in WT mice. QTmean-RR intervals display clear diurnal and circadian rhythms in both WT and KChIP2-/-. The amplitude of the circadian rhythm in QTmean-RR is 4.0 ± 0.3 and 3.1 ± 0.5 ms in WT and KChIP2-/-, respectively (p = 0.16). In conclusion, KChIP2 expression does not appear to underlie the circadian rhythm in repolarization duration.

MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish.

Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP.

A novel KCND3 gain-of-function mutation associated with early-onset of persistent lone atrial fibrillation.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K(+) Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. METHODS AND RESULTS: Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. CONCLUSION: Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility.

The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts.

BACKGROUND: Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. OBJECTIVE: To test the hypothesis that the voltage-sensitive dye di-4-ANEPPS modulates cardiac impulse propagation. METHODS: Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of di-4-ANEPPS on gap junctional conductance. RESULTS: In electrical mapping experiments, di-4-ANEPPS (7.5 µM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of di-4-ANEPPS was dose dependent. di-4-ANEPPS had no detectable effect on connexin 43-mediated gap junctional conductance in transfected HeLa cells. CONCLUSION: Our results demonstrate that the voltage-sensitive dye di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using di-4-ANEPPS in cardiac preparations.

Extracellular potassium inhibits Kv7.1 potassium channels by stabilizing an inactivated state.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1-50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC(50) of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2-5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

Pharmacological activation of Kv11.1 in transgenic long QT-1 rabbits.

Transgenic rabbits expressing pore mutants of K(V)7.1 display a long QT syndrome 1 (LQT1) phenotype. Recently, NS1643 has been described to increase I(Kr).We hypothesized that NS1643 would shorten the action potential duration (APD(90)) in LQT1 rabbits. Transgenic LQT1 rabbits were compared with littermate control (LMC) rabbits. In vivo electrocardiogram studies in sedated animals were performed at baseline and during 45 minutes of intravenous infusion of NS1643 or vehicle in a crossover design. Ex vivo monophasic action potentials were recorded from Langendorff-perfused hearts at baseline and during 45-minute perfusion with NS1643. Left ventricular refractory periods were assessed before and after NS1643 infusion. Genotype differences in APD accommodation were also addressed. In vivo NS1643 shortened the QTc significantly in LQT1 compared with vehicle. In Langendorff experiments, NS1643 significantly shortened the APD(90) in LQT1 and LMC [32.0 ± 4.3 milliseconds (ms); 21.0 ± 5.0 ms] and left ventricular refractory periods (23.7 ± 8.3; 22.6 ± 9.9 ms). NS1643 significantly decreased dp/dt (LQT1: 49% ± 3%; LMC: 63% ± 4%) and increased the incidence of arrhythmia. The time course of APD adaptation was impaired in LQT1 rabbits and unaffected by I(Kr) augmentation. In conclusion, K(V)11.1 channel activation shortens the cardiac APD in a rabbit model of inherited LQT1, but it comes with the risk of excessive shortening of APD.

Role of ERG1 isoforms in modulation of ERG1 channel trafficking and function.

The 'ether-a-go-go-related' gene type 1 (ERG1 or Kv11.1) protein is the product of the KCNH2 gene. Currents generated by ERG1 channels are important in a range of tissues including neuronal, smooth muscle, and cardiac tissues, as well as in cancer cells. There are five known isoforms of the ERG1 protein. Overlapping patterns of endogenous expression of ERG1 isoforms have been described in several tissue types. Abnormal changes in the relative abundance of ERG1 isoforms may result in disease. Recent studies have suggested that the different isoforms play a prominent role in expression and trafficking of ERG1 channels as well as in modulating the electrophysiological properties of the channels. This review focuses on the differences between the ERG1 isoforms and describes the physiological implications thereof. It is described how changes in the relative expression level of the isoforms may have significant physiological consequences by modulation of tissue excitability. Additionally, the review proposes a standardized nomenclature of ERG1 isoforms based on their structural features.

Differential expression of hERG1 channel isoforms reproduces properties of native I(Kr) and modulates cardiac action potential characteristics.

BACKGROUND: The repolarizing cardiac rapid delayed rectifier current, I(Kr), is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr). Marked heterogeneity in the kinetic properties of native I(Kr) has been described. We hypothesized that the heterogeneity of native I(Kr) can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD) and restitution. METHODOLOGY/PRINCIPAL FINDINGS: The results show that the heterogeneity of native I(Kr) can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased. CONCLUSIONS/SIGNIFICANCE: Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr) kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr). Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

Pharmacological activation of IKr impairs conduction in guinea pig hearts.

INTRODUCTION: The hERG (Kv11.1) potassium channel underlies cardiac I(Kr) and is important for cardiac repolarization. Recently, hERG agonists have emerged as potential antiarrhythmic drugs. As modulation of outward potassium currents has been suggested to modulate cardiac conduction, we tested the hypothesis that pharmacological activation of I(Kr) results in impaired cardiac conduction. METHODS AND RESULTS: Cardiac conduction was assessed in Langendorff-perfused guinea pig hearts. Application of the hERG agonist NS3623 (10 microM) prolonged the QRS rate dependently. A significant prolongation (16 +/- 6%) was observed at short basic cycle length (BCL 90 ms) but not at longer cycle lengths (BCL 250 ms). The effect could be reversed by the I(Kr) blocker E4031 (1 microM). While partial I(Na) inhibition with flecainide (1 microM) alone prolonged the QRS (34 +/- 3%, BCL 250 ms), the QRS was further prolonged by 19 +/- 2% when NS3623 was added in the presence of flecainide. These data suggest that the effect of NS3623 was dependent on sodium channel availability. Surprisingly, in the presence of the voltage sensitive dye di-4-ANEPPS a similar potentiation of the effect of NS3623 was observed. With di-4-ANEPPS, NS3623 prolonged the QRS significantly (26 +/- 4%, BCL 250 ms) compared to control with a corresponding decrease in conduction velocity. CONCLUSION: Pharmacological activation of I(Kr) by the hERG agonist NS3623 impairs cardiac conduction. The effect is dependent on sodium channel availability. These findings suggest a role for I(Kr) in modulating cardiac conduction and may have implications for the use of hERG agonists as antiarrhythmic drugs.

Predictive value of electrical restitution in hypokalemia-induced ventricular arrhythmogenicity.

The ventricular action potential (AP) shortens exponentially upon a progressive reduction of the preceding diastolic interval. Steep electrical restitution slopes have been shown to promote wavebreaks, thus contributing to electrical instability. The present study was designed to assess the predictive value of electrical restitution in hypokalemia-induced arrhythmogenicity. We recorded monophasic APs and measured effective refractory periods (ERP) at distinct ventricular epicardial and endocardial sites and monitored volume-conducted ECG at baseline and after hypokalemic perfusion (2.5 mM K(+) for 30 min) in isolated guinea pig heart preparations. The restitution of AP duration measured at 90% repolarization (APD(90)) was assessed after premature extrastimulus application at variable coupling stimulation intervals, and ERP restitution was assessed by measuring refractoriness over a wide range of pacing rates. Hypokalemia increased the amplitude of stimulation-evoked repolarization alternans and the inducibility of tachyarrhythmias and reduced ventricular fibrillation threshold. Nevertheless, these changes were associated with flattened rather than steepened APD(90) restitution slopes and slowed restitution kinetics. In contrast, ERP restitution slopes were significantly increased in hypokalemic hearts. Although epicardial APD(90) measured during steady-state pacing (S(1)-S(1) = 250 ms) was prolonged in hypokalemic hearts, the left ventricular ERP was shortened. Consistently, the epicardial ERP measured at the shortest diastolic interval achieved upon a progressive increase in pacing rate was reduced in the hypokalemic left ventricle. In conclusion, this study highlights the superiority of ERP restitution at predicting increased arrhythmogenicity in the hypokalemic myocardium. The lack of predictive value of APD(90) restitution is presumably related to different mode of changes in ventricular repolarization and refractoriness in a hypokalemic setting, whereby APD(90) prolongation may be associated with shortened ERP.

Characterization of hERG1a and hERG1b potassium channels-a possible role for hERG1b in the I (Kr) current.

I (Kr) is the fast component of the delayed rectifier potassium currents responsible for the repolarization of the cardiac muscle. The molecular correlate underlying the I (Kr) current has been identified as the hERG1 channel. Recently, two splice variants of the hERG1 alpha-subunit, hERG1a and hERG1b, have been shown to be co-expressed in human cardiomyocytes. In this paper, we present the electrophysiological characterization of hERG1a, hERG1b, and co-expressed hERG1a/b channels in a mammalian expression system using the whole-cell patch clamp technique. We also quantified the messenger RNA (mRNA) levels of hERG1a and hERG1b in human cardiac tissue, and based on the expressed ratios, we evaluated the resulting currents in Xenopus laevis oocytes. Compared to hERG1a channels, activation was faster for both hERG1b and hERG1a/b channels. The deactivation kinetics was greatly accelerated in the presence of hERG1b, whereas no difference in the time constant of inactivation was observed. The voltage-dependent recovery from inactivation was also similar. However, the time constant of recovery from inactivation was significantly faster for hERG1b channels compared to hERG1a and hERG1a/b. Quantification of hERG1a and hERG1b mRNA in the human heart showed that hERG1b mRNA constitutes, on average, 19% in the right atrium and 12% in the left ventricle of the total hERG1 mRNA. Expression of the observed ratios of hERG1a to hERG1b in X. laevis oocytes showed that these ratios are indeed sufficient to change the deactivation phenotype markedly. The present work suggests that hERG1b is likely to play a role in the formation of the native I (Kr) current.

Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core.

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.