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.

Altered phosphodiesterase (PDE)-cyclicAMP(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 mechanismunderlying 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.

Diabetes is a major risk factor for cardiovascular diseases, including cardiac arrhythmias, heart failure and sudden cardiac death. In animal models of diabetes, cardiac calcium handling is severely impaired. However, as diabetes is a multifactorial disease it is difficult to separate the effect of systemic and local factors in disease development. Here, we investigate the effect of locally disrupted cardiac insulin signaling on cardiac calcium handling in a mouse model of diabetic cardiomyopathy. We measured calcium transients in isolated cardiomyocytes from Cardiac Insulin Receptor Knock-Out (CIRKO) mice. Littermate wild-type (WT) mice were used as controls. Cardiomyocytes were field stimulated and intracellular calcium was monitored using the fluorescent indicator fluo2-LeakRes (34oC, pHo 7.4, [Ca2+]o = 1.0 mM). Calcium transient amplitude and decay kinetics were measured at 1 and 5 Hz. While no significant differences were observed between WT and KO cardiomyocytes, there was a tendency towards reduced calcium transient amplitude in the KO group (Amplitude (F/F0): WT, 1.6±0.7 (n=9); KO, 1.0±0.4 (n=16); p=0.05). Calcium transient decay was similar (tau (ms): WT, 183±44 (n=9); KO: 232±84 (n=17); p=0.15) and frequency-dependent acceleration of decay kinetics was observed in both groups (tau: p<0.01 for both WT and KO (1Hz vs 5 Hz)). In conclusion, at the cellular level the CIRKO mouse model of diabetic cardiomyopathy display a very mild calcium handling phenotype. These findings suggest that impaired cardiac insulin signaling per se plays only a minor role in the contractile dysfunction observed in systemic models of diabetes.

The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electrophysiological properties that can contribute to an arrhythmogenic substrate.

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

Diabetes is associated with a relatively high prevalence of sudden cardiac death but the underlying mechanism is not well understood. In experimental diabetes, cardiac expression of connexin43 is reduced. Downregulation of connexin43 is linked to a slowing of cardiac conduction which increases the risk of cardiac arrhythmias and thereby sudden cardiac death. It is known that hyperglycemia causes downregulation of connexin43 but it is unknown to which extend this occurs in whole hearts. Here, we test the hypothesis that acute hyperglycemia leads to slowing of cardiac conduction through downregulation of connexin43 in whole hearts. We measured ventricular conduction velocity in Langendorff perfused guinea pig hearts using a multi-electrode array under normo- and hyperglycemic conditions (5.5 and 30mM glucose respectively) for up to 4 hrs. In addition, one group of hearts was perfused with a mannitol solution as a control for changes in osmolarity. Expression of connexin43 was quantified by western blotting. In the control group, longitudinal conduction velocity remained stable over 4 hrs (63 cm/s) while transverse conduction decreased slightly (from 27 to 25 cm/s). Similar results were obtained from both the hyperglycemia and mannitol groups suggesting that conduction was unaffected by changes in glucose concentration and osmolarity. Accordingly, no differences were observed in the protein levels of connexin43 between the groups supporting the functional results. In conclusion, acute exposure to hyperglycemia does not appear to have any significant effects on cardiac conduction and does not modulate the expression of connexin43 in guinea pig hearts.

Purpose: Genetic factors contribute significantly to the pathogenesis of atrial fibrillation (AF) but molecular defects in the majority of patients with AF are unknown. The two-pore domain potassium channel, K2P3.1 (TASK-1) has atrial-specific expression and has been recently shown to be a major determinant of resting membrane potential in human atrial cardiomyocytes. We hypothesized that mutations in the KCNK3 gene that encodes TASK-1 might predispose to AF.

AIMS:Diminished repolarization reserve contributes to the arrhythmogenic substrate in many disease states. Pharmacological activation of K(+) channels has been suggested as a potential antiarrhythmic therapy in such conditions. Having previously demonstrated that I(K1) and I(Kr) can modulate cardiac conduction, we tested here the effects of pharmacological I(KATP) and I(Ks) activation on cardiac conduction and its dependence on the sodium current (I(Na)).METHODS AND RESULTS:Bath electrocardiograms (ECGs) recorded from Langendorff-perfused guinea pig ventricles revealed QRS prolongation during I(KATP) activation by pinacidil but not during I(Ks) activation by R-L3 relative to control. In contrast, when I(Na) was partially blocked by flecainide, R-L3 but not pinacidil prolonged the QRS relative to flecainide alone. Conduction velocity (θ) was quantified by optical mapping during epicardial pacing. Both longitudinal (θ(L)) and transverse (θ(T)) θ were reduced by pinacidil (by 10 ± 1 and 9 ± 3%, respectively) and R-L3 (by 11 ± 2% and 15 ± 4%, respectively). Flecainide decreased θ(L) by 33 ± 4% and θ(T) by 36 ± 5%. Whereas pinacidil did not further slow θ relative to flecainide alone, R-L3 decreased both θ(L) and θ(T).CONCLUSION:Pharmacological activation of I(KATP) and I(Ks) slows cardiac conduction; however, they demonstrate diverse effects on θ dependence on I(Na) blockade. These findings may have significant implications for the use of K(+) channel activators as antiarrhythmic drugs and for patients with Na(+) channel abnormalities or being treated with Na(+) channel blockers.

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 mu M) 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.

AIMSGenetic factors may be important in the development of atrial fibrillation (AF) in the young. KCNA5 encodes the potassium channel α-subunit KV1.5, which underlies the voltage-gated atrial-specific potassium current IKur. KCNAB2 encodes KVβ2, a β-subunit of KV1.5, which increases IKur. Three studies have identified loss-of-function mutations in KCNA5 in patients with idiopathic AF. We hypothesized that early-onset lone AF is associated with high prevalence of genetic variants in KCNA5 and KCNAB2.METHODS AND RESULTSThe coding sequences of KCNA5 and KCNAB2 were sequenced in 307 patients with mean age of 33 years at the onset of lone AF, and in 216 healthy controls. We identified six novel non-synonymous mutations [E48G, Y155C, A305T (twice), D322H, D469E, and P488S] in KCNA5 in seven patients. None were present in controls. We identified a significantly higher frequency of rare deleterious variants in KCNA5 in the patients than in controls. The mutations were analysed with confocal microscopy and whole-cell patch-clamp techniques. The mutant proteins Y155C, D469E, and P488S displayed decreased surface expression and loss-of-function in patch-clamp studies, whereas E48G, A305T, and D322H showed preserved surface expression and gain-of-function for KV1.5.CONCLUSIONThis study is the first to present gain-of-function mutations in KCNA5 in patients with early-onset lone AF. We identified three gain-of-function and three loss-of-function mutations. We report a high prevalence of variants in KCNA5 in these patients. This supports the hypothesis that both increased and decreased potassium currents enhance AF susceptibility.

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.

Background: The Kv7.1 channel underlies the IKs current and plays a key role in cardiac repolarization. Loss of IKs function can lead to the type 1 long QT syndrome (LQT1). The Kv7.1 activator R-L3 has been suggested as a potential antiarrhythmic therapy in LQT1. Having previously demonstrated that other K+ currents can modulate cardiac conduction, we tested here the effects of pharmacological IKs activation on cardiac conduction and its dependence on the sodium current (INa). Methods and Results: Conduction velocity (CV) was quantified by optical mapping during LV or RV pacing. Under control conditions transverse CV (CVT) was significantly greater (15±1 %, p<0.05) in RV than LV with no difference in longitudinal CV (CVL) between ventricles. During partial blockade of INa (flecainide, 1 µM), RV CVT decreased by 36±5 % while LV CVT decreased by 24±5 %. These data demonstrate greater RV conduction dependence on sodium channel availability. Partial IKs blockade by R-L3 (10 µM) significantly decreased both CVT (by 15±4 % in RV & 8±2 % in LV) and CVL (by 11±2% in RV & 6±1 % in LV) relative to control. Further, R-L3 exacerbated the effects of INa blockade: 1 µM flecainide now decreased CVT by 45±3% and 28±4% in RV and LV respectively. Conclusion: Pharmacological IKs activation by R-L3 slows cardiac conduction and shifts the conduction velocity - INa relationship downward. These findings may have significant implications for the use of Kv7.1 activator as antiarrhythmic drugs.

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 IC50 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.

One of the most widely used voltage-sensitive dyes for optically mapping cardiac conduction is di-4-ANEPPS. Previous studies suggest that di-4-ANEPPS broadens the QRS; however, little is known about its effects on myocardial conduction. We hypothesized that di-4-ANEPPS suppresses cardiac conduction velocity (CV). CV was quantified in Langendorff-perfused guinea pig hearts using unipolar electrode and optical recordings. Electrode recordings from the anterior epicardium revealed that di-4-ANEPPS (7.5 µM) slowed cardiac transverse CV significantly from 23±4 cm/s to 18±3 cm/s (p<0.05). To investigate a possible concentration dependent effect of di-4-ANEPPS, CV and anisotropy was quantified using optical signals recorded from the anterior epicardium of both the right and left ventricle (RV, LV) at different concentrations of di-4-ANEPPS. Increasing the concentration of di-4-ANEPPS from 1.9 to 15 µM reduced transverse CV by 7±2 cm/s in the RV (p<0.05) (n=4) and 4±2 cm/s in the LV (p<0.05) (n=4). The decrease in longitudinal CV trended towards significance in both the RV (14±7 cm/s, p=0.08) and the LV (15±9 cm/s, p=0.07). The anisotropic ratio of CV was not affected by di-4-ANEPPS concentration. Connexin43 conductance was not significantly changed by di-4-ANEPPS at 15 µM evident from dual patch clamp experiments on HeLa cell-pairs overexpressing rat connexin43 (n=5), suggesting that decreased gap junction conductance is not the underlying mechanism. These data suggest that the perfusion of di-4-ANEPPS into whole heart tissue slows CV in both the right and left ventricles and this effect does not appear to be connexin related. Investigators should take the effect of di-4-ANEPPS on conduction into account when interpreting data obtained with this dye.

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.

BACKGROUND AND PURPOSE Kv11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, Kv11.1a and Kv11.1b, with different kinetics, are expressed. Kv11.1 activators are potential therapeutic agents, but their effects have only been tested on the Kv11.1a isoform. In this study, the effects of two different Kv11.1 activators, NS1643 and RPR260243, were characterized on Kv11.1a and Kv11.1b channels.EXPERIMENTAL APPROACH Kv11.1a and Kv11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two‐electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243.KEY RESULTS NS1643 increased steady‐state currents through Kv11.1b several fold more than through Kv11.1a channels, without affecting EC50 values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for Kv11.1a. RPR260243 increased steady‐state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on Kv11.1b than on Kv11.1a. Effects of both compounds on heteromeric Kv11.1a/Kv11.1b channels were similar to those on Kv11.1a.CONCLUSIONS AND IMPLICATIONS Both NS1643 and RPR260243 displayed differential effects on Kv11.1a and Kv11.1b channels, the effects being relatively more pronounced on Kv11.1b channels. This affirms the importance of testing the effect of Kv11.1 activators on different channel isoforms.

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.

Activation of I-Kr Impairs Conduction. 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 mu M) 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 mu M). While partial I-Na inhibition with flecainide (1 mu M) 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. (J Cardiovasc Electrophysiol, Vol. 21, pp. 923-929, August 2010)