Lasse Skibsbye

Effects on Atrial Fibrillation in Aged Hypertensive Rats by Ca2+-Activated K+ Channel Inhibition

We have shown previously that inhibition of small conductance Ca2+-activated K+ (SK) channels is antiarrhythmic in models of acutely induced atrial fibrillation (AF). These models, however, do not take into account that AF derives from a wide range of predisposing factors, the most prevalent being hypertension. In this study we assessed the effects of two different SK channel inhibitors, NS8593 and UCL1684, in aging, spontaneously hypertensive rats to examine their antiarrhythmic properties in a setting of hypertension-induced atrial remodeling. Male spontaneously hypertensive rats and the normotensive Wistar-Kyoto rat strain were divided in 2×3 groups of animals aged 3, 8, and 11 months, respectively. The animals were randomly assigned to treatment with NS8593, UCL1684, or vehicle, and open chest in vivo experiments including burst pacing–induced AF were performed. The aging spontaneously hypertensive rats were more vulnerable to AF induction both by S2 stimulation and burst pacing. Vehicle affected neither the atrial effective refractory period nor AF duration. SK channel inhibition with NS8593 and UCL1684 significantly increased the atrial effective refractory period and decreased AF duration in both the normotensive and hypertensive strains with no decline in efficacy as age increased. In conclusion, SK channel inhibition with NS8593 and UCL1684 possesses antiarrhythmic properties in a rat in vivo model of paroxysmal AF with hypertension-induced atrial remodeling. The present results support the notion that SK channels may offer a promising new therapeutic target in the treatment of AF.

The Duration of Pacing-induced Atrial Fibrillation Is Reduced in Vivo by Inhibition of Small Conductance Ca2+-activated K+ Channels

Atrial fibrillation (AF) is associated with increased morbidity and is in addition the most prevalent cardiac arrhythmia. Compounds used in pharmacological treatment has traditionally been divided into Na+ channel inhibitors, β-blockers, K+ channel inhibitors, and Ca2+ channel inhibitors, whereas newer multichannel blockers such as amiodarone and ranolazine have been introduced later. This study was devoted to the evaluation of an acute pacing-induced in vivo model of AF in rats. Antiarrhythmic effects of well-known compounds such as lidocaine, dofetilide, and ranolazine were confirmed in this model. In addition, antiarrhythmic effects of different inhibitors of Ca2+-activated small conductance K+ (SK) channels were demonstrated. Intravenous application of 5 mg/kg of the negative SK channel modulator NS8593 reduced AF duration by 64.5%, and the lowest significantly effective dose was 1.5 mg/kg. A dose-effect relationship was established based on 6 different dose groups. Furthermore, it was demonstrated that the antiarrhythmic effect of NS8593 and other tested drugs was associated with an increase in atrial effective refractory period. The functional role of SK channels was confirmed by 2 other SK channel inhibitors, UCL1684 and apamin, thereby confirming the hypothesis that these channels might constitute a new promising target for antiarrhythmic treatment.

G-protein-coupled inward rectifier potassium current contributes to ventricular repolarization

AIMS The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. METHODS AND RESULTS Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. CONCLUSION Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

Antiarrhythmic Mechanisms of SK Channel Inhibition in the Rat Atrium.

Introduction: SK channels have functional importance in the cardiac atrium of many species, including humans. Pharmacological blockage of SK channels has been reported to be antiarrhythmic in animal models of atrial fibrillation; however, the exact antiarrhythmic mechanism of SK channel inhibition remains unclear. Objectives: We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability, thereby prolonging the effective refractory period and slowing the conduction velocity (CV). We therefore aimed at elucidating these properties of SK channel inhibition and the underlying antiarrhythmic mechanisms using microelectrode action potential (AP) recordings and CV measurements in isolated rat atrium. Automated patch clamping and two-electrode voltage clamp were used to access INa and IK,ACh, respectively. Results: The SK channel inhibitor N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) exhibited antiarrhythmic effects. ICA prevented electrically induced runs of atrial fibrillation in the isolated right atrium and induced atrial postrepolarization refractoriness and depolarized RMP. Moreover, ICA (1–10 &mgr;M) was found to slow CV; however, because of a marked prolongation of effective refractory period, the calculated wavelength was increased. Furthermore, at increased pacing frequencies, SK channel inhibition by ICA (10–30 &mgr;M) demonstrated prominent depression of other sodium channel–dependent parameters. ICA did not inhibit IK,ACh, but at concentrations above 10 &mgr;M, ICA use dependently inhibited INa. Conclusions: SK channel inhibition modulates multiple parameters of AP. It prolongs the AP duration and shifts the RMP towards more depolarized potentials through direct ISK block. This indirectly leads to sodium channel inhibition through accumulation of state dependently inactivated channels, which ultimately slows conduction and decreases excitability. However, a contribution from a direct sodium channel inhibition cannot be ruled. We here propose that the primary antiarrhythmic mechanism of SK channel inhibition is through direct potassium channel block and through indirect sodium channel inhibition.

The Role of CAV3 in Long-QT Syndrome Clinical and Functional Assessment of a Caveolin-3/Kv11.1 Double Heterozygote Versus Caveolin-3 Single Heterozygote

CITATION: Hedley, P. L. et al. 2013. The role of CAV3 in long QT : clinical and functional assessment of a caveolin-3/Kc11.1 compound heterozygote. Circulation: Cardiovascular Genetics, 6:452-461, doi:10.1161/CIRCGENETICS.113.000137.Background— Mutations in CAV3 , coding for caveolin-3, the major constituent scaffolding protein of cardiac caveolae, have been associated with skeletal muscle disease, cardiomyopathy, and most recently long–QT syndrome (LQTS) and sudden infant death syndrome. We examined the occurrence of CAV3 mutations in a large cohort of patients with LQTS. Methods and Results— Probands with LQTS (n=167) were screened for mutations in CAV3 using direct DNA sequencing. A single proband (0.6%) was found to be a heterozygous carrier of a previously described missense mutation, caveolin-3:p.T78M. The proband was also a heterozygous carrier of the trafficking-deficient Kv11.1:p.I400N mutation. The caveolin-3:p.T78M mutation was found isolated in 3 family members, none of whom had a prolonged QTc interval. Coimmunoprecipitations of caveolin-3 and the voltage-gated potassium channel subunit (Kv11.1) were performed, and the electrophysiological classification of the Kv11.1 mutant was carried out by patch-clamp technique in human embryonic kidney 293 cells. Furthermore, the T-wave morphology was assessed in mutation carriers, double mutation carriers, and nonmutation carriers by applying a morphology combination score. The morphology combination score was normal for isolated caveolin-3:p.T78M carriers and of LQT2 type in double heterozygotes. Conclusions— Mutations in CAV3 are rare in LQTS. Furthermore, caveolin-3:p.T78M did not exhibit a LQTS phenotype. Because no association has ever been found between LQTS and isolated CAV3 mutations, we suggest that LQTS9 is considered a provisional entity.Background—Mutations in CAV3, coding for caveolin-3, the major constituent scaffolding protein of cardiac caveolae, have been associated with skeletal muscle disease, cardiomyopathy, and most recently long–QT syndrome (LQTS) and sudden infant death syndrome. We examined the occurrence of CAV3 mutations in a large cohort of patients with LQTS. Methods and Results—Probands with LQTS (n=167) were screened for mutations in CAV3 using direct DNA sequencing. A single proband (0.6%) was found to be a heterozygous carrier of a previously described missense mutation, caveolin-3:p.T78M. The proband was also a heterozygous carrier of the trafficking-deficient Kv11.1:p.I400N mutation. The caveolin-3:p.T78M mutation was found isolated in 3 family members, none of whom had a prolonged QTc interval. Coimmunoprecipitations of caveolin-3 and the voltage-gated potassium channel subunit (Kv11.1) were performed, and the electrophysiological classification of the Kv11.1 mutant was carried out by patch-clamp technique in human embryonic kidney 293 cells. Furthermore, the T-wave morphology was assessed in mutation carriers, double mutation carriers, and nonmutation carriers by applying a morphology combination score. The morphology combination score was normal for isolated caveolin-3:p.T78M carriers and of LQT2 type in double heterozygotes. Conclusions—Mutations in CAV3 are rare in LQTS. Furthermore, caveolin-3:p.T78M did not exhibit a LQTS phenotype. Because no association has ever been found between LQTS and isolated CAV3 mutations, we suggest that LQTS9 is considered a provisional entity.

PKC and AMPK regulation of Kv1.5 potassium channels.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K+ current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.

GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

Abstract: G protein–coupled inwardly rectifying K+ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein–coupled receptors, including muscarinic M2 receptors and adenosine A1 receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD90), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD90 and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD90 and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A1 and M2 receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms.

Termination of Vernakalant-Resistant Atrial Fibrillation by Inhibition of Small-Conductance Ca 2+ -Activated K + Channels in Pigs

Background Evidence has emerged that small-conductance Ca2+-activated K+ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited by ventricular adverse effects and efficacy loss as AF progresses. Methods and Results A total of 43 pigs were used for the studies. AF reversion in conscious long-term tachypaced pigs: Pigs were subjected to atrial tachypacing (7 Hz) until they developed sustained AF that could not be reverted by vernakalant 4 mg/kg (18.8±3.3 days of atrial tachypacing). When the SK channel inhibitor AP14145 was tested in these animals, vernakalant-resistant AF was reverted to sinus rhythm, and reinduction of AF by burst pacing (50 Hz) was prevented in 8 of 8 pigs. Effects on refractory period and AF duration in open chest pigs: The effects of AP14145 and vernakalant on the effective refractory periods and acute burst pacing-induced AF were examined in anaesthetized open chest pigs. Both vernakalant and AP14145 significantly prolonged atrial refractoriness and reduced AF duration without affecting the ventricular refractoriness or blood pressure in pigs subjected to 7 days atrial tachypacing, as well as in sham-operated control pigs. Conclusions SK currents play a role in porcine atrial repolarization, and pharmacological inhibition of these with AP14145 demonstrates antiarrhythmic effects in a vernakalant-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs.

Two missense mutations in KCNQ1 cause pituitary hormone deficiency and maternally inherited gingival fibromatosis

Familial growth hormone deficiency provides an opportunity to identify new genetic causes of short stature. Here we combine linkage analysis with whole-genome resequencing in patients with growth hormone deficiency and maternally inherited gingival fibromatosis. We report that patients from three unrelated families harbor either of two missense mutations, c.347G>T p.(Arg116Leu) or c.1106C>T p.(Pro369Leu), in KCNQ1, a gene previously implicated in the long QT interval syndrome. Kcnq1 is expressed in hypothalamic GHRH neurons and pituitary somatotropes. Co-expressing KCNQ1 with the KCNE2 β-subunit shows that both KCNQ1 mutants increase current levels in patch clamp analyses and are associated with reduced pituitary hormone secretion from AtT-20 cells. In conclusion, our results reveal a role for the KCNQ1 potassium channel in the regulation of human growth, and show that growth hormone deficiency associated with maternally inherited gingival fibromatosis is an allelic disorder with cardiac arrhythmia syndromes caused by KCNQ1 mutations.Growth retardation is most commonly caused by genetic defects in the growth hormone pathway. Here, in families with growth retardation and gingival fibromatosis, the authors identify mutations in the potassium channel gene KCNQ1 that cause electrophysiological aberrations and altered ACTH secretion in vitro.

Refractoriness in human atria: Time and voltage dependence of sodium channel availability

BACKGROUND Refractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria. METHODS AND RESULTS Steady-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial fibrillation. The ex vivo measurements of action potential duration, effective refractory period and resting membrane potential were well-replicated in simulations using this new in silico model. Notably, the voltage threshold potential at which refractoriness was observed was not different between sinus rhythm and chronic atrial fibrillation tissues and was neither affected by changes in frequency (1 vs. 3Hz). CONCLUSIONS Our results suggest a preferentially voltage-dependent, rather than time-dependent, effect with respect to refractoriness at physiologically relevant rates in human atria. However, as the resting membrane potential is hyperpolarized in chronic atrial fibrillation, the voltage-dependence of excitability dominates, profoundly increasing the risk for arrhythmia re-initiation and maintenance in fibrillating atria. Our results thereby highlight resting membrane potential as a potential target in pharmacological management of chronic atrial fibrillation.