Characterization and influence of cardiac background sodium current in the atrioventricular node.

Background inward sodium current (IB,Na) that influences cardiac pacemaking has been comparatively under-investigated. The aim of this study was to determine for the first time the properties and role of IB,Na in cells from the heart's secondary pacemaker, the atrioventricular node (AVN). Myocytes were isolated from the AVN of adult male rabbits and mice using mechanical and enzymatic dispersion. Background current was measured using whole-cell patch clamp and monovalent ion substitution with major voltage- and time-dependent conductances inhibited. In the absence of a selective pharmacological inhibitor of IB,Na, computer modelling was used to assess the physiological contribution of IB,Na. Net background current during voltage ramps was linear, reversing close to 0mV. Switching between Tris- and Na(+)-containing extracellular solution in rabbit and mouse AVN cells revealed an inward IB,Na, with an increase in slope conductance in rabbit cells at -50mV from 0.54±0.03 to 0.91±0.05nS (mean±SEM; n=61 cells). IB,Na magnitude varied in proportion to [Na(+)]o. Other monovalent cations could substitute for Na(+) (Rb(+)>K(+)>Cs(+)>Na(+)>Li(+)). The single-channel conductance with Na(+) as charge carrier estimated from noise-analysis was 3.2±1.2pS (n=6). Ni(2+) (10mM), Gd(3+) (100µM), ruthenium red (100µM), or amiloride (1mM) produced modest reductions in IB,Na. Flufenamic acid was without significant effect, whilst La(3+) (100µM) or extracellular acidosis (pH6.3) inhibited the current by >60%. Under the conditions of our AVN cell simulations, removal of IB,Na arrested spontaneous activity and, in a simulated 1D-strand, reduced conduction velocity by ~20%. IB,Na is carried by distinct low conductance monovalent non-selective cation channels and can influence AVN spontaneous activity and conduction.

Inhibition of a TREK-like K+ channel current by noradrenaline requires both ß1- and ß2-adrenoceptors in rat atrial myocytes.

AIMS: Noradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss). METHODS AND RESULTS: Rat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca(2+) current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K(+) channel. The effect of noradrenaline on IKss was abolished by combined ß1-/ß2-adrenoceptor antagonism (1 µM propranolol or 10 µM ß1-selective atenolol and 100 nM ß2-selective ICI-118,551 in combination), but not by ß1- or ß2-antagonist alone. The action of noradrenaline could be mimicked by ß2-agonists (zinterol and fenoterol) in the presence of ß1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by ß1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined ß1-/ß2-antagonism, but not by atenolol alone. CONCLUSION: Noradrenaline inhibits a rat atrial TREK-like K(+) channel current via a PTX-sensitive mechanism involving co-operativity of ß1-/ß2-adrenoceptors that contributes to atrial APD prolongation.

Activation of glibenclamide-sensitive ATP-sensitive K+ channels during ß-adrenergically induced metabolic stress produces a substrate for atrial tachyarrhythmia.

BACKGROUND: Cardiac ATP-sensitive K(+) channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after ß-adrenoceptor stimulation. However, an increased atrial K(+)-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K(+) channel blockade on the electrophysiological responses to ß-adrenoceptor-induced metabolic challenge in intact atria. METHODS AND RESULTS: Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S(1)-S(2) protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10(-9) mol/L of the ß-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10(-6) mol/L). Perfusion with either of the ATP-sensitive K(+) channel blockers, glibenclamide (10(-5) mol/L) or tolbutamide (10(-3) mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10(-6) mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. CONCLUSIONS: Atrial ATP-sensitive K(+) channels activate in response to ß-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node.

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 µM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 µM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 µM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.

Atrial remodeling and the substrate for atrial fibrillation in rat hearts with elevated afterload.

BACKGROUND: Although arterial hypertension and left ventricular hypertrophy are considered good epidemiological indicators of the risk of atrial fibrillation (AF) in patients, the link between elevated afterload and AF remains unclear. We investigated atrial remodeling and the substrate for arrhythmia in a surgical model of elevated afterload in rats. METHODS AND RESULTS: Male Wistar rats (aged 3-4 weeks) were anesthetized and subjected to either partial stenosis of the ascending aorta (AoB) or sham operation (Sham). Experiments were performed on excised hearts 8, 14, and 20 weeks after surgery. Unipolar electrograms were recorded from the left atrial epicardial surface of perfused hearts using a 5×5 electrode array. Cryosections of left atrial tissue were retained for histological and immunocytochemical analyses. Compared to Sham, AoB hearts showed marked left atrial hypertrophy and fibrosis at 14 and 20 weeks postsurgery. The incidence and duration of pacing-induced AF was increased in hearts from AoB rats at 20 weeks postsurgery. The substrate for arrhythmia was associated with reduced vectorial conduction velocity and greater inhomogeneity in conduction but without changes in effective refractory period. Left atrial expression of the gap junction protein, connexin43, was markedly reduced in AoB compared with Sham hearts. CONCLUSIONS: Using a small-animal model, we demonstrate that elevated afterload in the absence of systemic hypertension results in increased inducibility of AF and left atrial remodeling involving fibrosis, altered atrial connexin43 expression, and marked conduction abnormalities.

ß-Adrenoceptor/PKA-stimulation, Na(+)-Ca(2+) exchange and PKA-activated Cl(-) currents in rabbit cardiomyocytes: a conundrum.

Investigations into the functional modulation of the cardiac Na(+)-Ca(2+) exchanger (NCX) by acute ß-adrenoceptor/PKA stimulation have produced conflicting results. Here, we investigated (i) whether or not ß-adrenoceptor activation/PKA stimulation activates current in rabbit cardiac myocytes under NCX-'selective' conditions and (ii) if so, whether a PKA-activated Cl(-)-current may contribute to the apparent modulation of NCX current (I(NCX)). Whole-cell voltage-clamp experiments were conducted at 37°C on rabbit ventricular and atrial myocytes. The ß-adrenoceptor-activated currents both in NCX-'selective' and Cl(-)-selective recording conditions were found to be sensitive to 10mM Ni(2+). In contrast, the PKA-activated Cl(-) current was not sensitive to Ni(2+), when it was activated downstream to the ß-adrenoceptors using 10µM forskolin (an adenylyl cyclase activator). When 10µM forskolin was applied under NCX-selective recording conditions, the Ni(2+)-sensitive current did not differ between control and forskolin. These findings suggest that in rabbit myocytes: (a) a PKA-activated Cl(-) current contributes to the Ni(2+)-sensitive current activated via ß-adrenoceptor stimulation under recording conditions previously considered selective for I(NCX); (b) downstream activation of PKA does not augment Ni(2+)-sensitive I(NCX), when this is measured under conditions where the Ni(2+)-sensitive PKA-activated Cl(-) current is not present.

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101.

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl(-) current (I(Cl.PKA)) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I(Cl.PKA) and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I(Cl.PKA) in a concentration- and voltage-dependent fashion (IC(50) at +100 mV=0.3 ± 1.5 µM and at -100 mV=5.1 ± 1.3 µM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I(Cl.PKA) (30 µM) was tested on other cardiac ion currents. Inward current at -120 mV, comprised predominantly of the inward-rectifier background K(+) current, I(K1), was reduced by ∼43% (n=5). Under selective recording conditions, the Na(+) current (I(Na)) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at -40 mV of ∼82%; n=8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca(2+) channel current (I(Ca,L)); fractional block at +10 mV of ∼49% and of ∼28% at -10 mV; n=11, with a ∼-3 mV shift in the voltage-dependence of I(Ca,L) activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I(Cl.PKA) channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.

Increased susceptibility to atrial tachyarrhythmia in spontaneously hypertensive rat hearts.

Although hypertension is the most prevalent risk factor for atrial fibrillation, there is currently no information available from animal models of hypertension regarding the development of atrial remodeling or increased susceptibility to atrial tachyarrhythmia. Therefore, we examined the susceptibility to atrial tachyarrhythmia and the development of atrial remodeling in excised perfused hearts from male spontaneously hypertensive rats in comparison with age-matched male Wistar-Kyoto normotensive controls at age 3 and 11 months, corresponding with early hypertension and pre-heart failure stages, respectively. The incidence and duration of left atrial tachyarrhythmia induced by burst pacing was greater in hearts from 11-month-old hypertensive animals than either in age-matched controls or in 3-month-old hypertensive rats, although there was no difference between hypertensive and normotensive hearts at 3 months. Thus, hypertension was associated with the development of an arrhythmic substrate. Atrial effective refractory period and the duration of monophasic action potentials recorded from the left atrium were not altered with either hypertension or age, although there were changes in the whole-cell Ca2+ current density of isolated left atrial myocytes. On the other hand, Masson's trichrome staining of wax-embedded sections of left atrium revealed markedly greater interstitial fibrosis in 11-month-old hypertensive rats compared with controls. These data constitute the first experimental evidence that hypertension is associated with the development of a substrate for atrial tachyarrhythmia involving left atrial fibrosis without changes in the atrial effective refractory period and demonstrate that the spontaneously hypertensive rat represents a suitable model for investigating hypertension-associated atrial remodeling.

Recent advances in understanding sex differences in cardiac repolarization.

A number of gender differences exist in the human electrocardiogram (ECG): the P-wave and P-R intervals are slightly longer in men than in women, whilst women have higher resting heart rates than do men, but a longer rate-corrected QT (QT(C)) interval. Women with the LQT1 and LQT2 variants of congenital long-QT syndrome (LQTS) are at greater risk of adverse cardiac events. Similarly, many drugs associated with acquired LQTS have a greater risk of inducing torsades de pointes (TdP) arrhythmia in women than in men. There are also male:female differences in Brugada syndrome, early repolarisation syndrome and sudden cardiac death. The differences in the ECG between men and women, and in particular those relating to the QT interval, have been explored experimentally and provide evidence of differences in the processes underlying ventricular repolarization. The data available from rabbit, canine, rat, mouse and guinea pig models are reviewed and highlight involvement of male:female differences in Ca and K currents, although the possible involvement of rapid and persistent Na current and Na-Ca exchange currents cannot yet be excluded. The mechanisms underlying observed differences remain to be elucidated fully, but are likely to involve the influence of gonadal steroids. With respect to the QT interval and risk of TdP, a range of evidence implicates a protective role of testosterone in male hearts, possibly by both genomic and non-genomic pathways. Evidence regarding oestrogen and progesterone is less unequivocal, although the interplay between these two hormones may influence both repolarization and pro-arrhythmic risk.

The neuroprotective agent sipatrigine blocks multiple cardiac ion channels and causes triangulation of the ventricular action potential.

Sipatrigine (BW 619C89), a blocker of neuronal Na+ and Ca2+ channels that is structurally related to lamotrigine, has been shown to be neuroprotective in models of cortical ischaemia. Although associated with cardiovascular effects in animal models in vivo, there is no published information concerning the effects of sipatrigine on cardiac ion currents and action potentials (AP). The aim of the present study was to examine the effects of sipatrigine on the delayed rectifier currents (I(Kr) and I(Ks)), the inward rectifier current (I(K1)), the L-type Ca2+ current (I(Ca,L)) and the fast Na+ current (I(Na)), as well as on AP duration at 30% (APD30) and 90% (APD90) repolarization, in guinea-pig isolated ventricular myocytes. Each of the currents was inhibited by sipatrigine, demonstrating the drug to be a relatively broad-spectrum blocker of cation channels in the heart. However, sipatrigine was a comparatively more potent inhibitor of I(Kr) (IC50 = 0.85 micromol/L) and I(Ks) (IC50 = 0.92 micromol/L) than of I(K1) (IC50 = 5.3 micromol/L), I(Ca,L) (IC50 = 6.0 micromol/L) and I(Na) (IC50 = 25.5 micromol/L). Consistent with block of I(Kr), I(Ks) and I(K1), sipatrigine (1-30 micromol/L) produced a concentration-dependent prolongation of APD90. Although lower concentrations of sipatrigine (< or = 3 micromol/L) caused APD(30) prolongation, higher concentrations (> or = 10 micromol/L) shortened APD30, consistent with an involvement of I(Ca,L) blockade. The contrasting effects of sipatrigine on APD30 and APD90 at higher concentrations resulted in a marked concentration-dependent triangulation of the AP. 5. The results of the present study demonstrate that sipatrigine, at concentrations previously shown to be neuroprotective in vitro, modulates cardiac K+, Ca2+ and Na+ currents and repolarization of the cardiac ventricular action potential.

Evidence for a novel K(+) channel modulated by alpha(1A)-adrenoceptors in cardiac myocytes.

Accumulating evidence suggests that steady-state K(+) currents modulate excitability and action potential duration, particularly in cardiac cell types with relatively abbreviated action potential plateau phases. Despite representing potential drug targets, at present these currents and their modulation are comparatively poorly characterized. Therefore, we investigated the effects of phenylephrine [PE; an alpha(1)-adrenoceptor (alpha(1)-AR) agonist] on a sustained outward K(+) current in rat ventricular myocytes. Under K(+) current-selective conditions at 35 degrees C and whole-cell patch clamp, membrane depolarization elicited transient (I(t)) and steady-state (I(ss)) outward current components. PE (10 microM) significantly decreased I(ss) amplitude, without significant effect on I(t). Preferential modulation of I(ss) by PE was confirmed by intracellular application of the voltage-gated K(+) channel blocker tetraethylammonium, which largely inhibited I(t) without affecting the PE-sensitive current (I(ss,PE)). I(ss,PE) had the properties of an outwardly rectifying steady-state K(+)-selective conductance. Acidification of the external solution or externally applied BaCl(2) or quinidine strongly inhibited I(ss,PE). However, I(ss,PE) was not abolished by anandamide, ruthenium red, or zinc, inhibitors of TASK acid-sensitive background K(+) channels. Furthermore, the PE-sensitive current was partially inhibited by external administration of high concentrations of tetraethylammonium and 4-aminopyridine, which are voltage-gated K(+) channel-blockers. Power spectrum analysis of I(ss,PE) yielded a large unitary conductance of 78 pS. I(ss,PE) resulted from PE activation of the alpha(1A)-AR subtype, involved a pertussis toxin-insensitive G-protein, and was independent of cytosolic Ca(2+). These results collectively demonstrate that alpha(1A)-AR activation results in the inhibition of an outwardly rectifying steady-state K(+) current with properties distinct from previously characterized cardiac K(+) channels.