Novel experimental results in human cardiac electrophysiology: measurement of the Purkinje fibre action potential from the undiseased human heart.

Data obtained from canine cardiac electrophysiology studies are often extrapolated to the human heart. However, it has been previously demonstrated that because of the lower density of its K(+) currents, the human ventricular action potential has a less extensive repolarization reserve. Since the relevance of canine data to the human heart has not yet been fully clarified, the aim of the present study was to determine for the first time the action potentials of undiseased human Purkinje fibres (PFs) and to compare them directly with those of dog PFs. All measurements were performed at 37 °C using the conventional microelectrode technique. At a stimulation rate of 1 Hz, the plateau potential of human PFs is more positive (8.0 ± 1.8 vs 8.6 ± 3.4 mV, n = 7), while the amplitude of the spike is less pronounced. The maximal rate of depolarization is significantly lower in human PKs than in canine PFs (406.7 ± 62 vs 643 ± 36 V/s, respectively, n = 7). We assume that the appreciable difference in the protein expression profiles of the 2 species may underlie these important disparities. Therefore, caution is advised when canine PF data are extrapolated to humans, and further experiments are required to investigate the characteristics of human PF repolarization and its possible role in arrhythmogenesis.

Effects of Chelidonium majus extracts and major alkaloids on hERG potassium channels and on dog cardiac action potential - a safety approach.

Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.

Abnormal repolarization as the basis for late potentials and fractionated electrograms recorded from epicardium in experimental models of Brugada syndrome.

OBJECTIVES: The aim of this study was to test the hypothesis that late potentials and fractionated electrogram activity are due to delayed depolarization within the anterior aspects of right ventricular (RV) epicardium in experimental models of Brugada syndrome (BrS). BACKGROUND: Clinical reports have demonstrated late potentials on signal-averaged electrocardiography (ECG) recorded in patients with BrS. Recent studies report the appearance of late potentials and fractionated activity on bipolar electrograms recorded in the epicardium of the RV outflow tract in patients with BrS. METHODS: Action potential and bipolar electrograms were recorded at epicardial and endocardial sites of coronary-perfused canine RV wedge preparations, together with a pseudo-ECG. The transient outward potassium current agonist NS5806 (5 µM) and the Ca(2+)-channel blocker verapamil (2 µM) were used to pharmacologically mimic the BrS genetic defect. RESULTS: Fractionated electrical activity was observed in RV epicardium, but not in endocardium, as a consequence of heterogeneities in the appearance of the second upstroke of the epicardial action potential, and discrete high-frequency spikes developed as a result of concealed phase 2 re-entry. In no case did we observe primary conduction delay as the cause of the BrS ECG phenotype or of late potential or fractionated electrogram activity. Quinidine (10 µM) and the phosphodiesterase-3 inhibitors cilostazol (10 µM) and milrinone (2.5 µM) restored electrical homogeneity, thus abolishing all late potentials and fractionated electrical activity. CONCLUSIONS: These data point to an alternative pathophysiological basis for late potentials and fractionated electrical activity recorded in the right ventricle in the setting of BrS. We demonstrate an association of such activity with abnormal repolarization and not with abnormal depolarization or structural abnormalities.

Mechanisms underlying the development of the electrocardiographic and arrhythmic manifestations of early repolarization syndrome.

Early repolarization pattern in the ECG has been associated with increased risk for ventricular tachycardia/fibrillation (VT/VF), particularly when manifest in inferior leads. This study examines the mechanisms underlying VT/VF in early repolarization syndrome (ERS). Transmembrane action potentials (APs) were simultaneously recorded from 2 epicardial sites and 1 endocardial site of coronary-perfused canine left-ventricular (LV) wedge preparations, together with a pseudo-ECG. Transient outward current (Ito) was recorded from epicardial myocytes isolated from the inferior and lateral LV of the same heart. J wave area (pseudo-ECG), epicardial AP notch magnitude and index were larger in inferior vs. lateral wall preparations at baseline and after exposure to provocative agents (NS5806+verapamil+acetylcholine (ACh)). Ito density was greater in myocytes from inferior vs. lateral wall (18.4 ± 2.3pA/pF vs. 11.6 ± 2.0pA/pF; p<0.05). A combination of NS5806 (7 µM) and verapamil (3 µM) or pinacidil (4 µM), used to pharmacologically model the genetic defects responsible for ERS, resulted in prominent J-point and ST-segment elevation. ACh (3 µM), simulating increased vagal tone, precipitated phase-2-reentry-induced polymorphic VT/VF. Using identical protocols, inducibility of arrhythmias was 3-fold higher in inferior vs. lateral wedges. Quinidine (10 µM) or isoproterenol (1 µM) restored homogeneity and suppressed VT/VF. Our data support the hypothesis that 1) ERS is caused by a preferential accentuation of the AP notch in the LV epicardium; 2) this repolarization defect is accentuated by elevated vagal tone; 3) higher intrinsic levels of Ito account for the greater sensitivity of the inferior LV wall to development of VT/VF; and 4) quinidine and isoproterenol exert ameliorative effects by reversing the repolarization abnormality.

Cellular mechanisms underlying the effects of milrinone and cilostazol to suppress arrhythmogenesis associated with Brugada syndrome.

BACKGROUND: Brugada syndrome is an inherited disease associated with vulnerability to ventricular tachycardia and sudden cardiac death in young adults. Milrinone and cilostazol, oral phosphodiesterase (PDE) type III inhibitors, have been shown to increase L-type calcium channel current (ICa) and modestly increase heart rate by elevating the level of intracellular cyclic adenosine monophosphate. OBJECTIVE: To examine the effectiveness of these PDE inhibitors to suppress arrhythmogenesis in an experimental model of Brugada syndrome. METHODS: Action potential (AP) and electrocardiographic recordings were obtained from epicardial and endocardial sites of coronary-perfused canine right ventricular wedge preparations. The Ito agonist NS5806 (5 µM) and Ca(2+) channel blocker verapamil (2 µM) were used to pharmacologically mimic Brugada phenotype. RESULTS: The combination induced all-or-none repolarization at some epicardial sites but not others, leading to ST-segment elevation as well as an increase in both epicardial and transmural dispersion of repolarization. Under these conditions, phase 2 reentry developed as the epicardial AP dome propagated from sites where it was maintained to sites at which it was lost, generating closely coupled extrasystoles and ventricular tachycardia. The addition of the PDE inhibitor milrinone (2.5 µM) or cilostazol (5-10 µM) to the coronary perfusate restored the epicardial AP dome, reduced dispersion, and abolished phase 2 reentry-induced extrasystoles and ventricular tachycardia. CONCLUSIONS: Our study identifies milrinone as a more potent alternative to cilostazol for reversing the repolarization defects responsible for the electrocardiographic and arrhythmic manifestations of Brugada syndrome. Both drugs normalize ST-segment elevation and suppress arrhythmogenesis in experimental models of Brugada syndrome.

L-364,373 (R-L3) enantiomers have opposite modulating effects on IKs in mammalian ventricular myocytes.

Activators of the slow delayed rectifier K⁺ current (IKs) have been suggested as promising tools for suppressing ventricular arrhythmias due to prolongation of repolarization. Recently, L-364,373 (R-L3) was nominated to activate IKs in myocytes from several species; however, in some studies, it failed to activate IKs. One later study suggested opposite modulating effects from the R-L3 enantiomers as a possible explanation for this discrepancy. Therefore, we analyzed the effect of the RL-3 enantiomers on IKs in ventricular mammalian myocytes, by applying standard microelectrode and whole-cell patch-clamp techniques at 37 °C. We synthesized 2 substances, ZS_1270B (right) and ZS_1271B (left), the 2 enantiomers of R-L3. In rabbit myocytes, ZS_1270B enhanced the IKs tail current by approximately 30%, whereas ZS_1271B reduced IKs tails by 45%. In guinea pig right ventricular preparations, ZS_1270B shortened APD90 (action potential duration measured at 90% repolarization) by 12%, whereas ZS_1271B lengthened it by approximately 15%. We concluded that R-L3 enantiomers in the same concentration range indeed have opposite modulating effects on IKs, which may explain why the racemic drug R-L3 previously failed to activate IKs. ZS_1270B is a potent IKs activator, therefore, this substance is appropriate to test whether IKs activators are ideal tools to suppress ventricular arrhythmias originating from prolongation of action potentials.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 µmol l(-1) BaCl2) and IKs block (1 µmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.

Diclofenac prolongs repolarization in ventricular muscle with impaired repolarization reserve.

BACKGROUND: The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti-inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle. METHODS: Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model. RESULTS: Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl(2) application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QT(c) interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT(c). Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 µM) decreased the amplitude of rapid (I(Kr)) and slow (I(Ks)) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I(Ca)) was slightly diminished, but the transient outward (I(to)) and inward rectifier (I(K1)) potassium currents were not influenced. CONCLUSIONS: Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve.

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue.

BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f) by 67% at -120 mV, while MEL57A (3 µM) reduced I(f) by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.

Electrophysiological effects of ivabradine in dog and human cardiac preparations: potential antiarrhythmic actions.

Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V(max)), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1-10 µM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V(max) block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V(max) block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat(-1) in dog ventricular muscle. In addition to a fast recovery of V(max) from inactivation (τ=41-46 ms) observed in control, a second slow component for recovery of V(max) was expressed (offset kinetics of V(max) block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 µM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.

Class I/B antiarrhythmic property of ranolazine, a novel antianginal agent, in dog and human cardiac preparations.

The aim of this study was to investigate the cellular electrophysiological effects of ranolazine on action potential characteristics. The experiments were carried out in dog and human cardiac preparations using the conventional microelectrode technique. In dog Purkinje fibres ranolazine produced a concentration- and frequency-dependent depression of the maximum rate of depolarization (V(max)) while action potential duration (APD) was shortened. In dog and human right ventricular papillary muscle ranolazine exerted no significant effect on APD, while it produced, like mexiletine, use-dependent depression of V(max) with relatively fast onset and offset kinetics. In dog midmyocardial preparations the drug did not exert statistically significant effect on repolarization at 10 µM, although a tendency toward prolongation was observed at 20 µM. A moderate lengthening of APD(90) by ranolazine was noticed in canine atrial preparations obtained from dogs in sinus rhythm and in tachypacing induced remodelled preparations. Use-dependent depression of V(max) was more pronounced in atria from dogs in sinus rhythm than those in remodelled atria or in the ventricle. These findings indicate that ranolazine, in addition to its known late sodium current blocking effect, also depresses peak I(Na) with class I/B antiarrhythmic characteristics. Although peak I(Na) inhibition by ranolazine is stronger in the atria, it is also substantial (at fast stimulation frequencies) in ventricular preparations. Ranolazine also decreased the dispersion of ventricular repolarization (the difference in APD(90) values between Purkinje fibres and papillary muscles), which can contribute to the antiarrhythmic property of the drug.

A multiscale investigation of repolarization variability and its role in cardiac arrhythmogenesis.

Enhanced temporal and spatial variability in cardiac repolarization has been related to increased arrhythmic risk both clinically and experimentally. Causes and modulators of variability in repolarization and their implications in arrhythmogenesis are however not well understood. At the ionic level, the slow component of the delayed rectifier potassium current (I(Ks)) is an important determinant of ventricular repolarization. In this study, a combination of experimental and computational multiscale studies is used to investigate the role of intrinsic and extrinsic noise in I(Ks) in modulating temporal and spatial variability in ventricular repolarization in human and guinea pig. Results show that under physiological conditions: i), stochastic fluctuations in I(Ks) gating properties (i.e., intrinsic noise) cause significant beat-to-beat variability in action potential duration (APD) in isolated cells, whereas cell-to-cell differences in channel numbers (i.e., extrinsic noise) also contribute to cell-to-cell APD differences; ii), in tissue, electrotonic interactions mask the effect of I(Ks) noise, resulting in a significant decrease in APD temporal and spatial variability compared to isolated cells. Pathological conditions resulting in gap junctional uncoupling or a decrease in repolarization reserve uncover the manifestation of I(Ks) noise at cellular and tissue level, resulting in enhanced ventricular variability and abnormalities in repolarization such as afterdepolarizations and alternans.