Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

Electrophysiological profile of propiverine – relationship to cardiac risk

Drugs that prolong the QT interval by blocking human ether-a-go-go (HERG) channels may enhance the risk of ventricular arrhythmia. The spasmolytic drug propiverine is widely used for the therapy of overactive bladder (OAB). Here, we have investigated the effects of propiverine on cardiac ion channels and action potentials as well as on contractile properties of cardiac tissue, in order to estimate its cardiac safety profile, because other drugs used in this indication had to be withdrawn due to safety reasons. Whole-cell patch clamp technique was used to record the following cardiac ion currents: rapidly and slowly activating delayed rectifier K⁺ current (I_Kr, I_Ks), ultra rapidly activating delayed rectifier K⁺ current (I_Kur), inwardly rectifying K⁺ current I_K1, transient outward K⁺ current (I_to), and L-type Ca²⁺ current (I_Ca,L). Action potentials in cardiac tissue biopsies were recorded with conventional microelectrodes. The torsade de pointes screening assay (TDPScreen^TM) was used for drug scoring. Propiverine blocked in a concentration-dependent manner HERG channels expressed in HEK293 cells, as well as native I_Kr current in ventricular myocytes of guinea pig (IC₅₀ values: 10 μM and 1.8 μM respectively). At high concentrations (100 μM), propiverine suppressed I_Ks. I_K1 and the transient outward current I_to and I_Kur were not affected. In guinea-pig ventricular and human atrial myocytes, propiverine also blocked I_Ca,L (IC₅₀ values: 34.7 μM and 41.7 μM, respectively) and reduced force of contraction. Despite block of I_Kr, action potential duration was not prolonged in guinea-pig and human ventricular tissue, but decreased progressively until excitation failed altogether. Similar effects were observed in dog Purkinje fibers. Propiverine obtained a low score in the TDPScreen^TM. In conclusion, in vitro and in vivo studies of propiverine do not provide evidence for an enhanced cardiovascular safety risk. We propose that lack of torsadogenic risk of propiverine is related to enhancement of repolarization reserve by block of I_Ca,L.     

Inhibitory effects of AMP 579, a novel cardioprotective adenosine A1/A2A receptor agonist, on native I Kr and cloned HERG current

We investigated the effects of 1S-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-imidazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide (AMP 579), a novel cardioprotective adenosine A₁/A_2A receptor agonist, on the rapid and slow components of the delayed rectifier K⁺ current (I_Kr and I_Ks) in guinea-pig ventricular myocytes and on the human ether-a-go-go-related gene (HERG) channel expressed in human embryonic kidney (HEK 293) cells. Whole-cell current and membrane potential were recorded using patch-clamp techniques. In guinea-pig ventricular myocytes, AMP 579 inhibited I_Kr in a concentration-dependent manner with IC₅₀ value of 15.2 M, when I_Kr was blocked by chromanol 293B. On the contrary, AMP 579 (10 M) did not affect I_Ks in the presence of the I_Kr blocker E-4031. The former effect of AMP 579 was unaffected by either the selective adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the non-selective adenosine A₁/A₂ receptor antagonist 8-sulphophenyltheophylline. Moreover, AMP 579-induced inhibition of I_Kr was not voltage- and frequency-dependent. In HEK 293 cells expressing HERG channels, AMP 579 (10 M) significantly blocked the HERG current at +10 mV by 34.9±7.0% (n=4, p<0.05), and the degree of inhibition was comparable with that observed in guinea-pig ventricular myocytes (36.8±6.0%, n=4). AMP 579 (10 M) significantly inhibited the L-type Ca²⁺ current (I_Ca) by 41.0±6.8% (n=5, p<0.05), which was unaffected by 8-sulphophenyl-theophylline. Consequently, despite its inhibitory actions on I_Kr or HERG current, the drug significantly shortened the action potential duration measured at 90% repolarization from 275.6±19.4 to 208.3±18.6 ms (n=4, p<0.05). Thus, AMP 579 inhibits both native I_Kr and cloned HERG channels with additional inhibitory effect of I_Ca, and such inhibitory effects may at least partially underlie the observed antifibrillatory action of the drug during myocardial ischemia/reperfusion.     

苄基四氢巴马汀对豚鼠心室肌细胞钾电流及钙、钠电流的作用

目的：研究苄基四氢巴马汀（BTHP）对心肌细胞的作用特点,以探讨其抗心律失常机制。方法：用全细胞膜片钳技术考察BTHP对心室肌细胞钾电流及钙、钠电流的作用。结果：BTHP 30 μmol.L^-1明显阻滞延迟整流钾电流（I_K包括：I_Kr及I_Ks）。可使I_Kr及I_Kr,tail的幅值下降,且对I_Kr阻滞作用呈频率依赖性;对I_Ks及I_Ks,tail幅值也有明显的抑制作用。BTHP 200　μmol.L^-1可明显阻滞I_Ca,L,使其电流幅值降低,但对I_K1,I_Ca,T,I_Na电流均无影响。结论：BTHP可明显阻滞心室肌细胞I_Kr,I_Ks,I_Ca,L电流,且对I_Kr阻滞作用呈频率依赖性。     

A Carbohydrate Fraction, AIP1, from Artemisia Iwayomogi Reduces the Action Potential Duration by Activation of Rapidly Activating Delayed Rectifier K+ Channels in Rabbit Ventricular Myocytes

We investigated the effects of a hot-water extract of Artemisia iwayomogi, a plant belonging to family Compositae, on cardiac ventricular delayed rectifier K⁺ current (I_K) using the patch clamp technique. The carbohydrate fraction AIP1 dose-dependently increased the heart rate with an apparent EC₅₀ value of 56.1±5.5 µg/ml. Application of AIP1 reduced the action potential duration (APD) in concentration-dependent fashion by activating I_K without significantly altering the resting membrane potential (IC₅₀ value of APD₅₀: 54.80±2.24, IC₅₀ value of APD₉₀: 57.45±3.47 µg/ml). Based on the results, all experiments were performed with 50 µg/ml of AIP1. Pre-treatment with the rapidly activating delayed rectifier K⁺ current (I_Kr) inhibitor, E-4031 prolonged APD. However, additional application of AIP1 did not reduce APD. The inhibition of slowly activating delayed rectifier K⁺ current (I_Ks) by chromanol 293B did not change the effect of AIP1. AIP1 did not significantly affect coronary arterial tone or ion channels, even at the highest concentration of AIP1. In summary, AIP1 reduces APD by activating I_Kr but not I_Ks. These results suggest that the natural product AIP1 may provide an adjunctive therapy of long QT syndrome.     

Effects of β-adrenoceptor stimulation on delayed rectifier K(+) currents in canine ventricular cardiomyocytes

BACKGROUND AND PURPOSE

While the slow delayed rectifier K⁺ current (I_Ks) is known to be enhanced by the stimulation of β-adrenoceptors in several mammalian species, phosphorylation-dependent regulation of the rapid delayed rectifier K⁺ current (I_Kr) is controversial.

EXPERIMENTAL APPROACH

In the present study, therefore, the effect of isoprenaline (ISO), activators and inhibitors of the protein kinase A (PKA) pathway on I_Kr and I_Ks was studied in canine ventricular myocytes using the whole cell patch clamp technique.

KEY RESULTS

I_Kr was significantly increased (by 30–50%) following superfusion with ISO, forskolin or intracellular application of PKA activator cAMP analogues (cAMP, 8-Br-cAMP, 6-Bnz-cAMP). Inhibition of PKA by Rp-8-Br-cAMP had no effect on baseline I_Kr. The stimulating effect of ISO on I_Kr was completely inhibited by selective β₁-adrenoceptor antagonists (metoprolol and CGP-20712A), by the PKA inhibitor Rp-8-Br-cAMP and by the PKA activator cAMP analogues, but not by the EPAC activator 8-pCPT-2''-O-Me-cAMP. In comparison, I_Ks was increased threefold by the activation of PKA (by ISO or 8-Br-cAMP), and strongly reduced by the PKA inhibitor Rp-8-Br-cAMP. The ISO-induced enhancement of I_Ks was decreased by Rp-8-Br-cAMP and completely inhibited by 8-Br-cAMP.

CONCLUSIONS AND IMPLICATIONS

The results indicate that the stimulation of β₁-adrenoceptors increases I_Kr, similar to I_Ks, via the activation of PKA in canine ventricular cells.     

Analysis of the contribution of I(to) to repolarization in canine ventricular myocardium

BACKGROUND AND PURPOSE

The contribution of the transient outward potassium current (I_to) to ventricular repolarization is controversial as it depends on the experimental conditions, the region of myocardium and the species studied. The aim of the present study was therefore to characterize I_to and estimate its contribution to repolarization reserve in canine ventricular myocardium.

EXPERIMENTAL APPROACH

Ion currents were recorded using conventional whole-cell voltage clamp and action potential voltage clamp techniques in canine isolated ventricular cells. Action potentials were recorded from canine ventricular preparations using microelectrodes. The contribution of I_to to repolarization was studied using 100 µM chromanol 293B in the presence of 0.5 µM HMR 1556, which fully blocks I_Ks.

KEY RESULTS

The high concentration of chromanol 293B used effectively suppressed I_to without affecting other repolarizing K⁺ currents (I_K1, I_Kr, I_p). Action potential clamp experiments revealed a slowly inactivating and a ‘late’ chromanol-sensitive current component occurring during the action potential plateau. Action potentials were significantly lengthened by chromanol 293B in the presence of HMR 1556. This lengthening effect induced by I_to inhibition was found to be reverse rate-dependent. It was significantly augmented after additional attenuation of repolarization reserve by 0.1 µM dofetilide and this caused the occurrence of early afterdepolarizations. The results were confirmed by computer simulation.

CONCLUSIONS AND IMPLICATIONS

The results indicate that I_to is involved in regulating repolarization in canine ventricular myocardium and that it contributes significantly to the repolarization reserve. Therefore, blockade of I_to may enhance pro-arrhythmic risk.     

Electrophysiological Effects of the Anti‐Cancer Drug Lapatinib on Cardiac Repolarization

Abstract: Lapatinib is one of several tyrosine kinase inhibitors used against solid tumour cancers such as breast and lung cancer. Although lapatinib is associated with a risk of QT prolongation, the effects of the drug on cellular cardiac electrical properties and on action potential duration (APD) have not been studied. To evaluate the potential effects of lapatinib on cardiac repolarization, we investigated its electrophysiological effects using a whole‐cell patch–clamp technique in transiently transfected HEK293 cells expressing human ether‐à‐go‐go (hERG; to examine the rapidly activating delayed rectifier K⁺ current, I_Kr), KCNQ1/KCNE1 (to examine the slowly activating delayed rectifier K⁺ current, I_Ks), KCNJ2 (to examine the inwardly rectifying K⁺ current, I_K1), or SCN5A (to examine the inward Na⁺ current, I_Na) and in rat cardiac myocytes (to examine the inward Ca²⁺ current, I_Ca). We also examined its effects on the APD at 90% (APD₉₀) in isolated rabbit Purkinje fibres. In ion channel studies, lapatinib inhibited the hERG current in a concentration‐dependent manner, with a half‐maximum inhibition concentration (IC₅₀) of 0.8 ± 0.09 μm . In contrast, at concentrations up to 3 μm , lapatinib did not significantly reduce the I_Na, I_K1 or I_Ca amplitudes; at 3 μm , it did slightly inhibit the I_Ks amplitude (by 19.4 ± 4.7%; p < 0.05). At 5 μm , lapatinib induced prolongation of APD₉₀ by 16.1% (p < 0.05). These results suggest that the APD₉₀‐prolonging effect of lapatinib on rabbit Purkinje fibres is primarily a result of inhibition of the hERG current and I_Ks, but not I_Na, I_K1 or I_Ca.     

Effect of the Class III Antiarrhythmic Agent E-4031 on the ATP-Sensitive Potassium Channel in Rabbit Ventricular Myocytes

Abstract: The class III antiarrhythymic drug E-4031. a known blocker of the delayed rectifier potassium channel (I_K), might also be capable of blocking the ATP-sensitive potassium channel (IKATP)- We examined this possibility by studying the effect of E-4031 on single IKATP channels in membrane patches excised from ventricular myocytes that were obtained by standard enzymatic dissociation techniques from New Zealand white rabbits. In inside-out patches, E-4031 caused a dose-dependent block of IKATP with an EC₅₀ of 31 ± 1 μM, Hill coefficient of 0.89±0.24 and no effect on channel conductance. Open dwell-time kinetics were fitted by two exponential components, with E-4031 causing reduction of the longer time constant. In outside-out patches, the concentration of E-4031 required to produce blockade was much higher. We conclude that E-4031 blocks the ATP-sensitive potassium channel and that it does so from within the cytoplasm, with one-to-one channel binding stoichiometry. Single channel conductance is unchanged, but the longer time constant for the open state is reduced, which suggests that E-4031 may be an open channel blocker of intermediate to slow time course.     

Pharmacological evidence for altered src kinase regulation of <Emphasis Type="Italic">I</Emphasis><Subscript>Ca,L</Subscript> in patients with chronic atrial fibrillation

A reduction in l-type Ca²⁺ current (I _Ca,L) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I _Ca,L is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I _Ca,L in cardiomyocytes. We studied α_1C l-type Ca²⁺ channel subunit expression and the regulation of I _Ca,L by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of α_1C l-type Ca²⁺ channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of α_1C did not differ between AF and sinus rhythm (SR; α_1C/calsequestrin: 1.0 ± 0.1 and 1.2 ± 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I _Ca,L from 3.0 ± 0.3 to 6.1 ± 0.8 pA/pF and from 2.8 ± 0.4 to 6.1 ± 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I _Ca,L was significantly lower. In this case, genistein lead to the same relative increase in I _Ca,L as in SR cells (from 1.46 ± 0.30 to 3.2 ± 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I _Ca,L by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 ± 0.4, 2.1 ± 0.2, and 2.7 ± 0.4 nmol/mg/min and 3.6 ± 0.4, 1.3 ± 0.2, and 2.4 ± 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I _Ca,L in AF is not due to a reduction in l-type Ca²⁺ channel protein expression. Indirect evidence for an impaired src kinase regulation of I _Ca,L together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I _Ca,L dysregulation in chronic AF.     

Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes

BACKGROUND AND PURPOSE

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of I_Kr blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds).

EXPERIMENTAL APPROACH

Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models.

KEY RESULTS

At clinically relevant concentrations (5–12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50–83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization.

CONCLUSION AND IMPLICATIONS

Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the I_Kr blocking drugs moxifloxacin and dofetilide or E4031.     

盐酸非洛普对豚鼠心室肌细胞延迟整流钾电流的抑制作用

目的　已知盐酸非洛普〔1 (2 ,6 二甲基苯氧基 ) 2 (3,4 二甲氧基苯乙氨基 )丙烷盐酸盐 ,DDPH〕对心肌钙电流和钠电流具有抑制作用 ,为全面了解其抗心律失常作用的离子机理 ,研究其对延迟整流钾电流的影响。方法　全细胞膜片钳技术记录豚鼠心室肌细胞快激活的延迟整流钾电流的尾电流(IKr tail)和慢激活的延迟整流钾电流 (IKs)及其尾电流 (IKs tail)。结果　DDPH(1～ 10 0 μmol·L- 1)浓度依赖性地抑制IKr tail,其IC50 为 7.0 (95 %可信限为4 .2 3～ 9.76 ) μmol·L- 1;DDPH 10 μmol·L- 1对IKr tail具有电压依赖性抑制作用。DDPH 10 ,30和 10 0 μmol·L- 1可浓度依赖性地抑制IKs及其IKs tail,使IKs从给药前的 (9.1± 0 .7)pA·pF- 1分别降至 (7.7± 1.7) ,(7.5± 1.8)和 (5 .6± 1.8)pA·pF- 1(P <0 .0 1) ;使IKs tail从给药前的 (1.4± 0 .2 )pA·pF- 1分别降至(1.1± 0 .2 ) ,(0 .9± 0 .2 )和 (0 .6± 0 .2 )pA·pF- 1(P <0 .0 5或P <0 .0 1) ;DDPH 30 μmol·L- 1对IKs tail具有电压依赖性抑制作用。结论　DDPH对豚鼠心室肌细胞延迟整钾电流具有抑制作用。     

The inotropic agents DPI 201-106 and BDF 9148 differentially affect potassium currents of guinea-pig ventricular myocytes

The inotropic agents DPI 201-106 and BDF 9148 increase action potential duration (APD) of heart muscle. This effect can be explained by inhibition of inactivation of sodium current, which is affected by both agents to a similar extent (Ravens et al. 1991, Br J Pharmacol 104:1019–1023). However, as DPI 201-106 prolongs APD of guinea-pig ventricle to a larger extent than BDF 9148, other currents may also be involved. The aim of the present study was to measure the effects of DPI 201-106 and BDF 9148 on the inward rectifier I_K1, and the two components of the delayed rectifier, I_Ks and I_Kr. The methyl-for-carbonitrile-substituted derivative BDF 8784 was included to study structure-activity relationships. Single-electrode whole-cell voltage-clamp technique was used to measure membrane currents of guinea-pig ventricular myocytes. Only DPI 201-106 reduced I_K1 at potentials both negative and positive to the reversal potential. Three M of DPI 201-106 reduced I_Ks, whereas 1 M of BDF 9148 had no effect on this current. These concentrations were equieffective with respect to positive inotropic action (Ravens et al. 1991, Br J Pharmacol 104:1019–1023). BDF 9148 did however block I_Ks at higher concentrations, as did BDF 8784. It is concluded that block of outward current by DPI 201-106, but insignificant effects of BDF 9148, are responsible for the differential effects of these compounds on APD at equieffective concentrations with respect to inotropy. Correspondence to: U. Ravens at the above address     

Response of IKr and hERG Currents to the Antipsychotics Tiapride and Sulpiride

The human ether-a-go-go-related gene (hERG) channel is important for repolarization in human myocardium and is a common target for drugs that prolong the QT interval. We studied the effects of two antipsychotics, tiapride and sulpiride, on hERG channels expressed in Xenopus oocytes and also on delayed rectifier K⁺ currents in guinea pig cardiomyocytes. Neither the amplitude of the hERG outward currents measured at the end of the voltage pulse, nor the amplitude of hERG tail currents, showed any concentration-dependent changes with either tiapride or sulpiride (3~300 µM). However, our findings did show that tiapride increased the potential for half-maximal activation (V_1/2) of HERG at 10~300 µM, whereas sulpiride increased the maximum conductance (G_max) at 3, 10 and 100 µM. In guinea pig ventricular myocytes, bath applications of 100 and 500 µM tiapride at 36℃ blocked rapidly activating delayed rectifier K⁺ current (I_Kr) by 40.3% and 70.0%, respectively. Also, sulpiride at 100 and 500 µM blocked I_Kr by 38.9% and 76.5%, respectively. However, neither tiapride nor sulpiride significantly affected the slowly activating delayed rectifier K⁺ current (I_Ks) at the same concentrations. Our findings suggest that the concentrations of the antipsychotics required to evoke a 50% inhibition of I_Kr are well above the reported therapeutic plasma concentrations of free and total compound.     

Current concepts in the management of long QT syndrome

Congenital and acquired long QT syndromes (LQTS) are diseases characterised by QT prolongation on the surface electrocardiogram (ECG) and a specific form of polymorphic ventricular tachycardia termed Torsade de Pointes (TdP). LQTS is caused by a net decrease in repolarising current, due to either gene mutation or drug action on late inward sodium current (I_Na), slowly activating delayed rectifier potassium current (I_Ks) and rapidly activating delayed rectifier potassium current (I_Kr). LQTS is associated with increased transmural dispersion of repolarisation (TDR) and phase 2 early afterdepolarisation (EAD), which are well-known risk factors for the development of TdP under conditions of QT prolongation. β-Adrenergic stimulation triggers the onset of TdP by inducing EAD and enhancing TDR. β-Adrenergic receptor blockade is the classic treatment for congenital forms of LQTS caused by gene mutation of ionic channels for I_Ks and I_Kr. Agents that shorten the QT interval, including a potassium supplement, I_Na blockers and I_Ks agonists, have been proposed to be useful in the treatment of LQTS. The strategies for the development of noncardiac, as well as cardiac, drugs with little risk of QT prolongation and TdP are also discussed.     

Ionic mechanisms underlying the negative chronotropic action of propofol on sinoatrial node automaticity in guinea pig heart

Background and Purpose

Propofol is a widely used intravenous anaesthetic agent, but has undesirable cardiac side effects, including bradyarrhythmia and its severe form asystole. This study examined the ionic and cellular mechanisms underlying propofol-induced bradycardia.

Experimental Approach

Sinoatrial node cells, isolated from guinea pig hearts, were current- and voltage-clamped to record action potentials and major ionic currents involved in their spontaneous activity, such as the hyperpolarization-activated cation current (I_f), T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively) and the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks, respectively). ECGs were recorded from Langendorff-perfused, isolated guinea pig hearts.

Key Results

Propofol (≥5 μM) reversibly decreased the firing rate of spontaneous action potentials and their diastolic depolarization rate. Propofol impaired I_f activation by shifting the voltage-dependent activation to more hyperpolarized potentials (≥1 μM), slowing the activation kinetics (≥3 μM) and decreasing the maximal conductance (≥10 μM). Propofol decreased I_Ca,T (≥3 μM) and I_Ca,L (≥1 μM). Propofol suppressed I_Ks (≥3 μM), but had a minimal effect on I_Kr. Furthermore, propofol (≥5 μM) decreased heart rates in Langendorff-perfused hearts. The sinoatrial node cell model reasonably well reproduced the negative chronotropic action of propofol.

Conclusions and Implications

Micromolar concentrations of propofol suppressed the slow diastolic depolarization and firing rate of sinoatrial node action potentials by impairing I_f activation and reducing I_Ca,T, I_Ca,L and I_Ks. These observations suggest that the direct inhibitory effect of propofol on sinoatrial node automaticity, mediated via multiple channel inhibition, underlies the propofol-induced bradycardia observed in clinical settings.     

Magnesium isoglycyrrhizinate inhibits L-type Ca<Superscript>2+</Superscript> channels,Ca<Superscript>2+</Superscript> transients,and contractility but not hERG K<Superscript>+</Superscript> channels

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K⁺ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca²⁺ currents (I_Ca-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (I_Kr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited I_Ca-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 0.22 mg/ml, and the maximal inhibitory effect was 61.10 ± 0.59%. MI at a concentration of 0.3 mg/ml reduced cell shortening by 24.12 ± 3.97% and the peak value of the Ca²⁺ transient by 36.54 ± 4.96%. MI had no significant influence on hERG K⁺ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of I_Ca-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on I_Kr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.     

Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents

Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K⁺ current (I _Kr and I _Ks) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 μM) suppressed I _Kr in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug–channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC₅₀ values of 0.11?±?0.01 and 0.76?±?0.04 μM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG—voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I _Ks was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 μM bisindolylmaleimide I or 0.1 μM PMA (PKC activator) was observed on I _Kr arguing against significant contribution of PKC to regulation of I _Kr. It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.     

Assessment of Testing Methods for Drug-Induced Repolarization Delay and Arrhythmias in an iPS Cell–Derived Cardiomyocyte Sheet: Multi-site Validation Study

A prospective comparison study across 3 independent research laboratories of a pure I_Kr blocker E-4031 was conducted by using the same batch of human iPS cell–derived cardiomyocytes in order to verify the utility and reliability of our original standard protocol. Field potential waveforms were recorded with a multi-electrode array system to measure the inter-spike interval and field potential duration. The effects of E-4031 at concentrations of 1 to 100 nM were sequentially examined every 10 min. In each facility, E-4031 significantly prolonged the field potential duration corrected by Fridericia’s formula and caused early afterdepolarizations occasionally resulting in triggered activities, whereas it tended to decrease the rate of spontaneous contraction. These results were qualitatively and quantitatively consistent with previous non-clinical in vitro and in vivo studies as well as clinical reports. There were inter-facility differences in some absolute values of the results, which were not observed when the values were normalized as percentage change. Information described in this paper may serve as a guide when predicting the drug-induced repolarization delay and arrhythmias with this new technology of stem cells.     

Evidence for multiple antiarrhythmic binding sites on the cardiac rapidly activating delayed rectifier K+ channel

We have previously shown that [³H]dofetilide binds with high affinity to sites associated with the guinea pig cardiac rapidly activating delayed rectifier K⁺ (I_Kr) channel and that class III antiarrhythmic agents, including dofetilide, clofilium, quinidine, sotalol, and sematilide, competitively displace [³H]dofetilide with IC₅₀ values that correlate with those for blockade of the I_Kr channel. In this report, we show that other class III antiarrhythmic agents, namely, E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonylamidobenzoyl)piperidine) and L-691, 121 (3,4-dihydro-1′-[2-(benzofurazan-5-yl)ethyl]-6-methanesfulonamidospiro[(2H)-1-benzopyran-2,4′-piperidin]-4-one), potently block guinea pig I_Kr channels with respective IC₅₀ values of 29 and 8 nM, yet have a low potency for displacement of [³H]dofetilide. Moreover, WIN 61773-2 [(R)(+)-4,5-dihydro-4-methyl-1-phenyl-3(2-phenylethyl)-(1H)-2,4-benzodiazepine monohydrochloride] biphasically displaces [³H]dofetilide according to a two site competitive binding model (site 1 = 21% displacement, IC₅₀ = 116 nM; site 2 = 79% displacement, IC₅₀ = 50 m?M) with correlation to I_Kr block in the first phase (IC₅₀ = 92 nM). These findings suggest that E--4031, L-691, 121, and WIN 61773-2 inhibit I_Kr channels by interacting at sites distinct from the high affinity [³H]dofetilide binding site. The partial displacement of [³H]dofetilide by low concentrations of WIN 61773-2, correlated with complete block of I_Kr, suggests allosteric modulation of the dofetilide binding site by this agent. ©1995 Wiley-Liss, Inc.