Hiroko Uemura

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K+ channel Kir6.1 forms K+ channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K+ channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K+ channel opener pinacidil did not induce K+ currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K+ channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.

Functional Roles of Cardiac and Vascular ATP-Sensitive Potassium Channels Clarified by Kir6.2-Knockout Mice

Abstract— ATP-sensitive potassium (KATP) channels were discovered in ventricular cells, but their roles in the heart remain mysterious. KATP channels have also been found in numerous other tissues, including vascular smooth muscle. Two pore-forming subunits, Kir6.1 and Kir6.2, contribute to the diversity of KATP channels. To determine which subunits are operative in the cardiovascular system and their functional roles, we characterized the effects of pharmacological K+ channel openers (KCOs, ie, pinacidil, P-1075, and diazoxide) in Kir6.2-deficient mice. Sarcolemmal KATP channels could be recorded electrophysiologically in ventricular cells from Kir6.2+/+ (wild-type [WT]) but not from Kir6.2−/− (knockout [KO]) mice. In WT ventricular cells, pinacidil induced an outward current and action potential shortening, effects that were blocked by glibenclamide, a KATP channel blocker. KO ventricular cells exhibited no response to KCOs, but gene transfer of Kir6.2 into neonatal ventricular cells rescued the electrophysiological response to P-1075. In terms of contractile function, pinacidil decreased force generation in WT but not KO hearts. Pinacidil and diazoxide produced concentration-dependent relaxation in both WT and KO aortas precontracted with norepinephrine. In addition, pinacidil induced a glibenclamide-sensitive current of similar magnitude in WT and KO aortic smooth muscle cells and comparable levels of hypotension in anesthetized WT and KO mice. In both WT and KO aortas, only Kir6.1 mRNA was expressed. These findings indicate that the Kir6.2 subunit mediates the depression of cardiac excitability and contractility induced by KCOs; in contrast, Kir6.2 plays no discernible role in the arterial tree.

Inhibitory effects of JTV-519, a novel cardioprotective drug, on potassium currents and experimental atrial fibrillation in guinea-pig hearts

We investigated the effects of JTV‐519 (4‐[3‐(4‐benzylpiperidin‐1‐yl)propionyl]‐7‐methoxy‐2,3,4,5‐tetrahydro‐1,4‐benzothiazepine monohydrochloride), a novel cardioprotective drug, on the repolarizing K+ currents in guinea‐pig atrial cells by use of patch‐clamp techniques. We also evaluated the effects of JTV‐519 on experimental atrial fibrillation (AF) in isolated guinea‐pig hearts. In atrial cells stimulated at 0.2 Hz, JTV‐519 in concentrations of 0.3 and 1 μM slightly prolonged the action potential duration (APD). The drug also reversed the action potential shortening induced by the muscarinic agonist carbachol in a concentration‐dependent manner. The muscarinic acetylcholine receptor‐operated K+ current (IK.ACh) was activated by the extracellular application of carbachol (1 μM), adenosine (10 μM) or by the intracellular loading of GTPγS (100 μM). JTV‐519 inhibited the carbachol‐, adenosine‐ and GTPγS‐induced IK.ACh with the IC50 values of 0.12, 2.29 and 2.42 μM, respectively, suggesting that the drug may inhibit IK.ACh mainly by blocking the muscarinic receptors. JTV‐519 (1 μM) inhibited the delayed rectifier K+ current (IK). Electrophysiological analyses indicated that the drug preferentially inhibits IKr (rapidly activating component) but not IKs (slowly activating component). In isolated hearts, perfusion of carbachol (1 μM) shortened monophasic action potential (MAP) and effective refractory period (ERP), and lowered atrial fibrillation threshold (AFT). Addition of JTV‐519 (1 μM) inhibited the induction of AF by prolonging MAP and ERP. We conclude that JTV‐519 can exert antiarrhythmic effects against AF by inhibiting repolarizing K+ currents. The drug may be useful for the treatment of AF in patients with ischaemic heart disease.

Inhibitory effects of aprindine on the delayed rectifier K+ current and the muscarinic acetylcholine receptor‐operated K+ current in guinea‐pig atrial cells

In order to clarify the mechanisms by which the class Ib antiarrhythmic drug aprindine shows efficacy against atrial fibrillation (AF), we examined the effects of the drug on the repolarizing K+ currents in guinea‐pig atrial cells by use of patch‐clamp techniques. We also evaluated the effects of aprindine on experimental AF in isolated guinea‐pig hearts. Aprindine (3 μM) inhibited the delayed rectifier K+ current (IK) with little influence on the inward rectifier K+ current (IK1) or the Ca2+ current. Electrophysiological analyses including the envelope of tails test revealed that aprindine preferentially inhibits IKr (rapidly activating component) but not IKs (slowly activating component). The muscarinic acetylcholine receptor‐operated K+ current (IK.ACh) was activated by the extracellular application of carbachol (1 μM) or by the intracellular loading of GTPγS. Aprindine inhibited the carbachol‐ and GTPγS‐induced IK.ACh with the IC50 values of 0.4 and 2.5 μM, respectively. In atrial cells stimulated at 0.2 Hz, aprindine (3 μM) per se prolonged the action potential duration (APD) by 50±4%. The drug also reversed the carbachol‐induced action potential shortening in a concentration‐dependent manner. In isolated hearts, perfusion of carbachol (1 μM) shortened monophasic action potential (MAP) and effective refractory period (ERP), and lowered atrial fibrillation threshold. Addition of aprindine (3 μM) inhibited the induction of AF by prolonging MAP and ERP. We conclude the efficacy of aprindine against AF may be at least in part explained by its inhibitory effects on IKr and IK.ACh.

Bradykinin B2‐receptor‐mediated modulation of membrane currents in guinea‐pig cardiomyocytes

1 In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)‐induced positive inotropic and chronotropic responses in isolated guinea‐pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques. 2 BK (0.1–1000 nM) increased the L‐type Ca2+ current (ICa), which was recorded from enzymatically‐dissociated atrial myocytes by the nystatin‐perforated patch method, in a concentration‐dependent fashion, and the calculated EC50 value for increasing ICa was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor‐operated K+ current (IK.ACh) that was activated by the muscarinic agonist carbachol (1 μM) with an EC50 value of 0.57 nM. Both the increase in ICa and the decrease in IK.ACh were blocked by HOE140, a selective bradykinin B2 receptor antagonist. 3 The BK‐induced inhibition of IK.ACh was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the IK.ACh inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK‐induced increase in ICa. 4 In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode. 5 We conclude that BK increases ICa and decreases IK.ACh in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2‐receptor‐mediated IK.ACh inhibition.

Histamine H1-receptor-mediated modulation of the delayed rectifier K+ current in guinea-pig atrial cells: opposite effects on IKs and IKr.

Histamine receptor‐mediated modulation of the rapid and slow components of the delayed rectifier K+ current (IK) was investigated in enzymatically‐dissociated atrial cells of guinea‐pigs using the whole cell configuration of the patch clamp technique. Histamine at a concentration of 10 μM enhanced IK recorded during strong depolarization to potentials ranging from +20 to +40 mV and inhibited IK recorded during mild depolarization to potentials ranging from −20 to −10 mV. The increase of IK was more prominent with longer depolarizing pulses, whereas the inhibition of IK was more marked with shorter depolarizing pulses, suggesting that histamine enhances IKs (the slow component of IK) and inhibits IKr (the rapid component of IK). The histamine‐induced enhancement of IKs and inhibition of IKr were abolished by 3 μM chlorpheniramine but not by 10 μM cimetidine, suggesting that these opposite effects of histamine on IKr and IKs are mediated by H1‐receptors. In the presence of 5 μM E‐4031, an IKr blocker, histamine hardly affected IK during mild depolarization although it enhanced IK during strong depolarization in a concentration‐dependent manner. Histamine increased IKs with EC50 value of 0.7 μM. In the presence of 300 μM indapamide, an IKs blocker, histamine hardly affected IKs but inhibited IKr in a concentration‐dependent manner. Histamine decreased IKr with IC50 value of 0.3 μM. Pretreatment with 100 nM calphostin C or 30 nM staurosporine, protein kinase C inhibitors, abolished the histamine‐induced enhancement of IKs, but failed to affect the histamine‐induced inhibition of IKr. We conclude that in guinea‐pig atrial cells H1‐receptor stimulation enhances IKs and inhibits IKr through different intracellular mechanisms.

A key role for the subunit SUR2B in the preferential activation of vascular KATP channels by isoflurane

It has been postulated that isoflurane, a volatile anaesthetic, produces vasodilatation through activation of ATP‐sensitive K+ (KATP) channels. However, there is no direct evidence for the activation of vascular KATP channels by isoflurane. This study was conducted to examine the effect of isoflurane on vascular KATP channels and compare it with that on cardiac KATP channels.

Influence of extracellular K+ concentrations on quinidine-induced K+ current inhibition in rat ventricular myocytes

Hypokalaemia is one of the important risk factors for development of torsades de pointes. We recently reported that hypokalaemia increased the electrocardiographic QT interval in rats treated with quinidine, but did not alter the arrhythmogenic potency of quinidine. In this study, we have investigated the influence of extracellular potassium concentration ([K+]o) on the inhibition of several types of cardiac potassium currents by quinidine. Such types of currents include the delayed rectifier potassium current (IK), the transient outward current (Ito), and the inward rectifier potassium current (IK1), as measured in isolated rat ventricular cells using patch‐clamp techniques.

Inhibitory effects of the antihistamines epinastine, terfenadine, and ebastine on potassium currents in rat ventricular myocytes.

We examined and compared the inhibitory effects of three non‐sedating antihistamines, terfenadine, ebastine, and epinastine, on delayed rectifier potassium current (IK) and transient outward potassium current (Ito) of rat isolated ventricular myocytes, using a patch clamp technique.

Interaction of class III antiarrhythmic drugs with muscarinic M2 and M3 receptors: radioligand binding and functional studies

We have recently reported that class III antiarrhythmic drugs inhibit the muscarinic acetylcholine (ACh) receptor-operated K+ current (IK, ACh) in guinea-pig atrial cells by different molecular mechanisms. The data obtained from the patch-clamp study suggest that d,l-sotalol inhibits IK, ACh by blocking the muscarinic receptors, whereas MS-551 inhibits the K+ current by blocking the muscarinic receptors and depressing the function of the K+ channel itself and/or the guanine nucleotide-binding protein (G protein). This study was undertaken to determine whether the class III antiarrhythmic drugs d,l-sotalol and MS-551 interact with the muscarinic receptors of cardiac and peripheral tissues. Both drugs inhibited concentration dependently the specific [3H]N-methylscopolamine ([3H]-NMS) binding to membrane preparations obtained from guinea-pig atria and submandibular glands. The competition curves of these drugs for [3H]-NMS binding to glandular membranes were monophasic, suggesting competition with [3H]-NMS at a single site. Although the competition curve of d,l-sotalol for [3H]-NMS binding to atrial membranes was monophasic, that of MS-551 was biphasic and showed high- and low-affinity states of binding. d,l-Sotalol showed slightly, but significantly, higher affinity for cardiac-type muscarinic receptors (M2) than for glandular-type muscarinic receptors (M3). The inhibition constant (Ki) for MS-551 in glandular membranes was also slightly greater than the high-affinity Ki value for the drug in atrial membranes. In guinea-pig left atria and ilea, d,l-sotalol shifted the concentration-response curves for the negative inotropic effect and the contracting effect of carbachol in a parallel manner. The slopes of Schild plot were not significantly different from unity, suggesting competitive antagonism, and the pA2 for d,l-sotalol in left atria was slightly greater than that in ilea. MS-551 also shifted the concentration response curve for the negative inotropic effect of carbachol in atrial preparations to a greater extent than that for the contracting effect in ileal preparations, although MS-551 failed to show a pure competitive antagonism. These results suggest that both d,l-sotalol and MS-551 interact with cardiac M2 and peripheral M3 receptors, and that at high concentrations they exert anticholinergic activity in cardiac and peripheral tissues.