Haruaki Nakaya

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.

Reflex chronotropic and inotropic effects of calcium channel-blocking agents in conscious dogs. Diltiazem, verapamil, and nifedipine compared.

In chronically instrumented, conscious dogs, rapid injection of equihypotensive doses of three calcium channel-blocking agents, verapamil (250 μg/kg), diltiazem (200 μg/kg) and nifedi-pine (50 μg/kg), produced disparate chronotropic and inotropic responses. Although they all decreased mean arterial pressure by about 10%, heart rate (93 ± 4 beats/min) was markedly increased to 175 ± 12 with nifedipine, to 163 ± 15 with verapamil, and only slightly increased to 118 ± 7 with diltiazem. Contractile responses measured before (left ventricular dP/dtmax, 2749 ± 131 mm Hg/sec) and during left ventricular ejection (endocardial dimension dD/dtmax, 57 ± 4 mm/sec) were increased by 24% and 14% with nifedipine, decreased by 26% and 22% with verapamil, and were unchanged with diltiazem. These chronotropic and inotropic responses to rapid intravenous administration of the three drugs were increased in a dose-dependent manner. Similar results also were observed after slow infusion of these drugs. To determine the extent to which autonomic reflexes participated in these cardiac responses, propranolol (0.5 mg/kg) or propranolol plus atropine (0.1–0.2 mg/kg) was administered prior to injection of each calcium channel-blocking agent. Propranolol abolished the positive inotropic response to nifedipine and potentiated the negative inotropic response to verapamil. Positive chronotropic responses to verapamil, nifedipine, and diltiazem were attenuated by propranolol plus atropine. These results suggest that equihypotensive doses of the three prominent calcium channel-blocking agents exert different degrees of autonomic reflex activation in awake, unsedated dogs. These reflexes, which modulate the direct effects of calcium channel-blocking agents on chronotropic and inotropic variables of the heart, may have important clinical implications.

ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC

We have cloned ClC‐3B, a novel alternative splicing variant of ClC‐3 (ClC‐3A) that is expressed predominantly in epithelial cells. ClC‐3B has a different, slightly longer C‐terminal end than ClC‐3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO‐1) domain of the epithelium‐specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC‐3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC‐3B alone showed diffuse immunoreactivity for ClC‐3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC‐3B, strong immunoreactivity for ClC‐3B appeared at the leading edges of membrane ruffles. Patch‐clamp experiments revealed that cotransfection of ClC‐3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl– channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC‐3B‐induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC‐3B and EBP50, ClC‐3B‐dependent ORCCs were activated via the protein kinase A‐dependent pathway. These findings indicate that ClC‐3B is itself a CFTR‐regulated ORCC molecule or its activator.

Inhibitory effect of bepridil on hKv1.5 channel current: comparison with amiodarone and E-4031.

Effects of bepridil on the depolarization-activated outward K(+) currents (I(out)) in rat atrial myocytes and the human cardiac K(+) (hKv1.5) channel current stably expressed in human embryonic kidney (HEK) 293 cells were examined, and compared with those of amiodarone and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulphonamide dihydrochloride dihydrate (E-4031). Membrane currents were recorded using patch-clamp techniques in enzymatically isolated rat atrial myocytes and HEK 293 cells expressing hKv1.5 channels. Bepridil potently inhibited I(out) elicited by depolarization pulses and prolonged the action potential in rat atrial cells. Bepridil also inhibited the hKv1.5 channel current with the IC(50) value of 6.6 microM. The inhibitory effects of bepridil on the currents in HEK 293 cells were voltage-dependent. Amiodarone weakly inhibited rat atrial I(out) and hKv1.5 channel current. In contrast, E-4031 at a concentration of 10 microM had little influence on these currents. Thus, bepridil inhibits hKv1.5 channel current and the inhibitory effect may be useful for the treatment of atrial fibrillation.

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 0.2u2003Hz, JTV‐519 in concentrations of 0.3 and 1u2003μM 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 (1u2003μM), adenosine (10u2003μM) or by the intracellular loading of GTPγS (100u2003μM). JTV‐519 inhibited the carbachol‐, adenosine‐ and GTPγS‐induced IK.ACh with the IC50 values of 0.12, 2.29 and 2.42u2003μM, respectively, suggesting that the drug may inhibit IK.ACh mainly by blocking the muscarinic receptors. JTV‐519 (1u2003μM) 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 (1u2003μM) shortened monophasic action potential (MAP) and effective refractory period (ERP), and lowered atrial fibrillation threshold (AFT). Addition of JTV‐519 (1u2003μM) 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.

Phosphorylation and functional regulation of CIC‐2 chloride channels expressed in Xenopus oocytes by M cyclin‐dependent protein kinase

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl− channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume‐sensitive Cl− channel, ClC‐2 is regulated by the M‐phase‐specific cyclin‐dependent kinase, p34cdc2/cyclin B. ClC‐2 channels were phosphorylated by p34cdc2/cyclin B in both in vitro and cell‐free phosphorylation assays. ClC‐2 phosphorylation was inhibited by olomoucine and abolished by a 632Ser‐to‐Ala (S632A) mutation in the C‐terminus, indicating that 632Ser is a target of phosphorylation by p34cdc2/cyclin B. Injection of activated p34cdc2/cyclin B attenuated the ClC‐2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC‐2 currents attenuated by p34cdc2/cyclin B were increased by application of the cyclin‐dependent kinase inhibitor, olomoucine (100 μm), an effect that was inhibited by calyculin A (5 nm) but not by okadaic acid (5 nm). A yeast two‐hybrid system revealed a direct interaction between the ClC‐2 C‐terminus and protein phosphatase 1. These data suggest that the ClC‐2 channel is also counter‐regulated by protein phosphatase 1. In addition, p34cdc2/cyclin B decreased the magnitude of ClC‐2 channel activation caused by cell swelling. As the activities of both p34cdc2/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC‐2 channel could be regulated physiologically by these factors.

Long-term endothelin a receptor blockade inhibits electrical remodeling in cardiomyopathic hamsters.

Background—The endothelin (ET) system is activated in failing hearts. Congestive heart failure frequently is associated with ventricular arrhythmias, which may result from electrical remodeling such as changes of ionic current density and heterogeneous action potential prolongation. We examined the effects of long-term ETA receptor blockade on the electrophysiological properties of ventricular cells, the surface ECG, and the survival in BIO 14.6 cardiomyopathic hamsters. Methods and Results—Membrane currents and action potentials were recorded from left ventricular cells isolated from normal F1&bgr; hamsters and cardiomyopathic BIO 14.6 hamsters untreated and chronically treated with TA-0201, an ETA receptor antagonist. In ventricular cells of untreated BIO 14.6 hamsters, the action potential duration was prolonged and the densities of the L-type Ca2+ current (ICa,L), the transient outward current (Ito), the delayed rectifier K+ current (IK), and the inward rectifier K+ current (IK1) were decreased compared with those of F1&bgr; hamsters. Long-term treatment with the ETA receptor antagonist significantly attenuated action potential duration prolongation and reduction of Ito, IK, and ICa,L in BIO 14.6 ventricular cells. Long-term ETA receptor blockade prevented the QT prolongation and ventricular arrhythmias and improved the survival rate in the cardiomyopathic hamsters. Conclusions—Long-term treatment with an ETA antagonist inhibits electrical remodeling such as downregulation of K+ and Ca2+ currents, action potential prolongation, and the increased QT interval and thereby suppresses ventricular arrhythmias in cardiomyopathic hearts. ETA receptor blockade may provide a new strategy for the prevention of ventricular arrhythmias associated with heart failure.

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 (3u2003μM) 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 (1u2003μM) or by the intracellular loading of GTPγS. Aprindine inhibited the carbachol‐ and GTPγS‐induced IK.ACh with the IC50 values of 0.4 and 2.5u2003μM, respectively. In atrial cells stimulated at 0.2u2003Hz, aprindine (3u2003μM) 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 (1u2003μM) shortened monophasic action potential (MAP) and effective refractory period (ERP), and lowered atrial fibrillation threshold. Addition of aprindine (3u2003μM) 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.

Inhibitory effect of the class III antiarrhythmic drug nifekalant on HERG channels: mode of action

Nifekalant is a class III antiarrhythmic drug that has been shown to be effective against ventricular tachyarrhythmias in experimental animals and humans. We examined the detailed electrophysiological effects of nifekalant on human-ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes. Nifekalant inhibited the HERG current in a concentration-dependent manner with an IC(50) value of 7.9 microM although the drug did not inhibit the minK current in Xenopus oocytes, suggesting selective inhibition of the rapid component of the delayed rectifier K(+) current (I(Kr)) in cardiomyocytes. Nifekalant showed a higher binding affinity for the open state than for the inactive state of HERG channels. Nifekalant inhibited HERG channels in a frequency-dependent manner. The onset of the blockade was rapid but the recovery from the block was slow. Nifekalant modified the voltage dependence and kinetics of HERG channel gating. Thus, nifekalant inhibits HERG channels in a voltage-dependent and frequency-dependent manner, and the inhibitory effect may underlie the clinical efficacy of the drug against ventricular tachyarrhythmias.