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Abstract We recently reported the reduced ATP-sensitive potassium (KATP) channel activities in the transgenic mouse heart overexpressing the vascular type KATP channel pore-forming subunit (Kir6.1). Although dysfunction of cardiac KATP channel has been nominated as a cause of cardiomyopathy in human, these transgenic mice looked normal as wild-type (WT) during the experiment period (~20 weeks). Extended observation period revealed unexpected deaths beginning from 30 weeks and about 50% of the transgenic mice died by 55 weeks. Surface ECG recordings from the transgenic mice at rest demonstrated the normal sinus rhythm and the regular ECG complex as well as the control WT mice except for prolonged QT interval. However, the stress ECG test with noradrenaline revealed abnormal intraventricular conduction delay and arrhythmogeneity in the transgenic mouse. Fibrotic changes in the heart tissue were remarkable in aged transgenic mice, and the cardiac fibrosis developed progressively at least from the age of 30 weeks. Gene expression analyses revealed the differentiation of cardiac fibroblasts to myofibroblasts with elevated cytokine expressions was initiated way in advance before the fibrotic changes and the upregulation of BNP in the ventricle. In sum, Kir6.1TG mice provide an electro-pathological disease concept originated from KATP channel dysfunction.

Genetic analysis of KCNJ8 has pointed a mutation (S422L) as a susceptible link to J wave syndrome (JWS). In vitro expression study indicated that the ATP-sensitive K+ (KATP) channel with the S422L mutation has the gain-of-function with reduced sensitivity to ATP. However, the electrophysiological impact of KCNJ8 has not been elucidated in vivo. Transgenic mouse strains overexpressing KCNJ8 S422L variant (TGmt) or WT (TGWT) in cardiomyocytes have been created to investigate the influence of KCNJ8 in cardiomyocytes and the JWS-related feature of the S422L variant on the cardiac electrophysiology. These TG strains demonstrated distinct changes in the J-ST segment of ECG with marked QT prolongation, which might be ascribed to the action potential prolongation resulting from the reduction of voltage-dependent K+ currents in ventricular cells. The pinacidil-induced KATP current was decreased in these TG myocytes and no obvious difference between TG and non-TG (WT) myocytes in the ATP sensitivity of the KATP channel was observed although the open probability of the KATP channels was significantly lower in TG myocytes than WT. These transgenic mouse strains with distinct ECG changes suggested that the S422L mutation in KCNJ8 gene is not a direct cause of JWS.

Although it has been reported that endotoxin-induced expression of Nox1 in the heart contributes to apoptosis in cardiomyocytes, functional role of Nox1 at the physiological expression level has not been elucidated. The aim of this study was to clarify the role of Nox1 under a hypoxic condition using wild-type (WT, Nox1+/Y) and Nox1-deficient (Nox1−/Y) mice. ECG recordings from anesthetized mice revealed that Nox1−/Y mice were more sensitive to hypoxia, resulting in bradycardia, compared to WT mice. Atrial and ventricular electrocardiograms recorded from Langendorff-perfused hearts revealed that hypoxic perfusion more rapidly decreased heart rate in Nox1−/Y hearts compared with WT hearts. Sinus node recovery times measured under a hypoxic condition were prolonged more markedly in the Nox1−/Y hearts. Sinoatrial node dysfunction of Nox1−/Y hearts during hypoxia was ameriolated by the pre-treatment with the Ca2+ channel blocker nifedipine or the K+ channel opener pinacidil. Spontaneous action potentials were recorded from enzymatically-isolated sinoatrial node (SAN) cells under a hypoxic condition. There was no significant difference in the elapsed times from the commencement of hypoxia to asystole between WT and Nox1−/Y SAN cells. These findings suggest that Nox1 may have a protective effect against hypoxia-induced SAN dysfunction.

Abstract.: The acetylcholine receptor–operated K+ (KACh) channel may be a novel target for atrial-specific antiarrhythmic therapy. Recently it has been demonstrated that tertiapin, a selective blocker of KACh channel, suppressed aconitine-induced atrial fibrillation (AF) in dogs. However, the precise mechanism by which the KACh-channel blocker inhibits the aconitine-induced AF remains unknown. This study was undertaken to determine the role of KACh channel in aconitine-induced AF in guinea pigs. Tertiapin terminated the aconitine-induced AF in anesthetized guinea pigs. The results of an in vitro electrophysiological experiment using atrial cells and atrial preparations suggest that aconitine might activate KACh channels in atrial cells, probably by intracellular Na+ accumulation, and inhibition of KACh channels by tertiapin might suppress AF by producing conduction block, probably due to further decrease in the resting membrane potential. Since it has been reported that constitutively active KACh channels can be observed in atrial cells of patients with chronic AF, aconitine-induced AF may be used as an experimental model for evaluation of drug effect on chronic AF. Keywords:: acetylcholine receptor–operated K+ channel, atrial fibrillation, aconitine, tertiapin

Insulin secretagogues are used for treatment of type 2 diabetes. We attempted to discover novel small molecules to stimulate insulin secretion by using in silico similarity search using sulfonylureas as query, followed by measurement of insulin secretion. Among 38 compounds selected by in silico similarity search, we found three diphenylsemicarbazides and one quinolone that stimulate insulin secretion. We focused on compound 8 (C8), which had the strongest insulin-secreting effect. Based on the structure-activity relationship of C8-derivatives, we identified diphenylthiosemicarbazide (DSC) 108 as the most potent secretagogue. DSC108 increased the intracellular Ca2+ level in MIN6-K8 cells. Competitive inhibition experiment and electrophysiological analysis revealed sulfonylurea receptor 1 (SUR1) to be the target of DSC108 and that this diphenylthiosemicarbazide directly inhibits ATP-sensitive K+ (KATP) channels. Pharmacokinetic analysis showed that DSC108 has a short half-life in vivo. Oral administration of DSC108 significantly suppressed the rises in blood glucose levels after glucose load in wild-type mice and improved glucose tolerance in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with impaired insulin secretion. Our data indicate that DSC108 is a novel insulin secretagogue, and is a lead compound for development of a new anti-diabetic agent.

Abstract.: Some class I antiarrhythmic drugs induce a sporadic hypoglycemia by producing insulin secretion via inhibition of ATP-sensitive K+ (KATP) channels of pancreatic β-cells. It remains undetermined whether amiodarone produces insulin secretion by inhibiting KATP channels. In this study, effects of amiodarone on KATP channels, L-type Ca2+ channel, membrane potential, and insulin secretion were examined and compared with those of quinidine in a β-cell line (MIN6). Amiodarone as well as quinidine inhibited the openings of the KATP channel in a concentration-dependent manner without affecting its unitary amplitude in inside-out membrane patches of single MIN6 cells, and the IC50 values were 0.24 and 4.9 μM, respectively. The L-type Ca2+ current was also inhibited by amiodarone as well as quinidine in a concentration-dependent manner. Although glibenclamide (0.1 μM) or quinidine (10 μM) significantly potentiated the insulin secretion from MIN6 cells, amiodarone (1 – 30 μM) failed to increase insulin secretion. Amiodarone (30 μM) and nifedipine (10 μM) significantly inhibited the increase in insulin secretion produced by 0.1 μM glibenclamide. Amiodarone (30 μM) produced a gradual decrease of the membrane potential, but did not produce repetitive electrical activity in MIN6 cells. Glibenclamide (1 μM) produced a slow depolarization, followed by spiking activity which was inhibited by 30 μM amiodarone. Thus, amiodarone is unlikely to produce hypoglycemia in spite of potent inhibitory action on KATP chan-nels in insulin-secreting cells, possibly due to its Ca2+ channel–blocking action. Keywords:: ATP-sensitive K+ channel, amiodarone, quinidine, insulin, β-cell

In guinea-pig cardiomyocytes, a cAMP-dependent Cl− current (ICl,cAMP) flows through a cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which belongs to a family of the ATP-binding cassette (ABC) proteins. Although several K+-channel openers and sulfonylurea ATP-sensitive K+ (KATP)–channel blockers reportedly inhibit ICl,cAMP, effects of nicorandil on the Cl− current have not been evaluated. This study was conducted to examine the effects of nicorandil on ICl,cAMP in isolated guinea-pig ventricular cells using patch clamp techniques. Nicorandil in concentrations higher than 300 μM enhanced the ICl,cAMP preactivated by 0.1 μM isoproterenol. The isoproterenol-induced ICl,cAMP was inhibited by 100 μM glibenclamide, but not by 100 μM pinacidil. SNAP (S-nitroso-N-acetyl-d,l-penicillamine, 10 μM), a nitric oxide (NO) donor, similarly enhanced the isoproterenol-induced ICl,cAMP. However, SG-86, a denitrated metabolite possessing K+ channel–opening action, failed to enhance the Cl− current. When the ICl,cAMP was activated by 3-isobutyl-1-methylxanthine (IBMX, 30 μM), either nicorandil or SNAP failed to enhance the isoproterenol-induced ICl,cAMP. Thus, nicorandil enhances ICl,cAMP in guinea-pig cardiomyocytes through an increase in intracellular cGMP, although direct modulation of ICl,cAMP by NO cannot be completely excluded. Keywords:: cAMP-dependent Cl− current, nicorandil, heart, nitric oxide (NO), cGMP

Mitochondrial ATP-sensitive K+ (mitoKATP) and Ca2+-activated K+ (mitoKCa) channels exist in cardiac myocytes, and they play key roles in cardioprotection. We have recently reported that K+ influx through mitoKATP or mitoKCa channels occurs independently of each other and confers cardioprotection in a similar manner. Activation of mitoKATP channel is augmented by protein kinase C (PKC), whereas mitoKCa channel is activated by protein kinase A (PKA). However, phosphatidylinositol 3-kinase (PI3-K) is linked to neither mitoKATP nor mitoKCa channels. We have demonstrated that bioactive substances modulate the opening of mitoKATP channels via a PKC-dependent pathway or opening of mitoKCa channels via a PKA-dependent pathway and thereby protecting the heart from ischemia/reperfusion injury. Several endogenous substances such as adenosine and bradykinin can reduce infarct size by activation of mitoKATP channels in a PKC-dependent manner. Adrenomedullin, a potent vasodilator peptide, potentiates the opening of mitoKCa channels by PKA activation. Treatment with adrenomedullin prior to ischemia results in the reduction of infarct size via a PKA-mediated activation of mitoKCa channels. Thus, some endogenous substances confer cardioprotection via PKA- or PKC-mediated activation of mitoKATP or mitoKCa channels. Keywords:: cardioprotection, mitochondrial ATP-sensitive K+ channel, mitochondrial Ca2+-activated K+ channel, protein kinase A, protein kinase C, cardiac disease

The phenomenon termed postconditioning, that is, brief episodes of ischemia/reperfusion at the onset of reperfusion reduce infarct size, is thought to involve the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Treatment with a drug activating PI3K at the onset of reperfusion may confer a similar cardioprotection. The sulfonylurea glimepiride has been shown to activate PI3K in human endothelial cells. We therefore tested in rabbit hearts whether glimepiride can produce postconditioning-mimetic actions. Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia and 120 min of reperfusion, and infarct size was determined by triphenyltetrazolium staining. Phosphorylation of Akt was analyzed by Western blotting. Glimepiride (10 μM) treatment for the first 10 min of reperfusion significantly reduced infarct size from 67.2 ± 1.3% in controls to 35.8 ± 4.5% (P

Abstract.: After the report of the Cardiac Arrhythmia Suppression Trial, a tabular framework of the Sicilian Gambit has been proposed to display actions of antiarrhythmic drugs on ion channels and receptors and to provide more rational pharmacotherapy of arrhythmias. However, because effects of antiarrhythmic drugs on If have not been thoroughly examined, we used patch clamp techniques to determine the effects of various antiarrhythmic drugs on the HCN (hyperpolarization-activated cyclic nucleotide–gated) channel currents. HCN4 channels, a dominant isoform of HCN channels in the heart, were expressed in HEK293 cells. Amiodarone and bepridil potently inhibited the HCN4 channel current with IC50 values of 4.5 and 4.9 μM, respectively, which were close to their therapeutic concentrations. The inhibitory effects of quinidine, disopyramide, cibenzoline, lidocaine, mexiletine, aprindine, propafenone, flecainide, propranolol, and verapamil on the HCN4 channel current were weak in their therapeutic concentrations, with IC50 values of 78.3, 249, 46.8, 276, 309, 43.7, 14.3, 1700, 50.5, and 44.9 μM, respectively, suggesting that the inhibitory effects on If would be clinically small. d,l-Sotalol hardly affected the HCN4 channel current. Information about the HCN4-channel effects of many antiarrhythmic drugs may be useful for determining the appropriate drug for treatment of various arrhythmias while minimizing adverse effects. Keywords:: antiarrhythmic drug, hyperpolarization-activated cyclic nucleotide–gated (HCN) channel subtype 4, pacemaker current, amiodarone, bepridil

Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K+ current (IK.ACh) and the delayed rectifier K+ current (IK) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (IKr) and slow component of IK (IKs). Azimilide inhibited the IK.ACh induced by carbachol (CCh, 1 µM), adenosine (10 µM), and intracellular loading of GTPγS (100 µM) in a concentration-dependent manner. The IC50 values of azimilide for inhibiting the CCh-, adenosine-, and GTPγS-induced IK.ACh were 1.25, 29.1, and 20.9 µM, respectively, suggesting that azimilide inhibits IK.ACh mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 – 10 µM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The IK.ACh inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF. Keywords:: azimilide, muscarinic acetylcholine receptor-operated K+ current, delayed rectifier K+ current, atrial fibrillation, action potential

Cardiac electrophysiological alterations induced by chronic exposure to reactive oxygen species and protective effects of dietary antioxidant have not been thoroughly examined. We recorded surface electrocardiograms (ECG) and evaluated cellular electrophysiological abnormalities in enzymatically-dissociated left ventricular (LV) myocytes in heart/muscle-specific manganesesuperoxide dismutase-deficient (H/M-Sod2-/-) mice, which exhibit dilated cardiomyopathy due to increased oxidative stress. We also investigated the influences of intake of apple polyphenols (AP) containingmainly procyanidinswith potent antioxidant activity. The QRS and QT intervals of ECG recorded in H/M-Sod2-/- mice were prolonged. The effective refractory period in the LV myocardium of H/M-Sod2-/- mice was prolonged, and susceptibility to ventricular tachycardia or fibrillation induced by rapid ventricular pacing was increased. Action potential duration in H/M-Sod2-/- LV myocytes was prolonged, and automaticity was enhanced. The density of the inwardly rectifier K+ current (IK1) was decreased in the LV cells of H/M-Sod2+ mice. The AP intake partially improved these electrophysiological alterations and extended the lifespan in H/M-Sod2+ mice. Thus, chronic exposure of the heart to oxidative stress produces a variety of electrophysiological abnormalities, increased susceptibility to ventricular arrhythmias, and action potential changes associated with the reduced density of IK1. Dietary intake of antioxidant nutrients may prevent oxidative stress-induced electrophysiological disturbances. [ABSTRACT FROM AUTHOR]

The therapeutic potential of molecular hydrogen (H2) is emerging in a number of human diseases and in their animal models, including in particular Parkinson's disease (PD). H2 supplementation of drinking water has been shown to exert disease-modifying effects in PD patients and neuroprotective effects in experimental PD model mice. However,H2 supplementation does not result in detectable changes in striatal H2 levels, indicating an indirect effect. Here we show that H2 supplementation increases gastric expression of mRNA encoding ghrelin, a growth hormone secretagogue, and ghrelin secretion, which are antagonized by the β1-adrenoceptor blocker, atenolol. Strikingly, the neuroprotective effect of H2 water was abolished by either administration of the ghrelin receptor-antagonist, D-Lys3 GHRP-6, or atenolol. Thus, the neuroprotective effect of H2 in PD is mediated by enhanced production of ghrelin. Our findings point to potential, novel strategies for ameliorating pathophysiology in which a protective effect of H2 supplementation has been demonstrated. [ABSTRACT FROM AUTHOR]

Objective: The aim was to examine the effects of lysophosphatidylcholine (LPC), an amphiphilic lipid metabolite in ischemic myocardium, on intracellular pH (pHi) regulatory systems in guinea pig papillary muscles. Methods: In CO2/HCO3−-buffered Tyrode solution, pHi, intracellular Na+ activity (aNai) and membrane potential of isolated guinea pig papillary muscles were measured using ion-selective microelectrode and conventional microelectrode. Standard ammonium prepulsing with 20 mM NH4Cl was used to produce an intracellular acid load, and effects of LPC on the pHi recovery from acidosis were evaluated in the absence and presence of a transport inhibitor. Results: LPC acidified the resting pHi by 0.03±0.01 pH units (n = 15, P<0.01) concomitantly with a slight decrease in resting membrane potential and an increase in aNai in quiescent preparations. The pHi recovery rate from an intracellular acid load was decreased to 83±4% of the control value by 30 μM LPC (n = 8, P<0.05) but not by 30 μM phosphatidylcholine (PC). In the presence of 10 μM 5-(N,N–hexamethylene) amiloride (HMA), a Na+–H+ exchange inhibitor, LPC still slowed pHi recovery from an intracellular acid load to 77±4% of the control (n = 5, P<0.05). However, LPC failed to alter the pHi recovery rate in the presence of 4,4′-diisothiocyanatostilbene–2,2′-disulfonic acid (DIDS, 0.5 mM), a Na+-HCO3− symport inhibitor. Conclusion: LPC impairs Na+–HCO3− symport but not Na+–H+ exchange, and LPC may potentiate its arrhythmogenic action by intensifying the intracellular acidosis in ischemic myocardium. [ABSTRACT FROM PUBLISHER]

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the IKr channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology. [ABSTRACT FROM AUTHOR]