Search

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.

Background Late and very late stent thrombosis after drug-eluting stent implantation is a major concern. The present study evaluated difference in the effects of sirolimus, paclitaxel and zotarolimus on endothelial cells. Methods Mouse endothelial cells were seeded in a 6-well plate. Cells were cultured with an antiproliferative drug at the expected concentrations for each well for 24hours before making 3 scratch lines with a pipette tip. After a 4.5hour incubation period, 3 reference scratch lines, vertically across the original scratch lines, were made in the same way. The experiment was repeated at least 6 times (6 plates). Measurements were performed at 9 crossings of each well. Wound healing ratio was calculated as 1−(distance of the first scratch/distance of the second scratch). % cell migration was calculated as (wound healing ratio at an expected drug concentration/wound healing ratio with no drug)×100. Average % cell migration at 54 crossings of 6 plates was calculated. Results Paclitaxel inhibited cell migration in a concentration-dependent manner. On the other hand, concentration-dependent inhibition was not observed for sirolimus or zotarolimus. Sirolimus showed a stronger inhibitory effect on migration of endothelial cells compared to zotarolimus. Conclusions The difference in the effect of antiproliferative drugs of drug-eluting stents on endothelial cells may be associated with relatively faster re-endothelialization of zotarolimus-eluting stent compared to the 1st generation DES.

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 Ca(2+) 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.

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.

Summary In enterohaemorrhagic Escherichia coli (EHEC) O157, there are two types of anaerobic nitric oxide (NO) reductase genes, an intact gene (norV) and a 204 bp deletion gene (norVs). Epidemiological analysis has revealed that norV-type EHEC are more virulent than norVs-type EHEC. Thus, to reveal the role of NO reductase during EHEC infection, we constructed isogenic norV-type and norVs-type EHEC mutant strains. Under anaerobic conditions, the norV-type EHEC was protected from NO-mediated growth inhibition, while the norVs-type EHEC mutant strain was not, suggesting that NorV of EHEC was effective in the anaerobic detoxification. We then investigated the role of NO reductase within macrophages. The norV-type EHEC produced a lower NO level within macrophages compared with the norVs-type EHEC. Moreover, the norV-type EHEC resulted in higher levels of Shiga toxin 2 (Stx2) within macrophages compared with the norVs-type EHEC. Finally, the norV-type EHEC showed a better level of survival than the norVs-type EHEC. These data suggest that the intact norV gene plays an important role for the survival of EHEC within macrophages, and is a direct virulence determinant of EHEC.

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

Erythropoietin reportedly has beneficial effects on the heart after myocardial infarction, but the underlying mechanisms of these effects are unknown. We here demonstrate that sonic hedgehog is a critical mediator of erythropoietin-induced cardioprotection in mice. Treatment of mice with erythropoietin inhibited left ventricular remodeling and improved cardiac function after myocardial infarction, independent of erythropoiesis and the mobilization of bone marrow–derived cells. Erythropoietin prevented cardiomyocyte apoptosis and increased the number of capillaries and mature vessels in infarcted hearts by upregulating the expression of angiogenic cytokines such as VEGF and angiopoietin-1 in cardiomyocytes. Erythropoietin also increased the expression of sonic hedgehog in cardiomyocytes, and inhibition of sonic hedgehog signaling suppressed the erythropoietin-induced increase in angiogenic cytokine expression. Furthermore, the beneficial effects of erythropoietin on infarcted hearts were abolished by cardiomyocyte-specific deletion of sonic hedgehog. These results suggest that erythropoietin protects the heart after myocardial infarction by inducing angiogenesis through sonic hedgehog signaling.

Background: The Japanese Rhythm Management Trial for Atrial Fibrillation (J-RHYTHM) study showed rhythm control was associated with fewer changes in the assigned treatment strategy compared to rate control in atrial fibrillation (AF). The aim was to describe how antiarrhythmics (AAs) were altered in the rhythm control arm and whether altering AAs would impact long-term outcomes. Methods and Results: Of 390 enrolled patients, 23.5% altered their AAs (drug alteration [DA] group). The hard endpoint (HE) was defined as a composite of death, stroke, embolism, major bleeding or heart failure hospitalization; soft endpoint (SE) was defined physical/psychological disability requiring alteration of treatment strategy. The patients were followed for 1.7 years. No significant difference was noted in the occurrence of HE (4.0% vs 6.5%, P=0.31), but DA-group patients had higher rates of SE (9.3% vs 18.4%, P=0.017) compared to single AA patients. The DA group was also associated with the occurrence of SE after adjustment (HR 1.90, P=0.042). When the DA group was subdivided according to the use of class III drugs or change of drugs between classes, there were no differences in outcomes. Conclusions: The need to change AA was associated with physical/psychological disabilities that seemed not to be relieved simply by changing AAs, and this should be considered as a marker for refractory paroxysmal AF requiring other strategies. (Circ J 2010; 74: 870 - 875)

Over the past decades there has been considerable progress in understanding the multifunctional roles of mitochondrial ion channels in metabolism, energy transduction, ion transport, signaling, and cell death. Recent data have suggested that some of these channels function under physiological condition, and others may be activated in response to pathological insults and play a key role in cytoprotection. This review outlines our current understanding of the molecular identity and pathophysiological roles of the mitochondrial ion channels in the heart with particular emphasis on cardioprotection against ischemia/reperfusion injury, and future research on mitochondrial ion channels.

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

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

Background Although previous clinical trials demonstrated the non-inferiority of a rate control to rhythm control strategy for management of atrial fibrillation (AF), the optimal treatment strategy for paroxysmal AF (PAF) remains unclear. Methods and Results A randomized, multicenter comparison of rate control vs rhythm control in Japanese patients with PAF (the Japanese Rhythm Management Trial for Atrial Fibrillation (J-RHYTHM) study) was conducted. The primary endpoint was a composite of total mortality, symptomatic cerebral infarction, systemic embolism, major bleeding, hospitalization for heart failure, or physical/psychological disability requiring alteration of treatment strategy. In the study, 823 patients with PAF were followed for a mean period of 578 days. The primary endpoint occurred in 64 patients (15.3%) assigned to rhythm control and in 89 patients (22.0%) to rate control (P=0.0128). No significant differences between the treatment strategies were observed in the incidences of death, stroke, bleeding and heart failure. Meanwhile, significantly fewer patients requested changes of assigned treatment strategy in the rhythm control vs the rate control group, which was accompanied by improvement in AF-specific quality of life scores. Conclusion The J-RHYTHM study showed that rhythm control was associated with fewer primary endpoints than rate control. However, mortality and cardiovascular morbidity were not affected by the treatment strategy (umin-CTR No. C000000106).

Female gender is a risk factor for drug-induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether-a-go-go-related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QTC intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17β-oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug-sensitivity of the hERG (IKr) channel in Langendorff-perfused guinea pig hearts, patch-clamped guinea pig cardiomyocytes and culture cells over-expressing hERG. We found that physiological concentrations of E2 partially suppressed IKr in a receptor-independent manner. E2-induced modification of voltage-dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug-binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QTC prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031-sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QTC prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.

The role of cardiac sarcolemmal ATP-sensitive K+ (KATP) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac KATP channel) gene, we investigated the pathophysiological role of KATP channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 ± 10 to 613 ± 84 ms (P < 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 ± 17 to 265 ± 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 ± 56 to 605 ± 108 ms (n= 6, P < 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K+ channel opener pinacidil (100 μm). Pinacidil induced a glibenclamide (1 μm)-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 ± 38 to 585 ± 91 ms (P < 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca2+ concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal KATP channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.

The angiotensin II type 1 (AT(1)) receptor is a G protein-coupled receptor that has a crucial role in the development of load-induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT(1) receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand-binding pocket. As an inverse agonist, candesartan suppressed the stretch-induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT(1) receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT(1) receptor undergoes a conformational switch that couples mechanical stress-induced activation and inverse agonist-induced inactivation.

抗不整脈薬による不整脈治療に対する考え方は，過去20年間で大きく変化した．現在，抗不整脈薬の治療対象は主に心房細動と重症心室性不整脈に絞られている．また，抗不整脈薬の分類も古典的なVaughan Williamsの分類からSicilian Gambitに移行しつつある．それと共に，直接的にイオンチャネル作用薬を用いて不整脈を治療するdownstream approachばかりでなく，不整脈の発生基盤となる電気的リモデリングを神経液性因子の調節によって抑制するupstream approachも提唱されるようになった．心室性不整脈の病因に関しても，先天性QT延長症候群やBrugada症候群などの様にイオンチャネルの遺伝子異常に起因するものも存在することが明らかとなり，病因に応じた治療が行われている．また，重症心室性不整脈に関してもIII群抗不整脈薬が主に用いられる様になり，救急医療における抗不整脈薬の使用法も変わりつつある．本稿では抗不整脈薬の分類，不整脈の発生機構と心室性不整脈の薬物療法について概説する．

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

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) 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.

Background and purpose: 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. Experimental approach: Effects of isoflurane on KATP channels were examined in aortic smooth muscle cells and cardiomyocytes of the mouse using patch clamp techniques. Effects of the anaesthetic on the KATP channels with different combinations of the inward rectifier pore subunits (Kir6.1 and Kir6.2) and sulphonylurea receptor subunits (SUR2A and SUR2B) reconstituted in a heterologous expression system were also examined. Key results: Isoflurane increased the coronary flow in Langendorff-perfused mouse hearts in a concentration-dependent manner, which was abolished by 10 μM glibenclamide. In enzymically-dissociated aortic smooth muscle cells, isoflurane evoked a glibenclamide-sensitive current (i.e. KATP current). In isolated mouse ventricular cells, however, isoflurane failed to evoke the KATP current unless the KATP current was preactivated by the K+ channel opener pinacidil. Although isoflurane readily activated the Kir6.1/SUR2B channels (vascular type), the volatile anesthetic could not activate the Kir6.2/SUR2A channels (cardiac type) expressed in HEK293 cells. Isoflurane activated a glibenclamide-sensitive current in HEK293 cells expressing Kir6.2/SUR2B channels. Conclusion and implications: Isoflurane activates KATP channels in vascular smooth muscle cells and produces coronary vasodilation in mouse hearts. SUR2B may be important for the activation of vascular-type KATP channels by isoflurane. British Journal of Pharmacology (2006) 149, 573–580. doi:10.1038/sj.bjp.0706891

Background Mega trials of rhythm vs rate control could not demonstrate the usefulness of available antiarrhythmic drugs, so a more effective and safer therapy for atrial fibrillation (AF) is now required. One candidate is the so-called "upstream therapy", which refers to the blockade of upstream modifying elements (renin - angiotensin system, cathecholamines, oxidative stress etc) that contribute to the arrhythmogenic substrate. Methods and Results The Japanese Rhythm Management Trial II for Atrial Fibrillation (J-RHYTHM II study) is a randomized comparative evaluation of an angiotensin II type 1 blocker (candesartan) and a dihydropiridine calcium blocker (amlodipine), both combined with antithrombotic therapy, as an antiarrhythmic therapy for the treatment of paroxysmal AF (PAF) associated with hypertension. To test the usefulness of this therapy, this study will reveal the recurrence rate of asymptomatic as well as symptomatic PAF during 1-year of treatment with candesartan or amlodipine, using daily transtelephonic monitoring. Conclusions The J-RHYTHM II study will follow 400 patients with PAF and hypertension who were treated at approximately 50 sites throughout Japan, and will provide clinically important information. (Circ J 2006; 70: 1318 - 1321)

Background Mitochondrial ATP-sensitive K+ (mitoKATP) channel plays a key role in cardioprotection. Hence, a sulfonylurea that does not block mitoKATP channels would be desirable to avoid damage to the heart. Accordingly, we examined the effects of sulfonylureas on the mitoKATP channel and mitochondrial Ca2+ overload. Methods Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoKATP channel activity. The mitochondrial Ca2+ concentration was measured by loading cells with rhod-2. Results The mitoKATP channel opener diazoxide (100 µM) reversibly increased flavoprotein oxidation to 31.8 ± 4.3% (n = 5) of the maximum value induced by 2,4-dinitrophenol. Glimepiride (10 µM) alone did not oxidize the flavoprotein, and the oxidative effect of diazoxide was unaffected by glimepiride (35.4 ± 3.2%, n = 5). Similarly, the diazoxide-induced flavoprotein oxidation was unaffected both by gliclazide (10 µM) and by tolbutamide (100 µM). Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca2+ overload, and the intensity of rhod-2 fluorescence increased to 197.4 ± 7.2% of baseline (n = 11). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca2+ overload (149.6 ± 5.1%, n = 11, p < 0.05 versus ouabain alone), and the effect was antagonized by the mitoKATP channel blocker 5-hydroxydecanoate (189.8 ± 27.8%, n = 5) and glibenclamide (193.1 ± 7.7%, n = 8). On the contrary, cardioprotective effect of diazoxide was not abolished by glimepiride (141.8 ± 7.8%, n = 6), gliclazide (139.0 ± 9.4%, n = 5), and tolbutamide (141.1 ± 4.5%, n = 7). Conclusions Our results indicate that glimepiride, gliclazide, and tolbutamide have no effect on mitoKATP channel, and do not abolish the cardioprotective effects of diazoxide. Therefore, these sulfonylureas, unlike glibenclamide, do not interfere with the cellular pathways that confer cardioprotection. Copyright © 2006 John Wiley & Sons, Ltd.

Bepridil, which is clinically useful in the treatment of arrhythmias, has been reported to inhibit sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels. However, the effect of bepridil on mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels remains unclear. The objective of the present study was to determine whether bepridil activates mitoK(ATP) channels and confers cardioprotection. SarcK(ATP) channels composed of Kir6.2+SUR2A in human embryonic kidney (HEK) 293 cells were examined using the patch-clamp technique. Flavoprotein fluorescence in guinea pig ventricular cells and matrix volume in isolated rat heart mitochondria were measured to assay mitoK(ATP) channel activity. Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was measured by loading cells with rhod-2 fluorescence. Coronary-perfused guinea pig ventricular muscles were subjected to 35-min no-flow ischemia followed by 60-min reperfusion. Bepridil (10 microM) completely inhibited the pinacidil-induced Kir6.2+SUR2A channel current expressed in HEK 293 cells. Bepridil reversibly oxidized the flavoprotein and increased mitochondrial matrix volume in a concentration-dependent manner. Furthermore, bepridil significantly attenuated the ouabain-induced increase of [Ca(2+)](m). Pretreatment with bepridil for 5 min before ischemia improved the recovery of developed tension measured after 60 min of reperfusion. These effects of bepridil were abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) and by the nonselective K(ATP) channel blocker glisoxepide (10 microM). Our results indicate that bepridil is an opener of mitoK(ATP) channels but an inhibitor of sarcK(ATP) channels and exerts a direct cardioprotective effect on native cardiac myocytes. This is the first report of a unique modulator of K(ATP) channels; bepridil would be expected to mitigate ischemic injury while blunting arrhythmias.

Background— The large-conductance Ca 2+ -activated K + (BK Ca ) channel in the cardiac inner mitochondrial membrane (mitoK Ca channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoK Ca channels remain to be answered. Methods and Results— Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoK Ca channel activity. The mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) and membrane potential (ΔΨ m ) were measured by loading cells with rhod-2 and JC-1, respectively. Cell death was assessed by trypan blue permeability. The BK Ca channel opener NS1619 reversibly increased the flavoprotein oxidation in a concentration-dependent manner. NS1619 (30 μmol/L) attenuated the ouabain (1 mmol/L)-induced elevation of [Ca 2+ ] m with accompanying depolarization of ΔΨ m . These effects of NS1619 were completely antagonized by the BK Ca channel blocker paxilline (2 μmol/L) but not by the mitochondrial ATP-sensitive K + (mitoK ATP ) channel blocker 5-hydroxydecanoate (500 μmol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 μmol/L), a mitoK ATP channel opener. The combined application of submaximally effective concentrations of NS1619 (10 μmol/L) and diazoxide (30 μmol/L) produced additive effects. NS1619 (30 μmol/L) blunted the rate of cell death during exposure to ouabain; this cardioprotective effect was prevented by paxilline. Activation of cAMP-dependent protein kinase by 8-bromoadenosine 3′5′-cyclic monophosphate (0.5 mmol/L) and forskolin (10 μmol/L) potentiated the NS1619-induced flavoprotein oxidation. Conclusions— Opening of mitoK Ca channels, which is modulated by cAMP-dependent protein kinase, depolarizes the ΔΨ m and attenuates the mitochondrial Ca 2+ overload. Our study further indicates that mitoK Ca channel activation confers cardioprotection in a manner similar to but independent of mitoK ATP channel activation.

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 A(1)/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 IC(50) value of 15.2 microM, when I(Kr) was blocked by chromanol 293B. On the contrary, AMP 579 (10 microM) 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 A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the non-selective adenosine A(1)/A(2) 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 microM) significantly blocked the HERG current at +10 mV by 34.9+/-7.0% (n=4, p0.05), and the degree of inhibition was comparable with that observed in guinea-pig ventricular myocytes (36.8+/-6.0%, n=4). AMP 579 (10 microM) significantly inhibited the L-type Ca(2+) current (I(Ca)) by 41.0+/-6.8% (n=5, p0.05), 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, p0.05). 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.

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca(2+) current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.

The anti-anginal drug nicorandil has been demonstrated to protect the myocardium against ischemic injury in both experimental and clinical studies. Although nicorandil seems to protect the myocardium via activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels, the mechanisms underlying its cardioprotection have remained elusive. We therefore examined whether nicorandil depolarizes the mitochondrial membrane and attenuates the mitochondrial Ca2+ overload. With the use of a Nipkow confocal system, the mitochondrial Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential (DeltaPsim) in rat ventricular myocytes were measured by loading cells with rhod-2 and JC-1 respectively. The number of cell hypercontractures resulting from mitochondrial Ca2+ overload was counted. Exposing cells to ouabain (1 mM) evoked mitochondrial Ca2+ overload and increased the intensity of rhod-2 fluorescence to 180+/-15% of baseline ( p0.001). Nicorandil (100 microM) significantly attenuated the ouabain-induced mitochondrial Ca2+ overload (129+/-4% of baseline; p0.001 vs. ouabain). Nicorandil decreased the DeltaPsim during application of ouabain, thereby reducing the intensity of JC-1 fluorescence to 89+/-2% of baseline ( p0.05). Exposure of myocytes to ouabain eventually resulted in cell hypercontracture (51+/-2%). This ouabain-induced cell hypercontracture was blunted by application of nicorandil (37+/-2%, p0.05 vs. ouabain). Moreover, these effects of nicorandil were abolished by 5-hydroxydecanoate (500 microM), a putative mitoKATP channel blocker, and by glibenclamide (10 microM), a nonselective KATP channel blocker. Our results suggest that nicorandil attenuates the matrix Ca2+ overload with accompanying depolarization of the mitochondrial membrane. Such effect might potentially be attributed to the mechanism of cardioprotection afforded by nicorandil.

We used Na(+)-Ca(2+) exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mice hearts after ischemia/reperfusion and the infarct size was significantly small in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.

ATP-sensitive potassium channel in the mitochondrial inner membrane (mitoKATP channel) rather than in the sarcolemma (sarcKATP channel) appears to play an important role in cardioprotection. We examined the effect of minoxidil, a potent antihypertensive agent and hair growth stimulator, on sarcKATP and mitoKATP channels in guinea-pig ventricular myocytes. Minoxidil activated a glybenclamide-sensitive sarcKATP channel current in the whole-cell recording mode with an EC50 of 182.6 μM. Minoxidil reversibly increased the flavoprotein oxidation, an index of mitoKATP channel activity, in a concentration-dependent manner. The EC50 for mitoKATP channel activation was estimated to be 7.3 μM; this value was notably ≈25-fold lower than that for sarcKATP channel activation. Minoxidil (10 μM) significantly attenuated the ouabain-induced increase of mitochondrial Ca2+ concentration, which was measured by loading cells with rhod-2 fluorescence. Furthermore, pretreatment with minoxidil (10 μM) before 20-min no-flow ischaemia significantly improved the recovery of developed tension measured after 60 min of reperfusion in coronary perfused guinea-pig ventricular muscles. These cardioprotective effects of minoxidil were completely abolished by the mitoKATP channel blocker 5-hydroxydecanoate (500 μM). Our results indicate that minoxidil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by the selective activation of mitoKATP channels. Keywords: KATP channel, minoxidil, mitochondria, cardioprotection Introduction Cardiac myocytes contain ATP-sensitive potassium (KATP) channels in both sarcolemmal plasma membrane (sarcKATP channels) and in mitochondrial inner membrane (mitoKATP channels) (Noma, 1983; Garlid et al., 1996; Liu et al., 1998). SarcKATP channels have been molecularly defined as an octameric complex of four pore-forming Kir6.2 and four SUR2A sulphonylurea receptors (Inagaki et al., 1996; Clement et al., 1997). On the other hand, the molecular cloning of mitoKATP channel has not yet been achieved, although recent studies using Kir6.1- and Kir6.2-deficient mice suggest that neither of these subunits is an essential component of the cardiac mitoKATP channel in mice (Miki et al., 2002; Suzuki et al., 2002). MitoKATP channels possess a distinct pharmacological profile, while sharing some pharmacological properties with sarcKATP channels. Notably, diazoxide opens mitoKATP channels ≈2000-fold more potently than sarcKATP channels in cardiac myocytes (Garlid et al., 1996). Consistent with this, Liu et al. (1998) have demonstrated that diazoxide oxidizes the mitochondrial matrix redox potential via opening of mitoKATP channels in rabbit hearts, whereas sarcKATP channels are resistant to diazoxide. Zang et al. (2001) have also demonstrated that diazoxide increases the open probability of reconstituted myocardial mitoKATP channels in lipid bilayers. Using diazoxide as a pharmacological tool, recent studies have suggested that mitoKATP channels rather than sarcKATP channels are involved in cardioprotection (Garlid et al., 1997; Liu et al., 1998; Sato et al., 2000b). However, diazoxide has been reported to inhibit succinate dehydrogenase (Schafer et al., 1969; Hanley et al., 2002), suggesting that the interpretation of the effect of diazoxide may not be straightforward. Accordingly, to further elucidate the functional role of mitoKATP channel, it is desirable to look at another mitoKATP channel-specific agent. Minoxidil (chemical structure shown in Figure 1) is a potent KATP channel opener, and has been shown to act as a vasodilating agent (Campese, 1981; Leblanc et al., 1989), and the drug is used externally for treatment of androgenetic alopecia at present (DeVillez, 1990). Hayashi et al. (1993) reported that a relatively high concentration of minoxidil opened the sarcKATP channel in guinea-pig ventricular myocytes. Contrarily, the effect of minoxidil on cardiac mitoKATP channel remains unclear. Although minoxidil has been shown to improve the contractile function after ischaemia–reperfusion in dog hearts (Yamamoto et al., 2002), cardioprotective action of minoxidil is not well understood. In the present study, we therefore examined the effects of minoxidil on mitoKATP channels by measuring flavoprotein fluorescence in guinea-pig ventricular myocytes. The results show that minoxidil confers cardioprotection via preferential activation of mitoKATP channels. Figure 1 Chemical structures of minoxidil sulphate, diazoxide, and nicorandil. Methods The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985). Cell preparation Adult guinea-pig ventricular myocytes were isolated by collagenase digestion, as previously described (Tohse et al., 1992). Once isolated, the cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum at room temperature until use. The cells used in the present experiments had a regular shape with clear cross-striation.

Disopyramide, a class la antiarrhythmic agent, has been reported to induce torsades de pointes (TdP) associated with excessive QT prolongation in electrocardiogram (ECG), especially when concomitantly administered with erythromycin, a macrolide antibiotic agent. In this study, we have evaluated the effects of erythromycin on action potential duration (APD) and potassium currents in rat ventricular myocytes in comparison with disopyramide. We have evaluated the relationship between in-vitro potassium current inhibition and in-vivo QT prolongation observed in a previous study. Action potentials and membrane potassium currents, including delayed rectifier current (IK) and transient outward current (Ito), were recorded using a whole-cell patch clamp method in enzymatically-dissociated ventricular cells. Erythromycin and disopyramide prolonged APD in a concentration-dependent manner. Disopyramide (10–100 μm) and erythromycin (100 μm) led to increases in the APD at 90% repolarization level. Disopyramide reduced IK (IC50 = 37.2 + 0.17 μm) and Ito (IC50 = 20.9 + 0.13 μm) while erythromycin reduced IK (IC50 = 60.1 + 0.29 μm) but not Ito. The observed prolongation of APD might be ascribed to the inhibition of potassium currents. Erythromycin produced the prolongation of APD and the inhibition of potassium currents with a lag time after addition of the drugs, which suggested that erythromycin might not reach potassium channels from outside the ventricular cells. The potency of disopyramide was almost equivalent under in-vitro and in-vivo conditions. However, potency of erythromycin in-vitro was far weaker than that in-vivo reported in a previous study, presumably due to a difference in the uptake of erythromycin into ventricular myocytes between in-vivo and in-vitro conditions. Therefore, when drug-induced risks of QT prolongation are to be evaluated, the difference of potencies between in-vitro and in-vivo should be taken into consideration.

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.

The Japanese Rhythm Management Trial for Atrial Fibrillation (J-RHYTHM study) is a randomized comparative evaluation of rate control and rhythm control, both combined with antithrombotic therapy, as therapeutic strategies for the treatment of atrial fibrillation (AF). This study differs from the earlier AFFIRM and RACE studies in that it has a composite primary end-point representing mortality and also physical/psychological disablement (total mortality, symptomatic cerebral infarction, systemic embolism, major bleeding, hospitalization for heart failure requiring intravenous administration of diuretics, and patient disablement). Patients' will to change the therapeutic strategy to the other is also considered as an end-point representing disablement under the assigned strategy. The secondary end-point includes quality of life scores and the efficacy and safety of drugs used in treating AF. The J-RHYTHM study emphasizes patient-reported experience and perception of AF-specific disablement, and the safety of antiarrhythmics available in Japan; it will follow 2600 patients treated at more than 150 sites in Japan for a 3-year period.