Shigeru Tanabe

ClC-3-independent, PKC-dependent Activity of Volume-sensitive Cl- Channel in Mouse Ventricular Cardiomyocytes

Volume-sensitive outwardly rectifying (VSOR) Cl- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl- channel in cardiomyocytes, the properties of VSOR Cl- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3-/-) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3+/+) and heterozygous (Clcn3+/-) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl- currents. We thus conclude that functional expression of VSOR Cl- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.

ClC-3-independent Sensitivity of Apoptosis to Cl– Channel Blockers in Mouse Cardiomyocytes

It has been shown that Cl^–/HCO₃^– exchangers and Cl^– channels, both of which are sensitive to stilbene derivatives, have essential roles in the mechanism of apoptosis induction. Staurosporine-induced apoptosis in neonatal mouse cardiomyocytes was prevented by a stilbene derivative, DIDS. To clarify whether Cl^–/HCO₃^– exchangers or Cl^– channels are targets of DIDS and whether ClC-3 is involved in the apoptotic process, staurosporine-induced reduction of cell viability, DNA laddering and caspase-3 activation were examined in cultured mouse ventricular myocytes derived from wild-type and ClC-3-deficient mice. Staurosporine-induced apoptosis and its DIDS sensitivity in ambient HCO₃^–-free conditions in which operation of Cl^–/HCO₃^– exchangers is minimized were indistinguishable from when HCO₃^– was present. Apoptosis was also prevented by application of a non-stilbene-derivative Cl^– channel blocker, NPPB, which cannot block Cl^–/HCO₃^– exchangers. Cardiomyocytes derived from ClC-3-deficient mice similarly underwent apoptosis after exposure to staurosporine; moreover, apoptosis was prevented by application of DIDS or NPPB. Thus, we conclude that in cardiomyocytes, apoptosis is critically dependent on operation not of Cl^–/HCO₃^– exchangers but of Cl^– channels which are distinct from ClC-3.

HCO(3)(-)-independent rescue from apoptosis by stilbene derivatives in rat cardiomyocytes.

Apoptosis of rat cardiomyocytes induced by staurosporine is prevented by a stilbene derivative (DIDS), which is a known blocker of both Cl ‐ / HCO 3 ‐ exchangers and Cl− channels. To clarify its target, staurosporine‐induced activation of caspase‐3, DNA laddering and cell death were examined in cultured rat cardiomyocytes. Removal of ambient HCO 3 ‐ , which minimizes the function of Cl ‐ / HCO 3 ‐ exchangers, failed to affect the preventive effect of DIDS on apoptosis. A carboxylate analog Cl− channel blocker, which does not block Cl ‐ / HCO 3 ‐ exchangers, also inhibited apoptotic events. Thus, rescue by DIDS of cardiomyocytes from apoptosis is mediated by blockage of Cl− channels.

Antiarrhythmic effects of a novel class III drug, KCB-328, on canine ventricular arrhythmia models

KCB-328 is a newly synthesized class III drug. To determine whether this drug has antiarrhythmic or proarrhythmic effects, we used canine ventricular arrhythmia models induced by coronary ligation and reperfusion, programmed electrical stimulation (PES), two-stage coronary ligation, digitalis, or epinephrine. KCB-328, in an intravenous infusion of 0.5 mg/kg/30 min, prolonged the QTc interval only 11%, but had antiarrhythmic effects on the reentry arrhythmias induced by PES (12 of 12 dogs with old myocardial infarction; p < 0.05). KCB-328, in an infusion of 1 mg/kg/h, suppressed the occurrence of fatal ventricular fibrillation (VF) induced by coronary ligation and reperfusion under either halothane anesthesia (p < 0.05) or pentobarbital anesthesia (p < 0.05). Under the halothane anesthesia, KCB-328 alone showed proarrhythmic effects [i.e., induction of ventricular premature contractions (VPCs)], but it did not induce a more severe effect such as torsades de pointes-type ventricular tachycardia (VT). In addition, KCB-328 had weak antiarrhythmic effects on the automaticity arrhythmias induced by 24-h coronary ligation but was effective neither on 48-h coronary ligation arrhythmias nor on the digitalis- and epinephrine-induced arrhythmias. Our results indicate that KCB-328 has powerful antiarrhythmic effects with fewer proarrhythmic potencies.

KCB-328: A Novel Class III Antiarrhythmic Agent with Little Reverse Frequency Dependence in Isolated Guinea Pig Myocardium

The effects of 1-(2-amino-4-methanesulfonamidophenoxy)-2-[N-(3,4-dimethoxypheneth yl)-N-methylamino] ethane hydrochloride (KCB-328), in comparison with those of dofetilide, were studied on the action potentials (APs) of isolated guinea pig papillary muscles. KCB-328 (0.003-3 microM) concentration-dependently prolonged the AP duration at 90% repolarization (APD90) at 1- and 3-Hz pacing, and the concentration-response relations at 1 and 3 Hz resemble each other. Dofetilide (0.001-1 microM) also produced the concentration-dependent prolongation of APD90 but more pronouncedly at 1 than at 3 Hz, demonstrating the reverse frequency-dependent effect. KCB-328 at 0.03, 0.1, 0.3, and 1 microM increased APD90 by 11 +/- 1, 19 +/- 1, 25 +/- 1, and 29 +/- 1% at 3 Hz and by 9 +/- 1, 19 +/- 2, 27 +/- 2, and 33 +/- 2% at 1 Hz, respectively. Prolongation of the effective refractory period (ERP) by each drug is parallel to those of APD90 at each pacing frequency. KCB-328 modified neither the maximal velocity of depolarization, amplitude of AP, and resting membrane potential in the fast APs, nor any parameters of the slow APs. In a separate experiment, the effects of KCB-328 on the ERP of contractile response (ERPc) of excised guinea-pig papillary muscles also were studied at 1 and 3 Hz. KCB-328 (0.01-10 microM) lengthened the ERPc in a concentration-dependent and frequency-independent manner as in the electrophysiologic results. This frequency-independent ERPc prolongation by KCB-328 was not influenced by increased extracellular K+ concentration from 4 to 10 mM. These results suggest that KCB-328 might be a selective class III agent with effects that are relatively frequency independent.

Synthesis and biological activity of KCB-328 and its analogues: novel class III antiarrhythmic agents with little reverse frequency dependence.

A series of 3,4-dimethoxyphenethylamine derivatives was prepared, and their prolongation effects on effective refractory period of contractile response (ERPc) and action potential duration (APD) in isolated guinea-pig papillary muscles at 1 Hz and 3 Hz were examined. SAR studies led to the identification of KCB-328 (51) which is a novel class III antiarrhythmic agent with little reverse frequency dependence.

Accumulation of autofluorescent storage material in brain is accelerated by ischemia in chloride channel 3 gene‐deficient mice

Autofluorescent storage material (ASM) is an aging pigment that accumulates during the normal course of senescence. Although the role of ASM has yet to be fully elucidated, ASM has been implicated in age‐related neurodegeneration. In this study, we determined the level of ASM in chloride channel 3 (ClC‐3) gene‐deficient (KO) mice both in response to aging and following mild global ischemia. To understand the mechanism of action of the ASM, mice subjected to ischemia were treated with the cyclooxygenase (COX) inhibitor indomethacin or with the noncompetitive glutamate receptor antagonist MK‐801. ClC‐3 KO mice displayed age‐related neurodegeneration of the neocortex as well as the hippocampus. The cortical layers in particular granular layers became thinner with aging. ASM accumulated in the brains of ClC‐3 KO mice was increased seven‐ to 50‐fold over that observed in the corresponding regions of their wild‐type littermates. Young wild‐type mice survived longer than age‐matched ClC‐3 KO mice after permanent global ischemia. However, in the case of older animals, the survival curves were similar. The ASM also increased four‐ to fivefold 10 days after mild global ischemia, an effect that was suppressed by treatment with indomethacin and MK‐801. These results suggest that temporary ischemia might trigger a process similar to aging in the brain, mimicking the effect of age‐related neurodegenerative diseases.