Cardioprotective Effect against Ischemia-Reperfusion Injury of PAK-200, a Dihydropyridine Analog with an Inhibitory Effect on Cl- but Not Ca2+ Current.

We examined the effects of a dihydropyridine analog, PAK-200, on guinea pig myocardium during experimental ischemia and reperfusion. In isolated ventricular cardiomyocytes, PAK-200 (1 µM) had no effect on the basal peak inward and steady-state currents but inhibited the isoprenaline-induced time-independent Cl- current. In the right atria, PAK-200 had no effect on the beating rate and the chronotropic response to isoprenaline. In an ischemia-reperfusion model with coronary-perfused right ventricular tissue, a decrease in contractile force and a rise in tension were observed during a period of 30-min no-flow ischemia. Upon reperfusion, contractile force returned to less than 50% of preischemic values. PAK-200 had no effect on the decline in contractile force during the no-flow ischemia but reduced the rise in resting tension. PAK-200 significantly improved the recovery of contractile force after reperfusion to about 70% of the preischemic value. PAK-200 was also shown to attenuate the decrease in tissue ATP during ischemia. Treatment of ventricular myocytes with an ischemia-mimetic solution resulted in depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca2+ concentrations. PAK-200 significantly delayed these changes. Thus, PAK-200 inhibits the cAMP-activated chloride current in cardiac muscle and may have protective effects against ischemia-reperfusion injury through novel mechanisms.

Mechanisms for the α-Adrenoceptor-Mediated Positive Inotropy in Mouse Ventricular Myocardium: Enhancing Effect of Action Potential Prolongation.

Mechanisms for the α-adrenoceptor-mediated positive inotropy in neonatal mouse ventricular myocardium were studied with isolated myocardial preparations. The phenylephrine-induced positive inotropy was suppressed by prazosin, nifedipine, and chelerythrine, a protein kinase C inhibitor, but not by SEA0400, a selective Na+/Ca2+ exchanger inhibitor. Phenylephrine increased the L-type Ca2+ channel current and prolonged the action potential duration, while the voltage-dependent K+ channel current was not influenced. In the presence of cromakalim, an ATP-sensitive K+ channel opener, the phenylephrine-induced prolongation of action potential duration, as well as the positive inotropy, were smaller than in the absence of cromakalim. These results suggest that the α-adrenoceptor-mediated positive inotropy is mediated by an increase in Ca2+ influx through the L-type Ca2+ channel, and the concomitant increase in action potential duration acts as an enhancing factor.

Negative Inotropic Effects of Class I Antiarrhythmics on Guinea Pig Ventricular Myocardium: Correlation with L-Type Ca2+ Channel Blockade.

The negative inotropic effects of nine Vaughan Williams class I antiarrhythmic drugs were examined in guinea pig ventricular tissue preparations. The drugs decreased the contractile force of papillary muscles with different potencies: the potency order was propafenone > aprindine > cibenzoline > flecainide > ranolazine > disopyramide > pilsicainide > mexiletine > GS-458967. The potency of drugs correlated with the reported IC50 values to block the L-type Ca2+ channel rather than the Na+ channel. The effects of drugs were roughly the same when examined under a high extracellular K+ solution, which inactivates the Na+ channel. Furthermore, the attenuation of the extracellular Ca2+-induced positive inotropy was strong with propafenone, moderate with cibenzoline, and weak with pilsicainide. These results indicate that the negative inotropic effects of class I antiarrhythmic drugs can be largely explained by their blockade of the L-type Ca2+ channel.

Inhibitory Effect of a Late Sodium Current Blocker, NCC-3902, on the Automaticity of the Guinea Pig Pulmonary Vein Myocardium.

The effect of blocking the persistent component of the sodium channel current (late INa) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late INa, but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late INa induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca2+ transients, but had no effect on Ca2+ sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late INa like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.

Intracellular Ca2+-Mediated Mechanisms for the Pacemaker Depolarization of the Mouse and Guinea Pig Sinus Node Tissue.

Intracellular Ca2+-mediated mechanisms for pacemaker depolarization were studied in sinus node tissue preparations from mice and guinea pigs. Microelectrode recordings revealed that the sinus node of the mouse, which had a higher beating rate, had a steeper slope of the pacemaker depolarization than that of the guinea pig. BAPTA and ryanodine, agents that interfere with intracellular Ca2+, significantly decreased the slope of the pacemaker depolarization in both species. In contrast, SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger (NCX), as well as change to low Na+ extracellular solution, significantly decreased the slope in the mouse, but not in the guinea pig. Niflumic acid, a blocker of the Ca2+ activated Cl- channel, decreased the slope in both species. Confocal microscopy revealed the presence of spontaneous Ca2+ oscillations during the interval between Ca2+ transients; such phenomenon was more pronounced in the mouse than in the guinea pig. Thus, although intracellular Ca2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node in both species, the NCX current was involved in the mouse but not in the guinea pig.

Positive Lusitropic Effect of Quercetin on Isolated Ventricular Myocardia from Normal and Streptozotocin-Induced Diabetic Mice.

The lusitropic effect of quercetin was examined on isolated ventricular myocardial tissue preparations from normal and streptozotocin-induced diabetic mice. The time required for 90% relaxation of the myocardium, which was prolonged in the diabetic mice, was shortened by quercetin in both normal and diabetic myocardia. This effect of quercetin was completely inhibited by cyclopiazonic acid but not by SEA0400. These results indicated that quercetin accelerates myocardial relaxation through activation of the sarco-endoplasmic reticulum Ca2+-ATPase.

Contribution of ATP-Mediated Positive Feedback to Sympathetic Nerve-Induced Positive Inotropy in Guinea Pig Ventricular Myocardium.

The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.

Fluorescence Discrimination of Pharmacological Effects on the Na+-Ca2+ Exchanger and Sarco-Endoplasmic Reticulum Ca2+-ATPase in Mouse Ventricular Cardiomyocytes.

We developed a method to evaluate the activity of the Na+-Ca2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) with fluorescence microscopy in mouse ventricular cardiomyocytes. In non-beating ventricular cardiomyocytes, α-adrenoceptor stimulation by phenylephrine caused a decrease in the cytoplasmic Ca2+ concentration, which was inhibited by SEA0400, an NCX inhibitor, but not cyclopiazonic acid, a SERCA inhibitor. ß-Adrenoceptor stimulation by isoprenaline caused a decrease in the cytoplasmic Ca2+ concentration, which was inhibited by cyclopiazonic acid but not SEA0400. Ellagic acid, a phenolic phytochemical, also decreased the basal Ca2+ concentration, which was inhibited by cyclopiazonic acid, but not SEA0400. Thus, this method using fluorescent microscopy and specific inhibitors would be useful for the evaluation of pharmacological agents acting on NCX and SERCA.

KB-R7943 Inhibits the Mitochondrial Ca2+ Uniporter but Not Na+-Ca2+ Exchanger in Cardiomyocyte-Derived H9c2 Cells.

The effect of KB-R7943, an inhibitor of the plasmalemmal Na+-Ca2+ exchanger, on mitochondrial Ca2+ transporters was examined with membrane-permeabilized cardiomyocyte-derived H9c2 cells expressing the fluorescent Ca2+ indicator, yellow cameleon 3.1, in the mitochondria. KB-R7943, as well as ruthenium red, inhibited the rise in mitochondrial Ca2+ on increasing the extramitochondrial Ca2+ concentration from 0 nM to 300 nM. CGP-37157, but not KB-R7943, inhibited the decline in mitochondrial Ca2+on return to Ca2+ free extramitochondrial solution. These results indicated that KB-R7943 has inhibitory effects on the mitochondrial Ca2+ uniporter, but not on the mitochondrial Na+-Ca2+ exchanger.

Differential effects of class I antiarrhythmic drugs on the guinea pig pulmonary vein myocardium: Inhibition of automatic activity correlates with blockade of a diastolic sodium current component.

The effects of class I antiarrhythmic drugs on the automaticity of isolated guinea pig pulmonary vein myocardia were investigated using microelectrode and voltage clamp methods. All of the drugs examined reduced the maximum rate of rise of automatic action potentials. The firing frequency and rate of diastolic depolarization were decreased by aprindine, flecainide and propafenone, but not by cibenzoline, disopyramide and pilsicainide, which correlated with blockade of the sodium current component induced by ramp depolarization mimicking the diastolic depolarization. In conclusion, class I antiarrhythmic drugs which block the diastolic sodium current component inhibit the automaticity of the pulmonary vein myocardium.

Involvement of the persistent Na+ current in the diastolic depolarization and automaticity of the guinea pig pulmonary vein myocardium.

The role of the Na+ current in the automaticity of the pulmonary vein myocardium was examined in isolated guinea pig pulmonary vein cardiomyocytes and tissue preparations. Tetrodotoxin inhibited the automaticity of pulmonary vein tissue preparations by suppressing the diastolic depolarization of the action potential. ATX-II, which increased the density of persistent component of the Na+ current (late INa), induced a depolarization of the resting membrane potential followed by spontaneous firing of action potentials. GS-458967, which inhibited the late INa, suppressed the diastolic depolarization and the firing of action potentials. Pilsicainide, which inhibited only the transient component of Na+ current (peak INa), had no effect on the firing frequency. GS-458967 had no effect on the contractile force of the working myocardium. In conclusion, late INa is involved in the diastolic depolarization and automaticity of the pulmonary vein myocardium. Late INa inhibitors appear to be effective therapeutic agents for atrial fibrillation with minimum adverse effects on the working myocardium.

Angiotensin II Induces Automatic Activity of the Isolated Guinea Pig Pulmonary Vein Myocardium through Activation of the IP3 Receptor and the Na⁺-Ca2+ Exchanger.

The automaticity of the pulmonary vein myocardium is known to be the major cause of atrial fibrillation. We examined the involvement of angiotensin II in the automatic activity of isolated guinea pig pulmonary vein preparations. In tissue preparations, application of angiotensin II induced an automatic contractile activity; this effect was mimicked by angiotensin I and blocked by losartan, but not by PD123,319 or carvedilol. In cardiomyocytes, application of angiotensin II induced an increase in the frequency of spontaneous Ca2+ sparks and the generation of Ca2+ transients; these effects were inhibited by losartan or xestospongin C. In tissue preparations, angiotensin II caused membrane potential oscillations, which lead to repetitive generation of action potentials. Angiotensin II increased the diastolic depolarization slope of the spontaneous or evoked action potentials. These effects of angiotensin II were inhibited by SEA0400. In tissue preparations showing spontaneous firing of action potentials, losartan, xestospongin C or SEA0400 decreased the slope of the diastolic depolarization and inhibited the firing of action potentials. In conclusion, in the guinea pig pulmonary vein myocardium, angiotensin II induces the generation of automatic activity through activation of the IP3 receptor and the Na⁺-Ca2+ exchanger.

Resistance of Fetal Guinea Pig Ventricular Myocardium to Hypoxia: Maintained Intracellular ATP Prevents the Opening of ATP-Sensitive Potassium Channels.

The presence and function of the ATP-sensitive potassium channel current (IKATP) were examined in the guinea pig myocardium to clarify the mechanisms for the resistance of the fetal myocardium to hypoxia. Experimental hypoxia markedly reduced the action potential duration and contractile force in isolated ventricular myocardium from the adult, but only moderately in those from the fetus. In isolated ventricular cardiomyocytes, the density of the IKATP activated by cromakalim, as well as their sensitivity to intracellular ATP concentration, were not different between the fetus and adult. The tissue ATP content was similar between the fetal and adult myocardium under normal condition, but the hypoxia-induced decrease was smaller in the fetus. Confocal microscopic analysis revealed that the mitochondria in the fetal cardiomyocyte is less in quantity than that in the adult and is more localized to the cell center. These results indicate that IKATP in the fetal guinea pig myocardium has a current density and ATP sensitivity similar to those of the adult, but is not activated under hypoxic conditions because the energy metabolism of the fetal myocardium is less dependent on oxidative phosphorylation.

Pathological prolongation of action potential duration as a cause of the reduced alpha-adrenoceptor-mediated negative inotropy in streptozotocin-induced diabetic mice myocardium.

Effect of pathological prolongation of action potential duration on the α-adrenoceptor-mediated negative inotropy was studied in streptozotocin-induced diabetic mice myocardium. In streptozotocin-treated mouse ventricular myocardium, which had longer duration of action potential than that in control mice, the negative inotropic response induced by phenylephrine was smaller than that in control mice. 4-Aminopyridine prolonged the action potential duration and decreased the negative inotropy in control mice. Cromakalim shortened the action potential duration and increased the negative inotropy in streptozotocin-treated mice. These results suggest that the reduced α-adrenoceptor-mediated inotropy in the diabetic mouse myocardium is partly due to its prolonged action potential.

Fluorescence Analysis of the Mitochondrial Effect of a Plasmalemmal Na+/Ca2+ Exchanger Inhibitor, SEA0400, in Permeabilized H9c2 Cardiomyocytes.

We investigated the effect on mitochondrial Ca2+ of SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX) which reduces mitochondrial Ca2+ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca2+ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca2+ concentration caused by an increase in extramitochondrial Ca2+ concentration was inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca2+ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.

Cardiac overexpression of Epac1 in transgenic mice rescues lipopolysaccharide-induced cardiac dysfunction and inhibits Jak-STAT pathway.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the ß-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

Permissive role of reduced inwardly-rectifying potassium current density in the automaticity of the guinea pig pulmonary vein myocardium.

The electrophysiological properties underlying the automaticity of the guinea pig pulmonary vein myocardium were studied. About 30% of the isolated pulmonary vein tissue preparations showed spontaneous electrical activity, as shown by glass microelectrode recordings from their myocardial layer. The remaining quiescent preparations had a resting membrane potential less negative than that in the left atria. Blockade of the acetylcholine activated potassium current (IK-ACh) by tertiapin induced a depolarizing shift of the resting membrane potential and automatic electrical activity in the pulmonary vein, but not in the atria. The tertiapin-induced electrical activity, as well as the spontaneous activity, was inhibited by the application of carbachol or by chelation of intracellular Ca2+ by BAPTA. The isolated pulmonary vein cardiomyocytes had an IK-ACh density similar to that of the atrial cardiomyocytes, but a lower density of the inwardly-rectifying potassium current (IK1). Spontaneous Ca2+ transients were observed in about 30% of the isolated pulmonary vein cardiomyocytes, but not in atrial cardiomyocytes. The Ca2+ transients in the pulmonary vein cardiomyocytes were induced by tertiapin and inhibited by carbachol. These results indicate that the pulmonary vein cardiomyocytes have a reduced density of the inwardly-rectifying potassium current, which plays a permissive role in their intracellular Ca2+-dependent automaticity.

Involvement of alpha- and beta-adrenoceptors in the automaticity of the isolated guinea pig pulmonary vein myocardium.

We examined the involvement of adrenoceptors in the automaticity of the pulmonary vein myocardium, which probably plays a crucial role in the generation of atrial fibrillation. The automatic activity of the myocardium in guinea pig pulmonary vein tissue preparations were monitored by contractile force or membrane potential measurement. In quiescent preparations, application of noradrenaline induced an automatic activity. The firing frequency was reduced by prazosin or atenolol. Methoxamine induced an automatic activity of low frequency, which was accelerated by further application of isoproterenol. In preparations driven at a constant frequency, noradrenaline, in the presence of atenolol, caused a depolarizing shift of the resting membrane potential and an increase in the slope of the diastolic depolarization. In contrast, in the presence of prazosin, noradrenaline had no effect on the slope, but caused acceleration of the late repolarization and a hyperpolarizing shift of the maximum diastolic potential. At clinically relevant concentrations, carvedilol significantly inhibited the noradrenaline-induced activity but bisoprolol did not. It was concluded that α1- and ß1-adrenoceptor stimulation enhance automaticity through different mechanisms in the guinea pig pulmonary vein myocardium. Dual blockade of these adrenoceptors appears to be effective for suppressing noradrenaline-induced pulmonary vein automaticity and probably atrial fibrillation.

Enhancement of Automaticity by Mechanical Stretch of the Isolated Guinea Pig Pulmonary Vein Myocardium.

The effects of mechanical stretch on the automaticity of the guinea pig isolated pulmonary vein preparations were studied in microelectrode experiments. The application of cumulative mechanical stretch to the preparations resulted in increases in the firing rate of spontaneous electrical activity in the myocardial layer. This effect was significantly inhibited by stretch-activated channel blockers such as gadolinium or streptomycin. These results suggest that acute mechanical stretch enhances the automaticity of the guinea pig pulmonary vein myocardium through the opening of the stretch-activated channels.

Manifestation of automaticity in the pulmonary-vein myocardium of rats with abdominal aorto-venocaval shunt.

Effect of abdominal aorto-venocaval shunt (AVS) on the automaticity of the pulmonary-vein myocardium was studied in the rat. Spontaneous electrical activity was observed in one third of the isolated pulmonary-vein preparations from the AVS rats, but scarcely in those from sham-operated rats; the activity was induced by tertiapin and suppressed by carbachol or chelation of intracellular Ca(2+). The evoked action potentials in AVS rats had less negative resting membrane potential and longer action potential duration than those in sham-operated rats. These results suggest that the automaticity of the rat pulmonary-vein myocardium is manifested under chronic volume overload.