ALOX12 mutation in a family with dominantly inherited bleeding diathesis.

The arachidonic acid (AA) cascade plays a significant role in platelet aggregation. AA released from membrane phospholipids is metabolized by cyclooxygenase (COX) pathway to thromboxane A2 (TXA2) or by 12S-lipoxygenase (ALOX12) to 12-hydroperoxyeicosatetraenoic acid (12-HPETE). In contrast to a well-known role of the COX pathway in platelet aggregation, the role of ALOX12 is not well understood. Platelets of ALOX12-deficient mice exhibit increased sensitivity for ADP-induced aggregation. However, recent evidence strongly suggests a significant role of ALOX12 in platelet aggregation and calcium signaling. 12-HPETE potentiates thrombin- and thromboxane-induced platelet aggregation, and calcium signaling. Inhibition experiments of ALOX12 demonstrated decreased platelet aggregation and calcium signaling in stimulated platelets. We studied a family with a dominantly inherited bleeding diathesis using next-generation sequencing analysis. Platelet aggregation studies revealed that the proband's platelets had defective aggregation responses to ADP, TXA2 mimetic U46619, collagen, and AA, normal affinity of TXA2 receptor for U46619, and normal induction of GTPase activity upon stimulation with U46619. However, the production of inositol 1,4,5-triphosphate (IP3) was only increased up to 30% of the control upon U46619 stimulation, suggesting a defect in phospholipase C-ß2 (PLCB2) activation downstream from TXA2 receptors. Affected family members had no mutation of PLCB2, but had a heterozygous c.1946A > G (p.Tyr649Cys) mutation of ALOX12. ALOX12 activity in platelets from the affected members was decreased to 25-35% of the control. Our data strongly suggested that a heterozygous c.1946A > G ALOX12 mutation was a disease-causing mutation; however, further experiments are required to confirm the pathogenesis of ALOX12 mutation in platelet aggregation.

Insulin suppresses IKs (KCNQ1/KCNE1) currents, which require ß-subunit KCNE1.

Abnormal QT prolongation in diabetic patients has become a clinical problem because it increases the risk of lethal ventricular arrhythmia. In an animal model of type 1 diabetes mellitus, several ion currents, including the slowly activating delayed rectifier potassium current (IKs), are altered. The IKs channel is composed of KCNQ1 and KCNE1 subunits, whose genetic mutations are well known to cause long QT syndrome. Although insulin is known to affect many physiological and pathophysiological events in the heart, acute effects of insulin on cardiac ion channels are poorly understood at present. This study was designed to investigate direct electrophysiological effects of insulin on IKs (KCNQ1/KCNE1) currents. KCNQ1 and KCNE1 were co-expressed in Xenopus oocytes, and whole cell currents were measured by a two-microelectrode voltage-clamp method. Acute application of insulin suppressed the KCNQ1/KCNE1 currents and phosphorylated Akt and extracellular signal-regulated kinase (ERK), the two major downstream effectors, in a concentration-dependent manner. Wortmannin (10(-6) M), a phosphoinositide 3-kinase (PI3K) inhibitor, attenuated the suppression of the currents and phosphorylation of Akt by insulin, whereas U0126 (10(-5) M), a mitogen-activated protein kinase kinase (MEK) inhibitor, had no effect on insulin-induced suppression of the currents. In addition, insulin had little effect on KCNQ1 currents without KCNE1, which indicated an essential role of KCNE1 in the acute suppressive effects of insulin. Mutagenesis studies revealed amino acid residues 111-118 within the distal third C-terminus of KCNE1 as an important region. Insulin has direct electrophysiological effects on IKs currents, which may affect cardiac excitability.

Endocytic regulation of voltage-dependent potassium channels in the heart.

Understanding the regulation of cardiac ion channels is critical for the prevention of arrhythmia caused by abnormal excitability. Ion channels can be regulated by a change in function (qualitative) and a change in number (quantitative). Functional changes have been extensively investigated for many ion channels including cardiac voltage-dependent potassium channels. By contrast, the regulation of ion channel numbers has not been widely examined, particularly with respect to acute modulation of ion channels. This article briefly summarizes stimulus-induced endocytic regulation of major voltage-dependent potassium channels in the heart. The stimuli known to cause their endocytosis include receptor activation, drugs, and low extracellular [K(+)], following which the potassium channels undergo either clathrin-mediated or caveolin-mediated endocytosis. Receptor-mediated endocytic regulation has been demonstrated for Kv1.2, Kv1.5, KCNQ1 (Kv7.1), and Kv4.3, while drug-induced endocytosis has been demonstrated for Kv1.5 and hERG. Low [K(+)](o)-induced endocytosis might be unique for hERG channels, whose electrophysiological characteristics are known to be under strong influence of [K(+)](o). Although the precise mechanisms have not been elucidated, it is obvious that major cardiac voltage-dependent potassium channels are modulated by endocytosis, which leads to changes in cardiac excitability.

Azelnidipine treatment reduces the expression of Cav1.2 protein.

AIMS: Azelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. MAIN METHODS: HEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavß2c. KEY FINDINGS: The surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavß2c subunits that function as a chaperone of Cav1.2. SIGNIFICANCE: This study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

KCNQ1 is internalized by activation of α1 adrenergic receptors.

KCNQ1 (Kv7.1 or KvLQT1) plays important physiological roles in various tissues forming potassium channels with KCNE subunits. Among the channels formed by KCNQ1 and KCNE subunits, the best studied is the slow delayed rectifier potassium channel in the heart, the IKs (KCNQ1/KCNE1) channel, which is critical for repolarization of cardiac action potential. The KCNQ1 channel is internalized by Nedd4/Nedd4-like ligase-dependent ubiquitination. It is also reported that phosphorylation of KCNE1 by PKC results in internalization of the KCNQ1/KCNE1 channel. Because we have observed down-regulation of KCNQ1/KCNE1 currents by activation of the α1-adrenergic receptor (α1AR) that activates PKC, this study investigated whether α1AR causes internalization of the KCNQ1 protein. We fused HaloTag to the extracellular region of KCNQ1 (Halo-KCNQ1) and co-expressed it with α1ARs in HEK293 cells. The KCNQ1 protein on the cell surface was selectively labeled with membrane-impermeable HaloTag ligands, and changes in its localization were monitored by confocal fluorescence microscopy. Activation of α1AAR and α1BAR caused marked internalization of KCNQ1, which was not KCNE1-dependent. Internalization of KCNQ1 by α1AR activation was inhibited by disruption of the PY motif or the YXXΦ motif in the C-terminus. Double staining for the receptor and the channel revealed that KCNQ1 internalization was independent of α1AR internalization. Our results suggest that α1AR-mediated direct internalization of KCNQ1 is AP2/clathrin-dependent and may be triggered by ubiquitination of KCNQ1 via the AMP dependent kinase (AMPK)/Nedd4-2 pathway. When phenylephrine was applied to rat neonatal cardiomyocytes transfected with KCNQ1 and α1AR, the KCNQ1 protein was internalized. The internalization of KCNQ1 by α1AR would affect pathophysiology in a variety of tissues expressing KCNQ1, which merits further in vivo study.

Signal transduction and Ca2+ signaling in contractile regulation induced by crosstalk between endothelin-1 and norepinephrine in dog ventricular myocardium.

In certain cardiovascular disorders, such as congestive heart failure and ischemic heart disease, several endogenous regulators, including norepinephrine (NE) and endothelin-1 (ET-1), are released from various types of cell. Because plasma levels of these regulators are elevated, it seems likely that cardiac contraction might be regulated by crosstalk among these endogenous regulators. We studied the regulation of cardiac contractile function by crosstalk between ET-1 and NE and its relationship to Ca2+ signaling in canine ventricular myocardium. ET-1 alone did not affect the contractile function. However, in the presence of NE at subthreshold concentrations (0.1 to 1 nmol/L), ET-1 had a positive inotropic effect (PIE). In the presence of NE at higher concentrations (100 to 1000 nmol/L), ET-1 had a negative inotropic effect. ET-1 had a biphasic inotropic effect in the presence of NE at an intermediate concentration (10 nmol/L). The PIE of ET-1 was associated with an increase in myofilament sensitivity to Ca2+ ions and a small increase in Ca2+ transients, which required the simultaneous activation of protein kinase A (PKA) and PKC. ET-1 elicited translocation of PKCepsilon from cytosolic to membranous fraction, which was inhibited by the PKC inhibitor GF 109203X. Whereas the Na+-H+ exchange inhibitor Hoe 642 suppressed partially the PIE of ET-1, detectable alteration of pHi did not occur during application of ET-1 and NE. The negative inotropic effect of ET-1 was associated with a pronounced decrease in Ca2+ transients, which was mediated by pertussis toxin-sensitive G proteins, activation of protein kinase G, and phosphatases. When the inhibitory pathway was suppressed, ET-1 had a PIE even in the absence of NE. Our results indicate that the myocardial contractility is regulated either positively or negatively by crosstalk between ET-1 and NE through different signaling pathways whose activation depends on the concentration of NE in the dog.

Differential action of a protein tyrosine kinase inhibitor, genistein, on the positive inotropic effect of endothelin-1 and norepinephrine in canine ventricular myocardium.

Experiments were carried out in isolated canine ventricular trabeculae and acetoxymethylester of indo-1-loaded single myocytes to elucidate the role of protein tyrosine kinase (PTK) in the inotropic effect of endothelin-1 (ET-1) induced by crosstalk with norepinephrine (NE). The PTK inhibitor genistein was used as a pharmacological tool. Genistein but not daidzein inhibited the positive inotropic effect and the increase in Ca(2+) transients induced by ET-1 by crosstalk with NE at low concentrations. Genistein and daidzein antagonized the negative inotropic effect and the decrease in Ca(2+) transients induced by ET-1 by crosstalk with NE at high concentrations, but genistein did not affect the antiadrenergic effect of carbachol. Genistein but not daidzein enhanced the positive inotropic effect and the increase in Ca(2+) transients induced by NE via beta-adrenoceptors, while the enhancing effect of genistein was abolished by the protein tyrosine phosphatase inhibitor vanadate. These findings indicate that genistein (1) induces a positive inotropic effect in association with an increase in Ca(2+) transients, (2) inhibits the positive inotropic effect of ET-1 induced by crosstalk with NE, and (3) enhances the positive inotropic effect of NE induced via beta-adrenoceptors by inhibition of PTK. In addition, genistein inhibits the negative inotropic effect of ET-1 induced by crosstalk with NE through a PTK-unrelated mechanism. PTK may play a crucial role in the receptor-mediated regulation of cardiac contractile function in canine ventricular myocardium.

Differential inhibition by the Rho kinase inhibitor Y-27632 of the increases in contractility and Ca2+ transients induced by endothelin-1 in rabbit ventricular myocytes.

The role of Rho kinase activation in the regulation of cardiac contractility and Ca(2+) signaling remains unclear, whereas its role in smooth muscle regulation has been well documented. To study the potential role of Rho kinase in the regulation of cardiac contractility and Ca(2+) transients induced by endothelin-1 (ET-1) and isoproterenol, we used the Rho kinase inhibitor Y-27632 in rabbit ventricular myocardium and myocytes loaded with indo-1/AM. Y-27632 (3-30 microM) inhibited significantly the baseline contractility and Ca(2+) transients. Furthermore, Y-27632 suppressed the increase in contractility and Ca(2+) transients induced by ET-1 in a concentration-dependent manner, when it was used in a concentration at which it did not affect the effects of isoproterenol via beta-adrenoceptors. In the presence of Y-27632, ET-1 increased cell shortening in the absence of an increase in Ca(2+) transients. This is an indication that the increase in myofilament Ca(2+) sensitivity induced by ET-1 is less susceptible to the inhibitory action of Y-27632. These findings imply that the Rho kinase activation may partially contribute to the ET-1-induced regulation of contractility, primarily due to an ET-1-induced increase in Ca(2+) transients in rabbit ventricular myocardium.

Differential inhibition by TAK-044 of the inotropic effects of endothelin-1 and endothelin-3.

The influence of a nonselective antagonist of endothelin receptors, TAK-044 (cyclo-[d-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-l-alanyl-l-alpha-aspartyl-d-2-(2-thienyl)glycyl-l-leucyl-d-tryptophyl] disodium), on the positive inotropic effect of endothelin-1 and endothelin-3 was investigated in isolated rabbit myocardium. While TAK-044 produced a concentration-dependent rightward shift of the concentration-response curve for endothelin-1 and endothelin-3, the effect of endothelin-3 was hundred times more sensitive to TAK-044 than that of endothelin-1. The combination of FR139317 ([2-(R)-[2(R)-[2(S)-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl] amino-3-(2-pyridyl)propionic acid]) and BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine) mimicked the inhibitory action of TAK-044 on the positive inotropic effect of endothelin-3 but enhanced the effect of endothelin-1. In a receptor-binding assay, TAK-044 was four times more potent in antagonizing the specific binding of endothelin-1 than that of endothelin-3. Endothelin-1 may activate receptor subtypes that trigger both positive and negative inotropic effects, the latter being more susceptible to the antagonistic action of TAK-044, which may explain in part the differential antagonistic action of TAK-044 on the inotropic effect of endothelin-1 and endothelin-3.

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes.

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.

Wortmannin inhibits the increase in myofilament Ca(2+) sensitivity induced by cross-talk of endothelin-1 with norepinephrine in canine ventricular myocardium.

Endothelin-1 (ET-1) modulates cardiac contractility by cross-talk with norepinephrine (NE) in canine ventricular myocardium. The present experiments were performed to investigate the influence of wortmannin that has inhibitory action on phosphatidylinositol 3-kinase (PI3-K) (IC50 = 3 nM) and myosin light chain kinase (MLCK) (IC50 = 200 nM) on Ca(2+) signaling and the inotropic effects of ET-1 induced by cross-talk with NE. Experiments were carried out in isolated canine ventricular trabeculae and indo-1/AM-loaded single ventricular cardiomyocytes. ET-1 alone elicited a transient small negative inotropic effect (NIE). In the presence of NE at low (1-10 nM) and high (100 nM) concentrations, ET-1 induced a long-lasting positive inotropic effect (PIE) or a marked sustained NIE, respectively. Wortmannin up to 300 nM did not affect the contractility; and at 1 microM and higher, it decreased the basal contraction without suppressing Ca(2+) transients. Wortmannin (1 microM) inhibited the long-lasting PIE of ET-1 without affecting the ET-1-induced increase in Ca(2+) transients. Wortmannin at the same concentration did not affect the ET-1-induced transient and sustained NIE and the PIE mediated by beta-adrenoceptor stimulation. These results imply that wortmannin exerts selective inhibitory action on the increase in myofilament Ca(2+) sensitivity induced by cross-talk of ET-1 with NE probably through an inhibition of MLCK in canine ventricular myocardium.

Receptor subtypes mediating the inotropic effects and Ca(2+) signaling induced by endothelin-1 through crosstalk with norepinephrine in canine ventricular myocardium.

In canine ventricular myocardium, endothelin-1 (ET-1) alone induced only a weak transient negative inotropic effect (NIE). However, ET-1 induced a marked sustained positive inotropic effect (PIE) subsequent to a transient NIE in the presence of norepinephrine (NE) at low concentrations (0.1 - 1 nM) and elicited a pronounced sustained NIE in the presence of NE at high concentrations (around 100 nM). Thus, the extent of beta-adrenoceptor stimulation induced by NE played a crucial role in determining the characteristics of the inotropic effects of ET-1. The characteristics of ET receptor subtypes involved in contractile regulation and Ca(2+) signaling induced by ET-1 were determined. The ET-1-induced transient NIE and decrease in Ca(2+) transients were abolished by the selective ET(A)-receptor antagonist FR319317, but not by the selective ET(B)-receptor antagonist BQ-788. The sustained PIE and the increase in Ca(2+) transients induced by ET-1 were abolished by FR319317, but not inhibited by BQ-788. In contrast, the sustained NIE of ET-1 was abolished by the non-selective ET antagonist TAK-044, markedly attenuated by FR319317, and partially inhibited by BQ-788. ET-1 alone elicited a PIE in the presence of BQ-788, which indicates that the activation of ET(B)-receptors counteracts the development of the PIE of ET-1. The current findings indicate that both ET(A) and ET(B) receptors are involved in the regulation of Ca(2+) signaling and contractility in canine ventricular myocardium.

Inhibitory action of the phosphatase inhibitor cantharidin on the endothelin-1-induced and the carbachol-induced negative inotropic effect in the canine ventricular myocardium.

In the canine ventricular myocardium, endothelin-1 and the muscarinic agonist carbachol scarcely affect the basal force of contraction but do induce a pronounced negative inotropic effect in the presence of beta-adrenoceptor agonists. Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin inhibits the negative inotropic effect induced by endothelin-1 and carbachol in isolated canine ventricular trabeculae. In the presence of 100 nM norepinephrine, endothelin-1 (10 nM) and carbachol (30 nM) decreased the norepinephrine-induced positive inotropic effect to about 40% of the norepinephrine-induced maximal response. Cantharidin at 10 microM affected neither the basal force of contraction nor the positive inotropic effect of 100 nM norepinephrine, but it did attenuate markedly the negative inotropic effect of endothelin-1. By contrast, the negative inotropic effect of carbachol was not affected by 10 microM cantharidin. At 30 microM, cantharidin induced a positive inotropic effect and enhanced the positive inotropic effect of norepinephrine by approximately 60%. Cantharidin (30 microM) markedly attenuated the negative inotropic effect of 30 nM carbachol and partially decreased the negative inotropic effect of 100 nM carbachol. The present results indicate that the activation of phosphatase that is susceptible to cantharidin is involved in both the endothelin-1-induced and the carbachol-induced negative inotropic effect. The observation that the negative inotropic effect of endothelin-1 is inhibited by cantharidin at 10 microM and that cantharidin does not affect the negative inotropic effect of carbachol supports the view that the extent of the contribution of phosphatase activation may be higher in the endothelin-1-induced negative inotropic effect than in the carbachol-induced negative inotropic effect.

Differential inotropic effects of endothelin-1, angiotensin II, and phenylephrine induced by crosstalk with cAMP-mediated signaling process in dog ventricular myocardium.

Endothelin-1 (ET-1), angiotensin II (Ang II), and phenylephrine, an alpha1-adrenoceptor agonist, share the common signaling process, resulting in activation of Gq protein-coupled receptor (GqPCR) to activate the hydrolysis of phosphoinositide (PI). They do not elicit any inotropic effect in isolated dog ventricular muscle. In the presence of forskolin or IBMX (3-isobutyl-1-methylxanthine), ET-1 produced a dual effect, that is, a positive inotropic effect (PIE) and/or a negative inotropic effect (NIE) depending on concentrations of forskolin or IBMX present simultaneously with ET-1. Phenylephrine produced a definite PIE and Ang II induced a small and transient PIE in the presence of forskolin or IBMX, but they did not elicit a NIE. Facilitation of Ca2+ influx via L-type Ca2+ channel may play a crucial role in the crosstalk because GqPCR agonists produced, likewise a PIE in the presence of Bay k 8644. GqPCR agonists failed to induce a PIE in the presence of dihydroouabain or elevated [Ca2+]o. These findings indicate that the accumulation of cAMP or activation of L-type Ca2+ channels markedly modulates the inotropic response to GqPCR agonists in a manner that leads to a PIE in dog ventricular myocardium. In addition, ET-1, but not Ang II or phenylephrine, activates the signal transduction process that results in a NIE.