Role of Ca²⁺/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca²⁺current during endothelin-1 stimulation

Abstract

current (ICa) is one of the important determinants of cardiac excitation-contraction coupling, the effect of ET-1 on the I Ca is not always clear. The controversial results appear to be due to different patchclamp methods. The present study measured the effect of ET-1 on the I Ca of rat ventricular myocytes using the perforated patch-clamp technique. The holding potential was set to Ϫ40 mV, and depolarization was applied every 10 s. ET-1 (10 nM) increased the I Ca in a monophasic manner. The current reached a steady state 15 min after the application of ET-1, when the measurement was done. Endothelin receptor subtype expression was also investigated using Western immunoblotting. ET A-receptor protein was expressed, but ETB-receptor protein was not expressed, in the cell membranes of rat ventricular myocytes. The effect of ET-1 on the I Ca was inhibited by a selective ET A-receptor antagonist, BQ-123, but not by a selective ETB-receptor antagonist, BQ-788. The effect was inhibited by protein kinase C (PKC) inhibitor chelerythrine and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93, but not by its inactive analog KN-92. The effect of ET-1 was also blocked by another CaMKII inhibitor, autocamtide-2-related inhibitory peptide. These results suggest that ET-1 increases the I Ca via the ETA-receptor-PKC-CaMKII pathway. perforated patch clamp; endothelin A receptor; KN-93; BQ-123 ENDOTHELIN-1 (ET-1) IS KNOWN as a strong vasoconstrictive peptide. Production of ET-1 is increased under pathophysiological conditions in the cardiovascular system, such as heart failure (30), acute myocardial infarction (21), and hypertension (26). This peptide also directly affects cardiac muscle and shows a positive inotropic effect. However, the intracellular mechanism of the inotropic effect remains unclear. ET-1 induces phosphoinositide hydrolysis and activates protein kinase C (PKC) (11,12,28). Therefore, ET-1 increases intracellular pH by PKCinduced activation of the Na/H exchanger. Intracellular alkalization would increase contractility, because Ca 2ϩ sensitivity of the contractile elements is increased in a pH-dependent manner (17). However, there is a discrepancy between the change in intracellular pH and that in contractility when ET-1 is applied (13, 14). ET-1 increases the L-type Ca 2ϩ current (I Ca ), which is also one of the key determinants of cardiac muscle contraction, although the effect of ET-1 on I Ca is controversial (5,12,29,31,32,36). There are two distinct endothelin receptor subtypes: ET A and ET B . Stimulation of the ET A receptor induces vasoconstriction in smooth muscle cells, while that of ET B receptor induces vasorelaxation through the nitric oxide production in endothelial cells. The ET A receptor is thought to be dominant in cardiac muscle. The ET A receptor is one of the G proteincoupled receptors, and it couples with G q , which is the same as AT 1 receptor and ␣ 1A -adrenoceptor. ␣ 1A -Adrenoceptor stimulation increases the I Ca via the activation of PKC and Ca 2ϩ / calmodulin-depen...

Fluorescence Resonance Energy Transfer–Based Sensor Camui Provides New Insight Into Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Intact Cardiomyocytes

“…These findings are in good agreement with a previously published study on activation of CaMKII by AngII. 5 Interestingly, while other studies have established a link between ET-1 signaling and CaMKII activity, 28-30 our data is the first to show that ET-1 regulates CaMKII in large part by oxidation and autonomous activation. In contrast to AngII and ET-1, we show here that PE and Iso activate CaMKII through a pathway that is largely dependent on Ca 2+ transient signaling and to a variable degree T286 phosphorylation, as demonstrated by reduced CaMKII activation of the T286A Camui mutant.…”

Angiotensin II induces afterdepolarizations via reactive oxygen species and calmodulin kinase II signaling

“…Thus, it is very likely that NADPH oxidase-ROS-CaMKII pathway could represent a common mechanism accounting for arrhythmias induced by the agonists of above receptors. Supporting this assumption, endothelin-1 has been reported to cause ventricular tachyarrhythmias via prolongation of APD and formation of EADs [34–36], promote mitochondrial ROS production triggered by NADPH oxidase [37], and activates I Ca,L by CaMKII [38]. Further studies are needed to provide direct link between these individual steps.…”

Role of Endothelin in the Induction of Cardiac Hypertrophy In Vitro

“…A previous study shows that direct application of IP 3 increased the Ca 2+ spark frequency of isolated ventricular myocyte in which IP 3 inhibitor could abolish the acute effect of ET-1 on increasing the amplitude of intracellular Ca 2+ transients [44]. CaMKII inhibition could also attenuate ET-1 increasing cardiac L-type Ca 2+ current [45]. Therefore, if the mechanical stretch-activated inotropic response occurs mainly through ET-1, both CaMKII and IP 3 activation should also be involved.…”

“…Consequently, increased NCX leads to increased intracellular Ca 2+ which then activates both CaMKII and calcineurin-NFAT signaling cascades [48]. Between the two signaling pathways, only CaMKII activation can possibly induce an increase in intracellular Ca 2+ transients [45] and cardiac hypertrophy [49]. However, previous reports on the negative effect of CaMKII on calcineurin dependent NFAT-nuclear translocation [50] suggest that the physiological role of CaMKII likely regulates calcineurin-NFAT signaling activity.…”