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The IK1/Kir2.1 channel agonist zacopride prevents and cures acute ischemic arrhythmias in the rat.

ABSTRACT: Arrhythmogenesis in acute myocardial infarction (MI) is associated with depolarization of resting membraine potential (RMP) and decrease of inward rectifier potassium current (IK1) in cardiomyocytes. However, clinical anti-arrhythmic agents that primarily act on RMP by enhancing the IK1 channel are not currently available. We hypothesized that zacopride, a selective and moderate agonist of the IK1/Kir2.1 channels, prevents and cures acute ischemic arrhythmias. To test this viewpoint, adult Sprague-Dawley (SD) rats were subjected to MI by ligating the left main coronary artery. The antiarrhythmic effects of zacopride (i.v. infusion) were observed in the settings of pre-treatment (zacopride given 3 min prior to coronary occlusion), post-treatment (zacopride given 3 min after coronary occlusion) and therapeutic treatment (zacopride given 30 s after the onset of the first sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) post MI). In all the three treatment modes, zacopride (15 ?g/kg) inhibited MI-induced ventricular tachyarrhythmias, as shown by significant decreases in the premature ventricular contraction (PVC) and the duration and incidence of VT or VF. In Langendorff perfused rat hearts, the antiarrhythmic effect of 1 ?mol/L zacopride were reversed by 1 ?mol/L BaCl2, a blocker of IK1 channel. Patch clamp results in freshly isolated rat ventricular myocytes indicated that zacopride activated the IK1 channel and thereby reversed hypoxia-induced RMP depolarization and action potential duration (APD) prolongation. In addition, zacopride (1 ?mol/L) suppressed hypoxia- or isoproterenol- induced delayed afterdepolarizations (DADs). In Kir2.x transfected Chinese hamster ovary (CHO) cells, zacopride activated the Kir2.1 homomeric channel but not the Kir2.2 or Kir2.3 channels. These results support our hypothesis that moderately enhancing IK1/Kir2.1 currents as by zacopride rescues ischemia- and hypoxia- induced RMP depolarization, and thereby prevents and cures acute ischemic arrhythmias. This study brings a new viewpoint to antiarrhythmic theories and provides a promising target for the treatment of acute ischemic arrhythmias.

SUBMITTER: Zhai XW

PROVIDER: S-EPMC5436763 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The IK1/Kir2.1 channel agonist zacopride prevents and cures acute ischemic arrhythmias in the rat.

Zhai Xu-Wen XW Zhang Li L Guo Yun-Fei YF Yang Ying Y Wang Dong-Ming DM Zhang Yan Y Li Pan P Niu Yi-Fan YF Feng Qi-Long QL Wu Bo-Wei BW Cao Ji-Min JM Liu Qing-Hua QH

PloS one 20170518 5

Arrhythmogenesis in acute myocardial infarction (MI) is associated with depolarization of resting membraine potential (RMP) and decrease of inward rectifier potassium current (IK1) in cardiomyocytes. However, clinical anti-arrhythmic agents that primarily act on RMP by enhancing the IK1 channel are not currently available. We hypothesized that zacopride, a selective and moderate agonist of the IK1/Kir2.1 channels, prevents and cures acute ischemic arrhythmias. To test this viewpoint, adult Sprag ...[more]

PMID: 28542320

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Project description:Ventricular tachycardia is the leading cause of sudden arrhythmic death in the U.S. Recently, the moderate IK1 channel activator, zacopride, was shown to suppress triggered ventricular tachycardia in rats. Nonetheless, concerns were raised about the possibility of pro-arrhythmic activity after IK1 channel stimulation based on the promising anti-arrhythmic strategy of IK1 blockade in other animal models. Therefore, the goal of the current study was to investigate the ex-vivo effects of zacopride on triggered arrhythmia and contractility in ventricular human myocardium in order to validate data that was solely obtained from animal models. Application of 100nmol/L isoproterenol and 0.5mmol/L caffeine led to triggered arrhythmia in isolated cardiac muscles from non-failing and end-stage failing hearts. However, the occurrence of arrhythmia in muscles of non-failing hearts was markedly higher than those of end-stage failing hearts. Interestingly, zacopride eliminated the ex-vivo triggered arrhythmia in these muscles of non-failing and failing hearts in a concentration-dependent manner, with an effective IC50 in the range of 28-40?mol/L. Conversely, in the absence of isoproterenol/caffeine, zacopride led to a negative inotropic effect in a concentration-dependent manner. Reduced cardiac contraction was clearly observed at high zacopride concentration of 200?mol/L, along with the occurrence of contractile alternans in muscles of non-failing and failing hearts. Zacopride shows promising antiarrhythmic effects against triggered arrhythmia in human ventricular myocardium. However, in the absence of Ca2+ overload/arrhythmia, zacopride, albeit at high concentrations, decreases the force of contraction and increases the likelihood of occurrence of contractile alternans, which may predispose the heart to contractile dysfunction and/or arrhythmia. Overall, our results represent a key step in translating this drug from the benchtop to the bedside in the research area.

Project description:Intracellular Ca2+ overload, prolongation of the action potential duration (APD), and downregulation of inward rectifier potassium (IK1) channel are hallmarks of electrical remodeling in cardiac hypertrophy and heart failure (HF). We hypothesized that enhancement of IK1 currents is a compensation for IK1 deficit and a novel modulation for cardiac Ca2+ homeostasis and pathological remodeling. In adult Sprague-Dawley (SD) rats in vivo, cardiac hypertrophy was induced by isoproterenol (Iso) injection (i.p., 3 mg/kg/d) for 3, 10, and 30 days. Neonatal rat ventricular myocytes (NRVMs) were isolated from 1 to 3 days SD rat pups and treated with 1 μmol/L Iso for 24 h in vitro. The effects of zacopride, a selective IK1/Kir2.1 channel agonist, on cardiac remodeling/hypertrophy were observed in the settings of 15 μg/kg in vivo and 1 μmol/L in vitro. After exposing to Iso for 3 days and 10 days, rat hearts showed distinct concentric hypertrophy and fibrosis and enhanced pumping function (P < 0.01 or P < 0.05), then progressed to dilatation and dysfunction post 30 days. Compared with the age-matched control, cardiomyocytes exhibited higher cytosolic Ca2+ (P < 0.01 or P < 0.05) and lower SR Ca2+ content (P < 0.01 or P < 0.05) all through 3, 10, and 30 days of Iso infusion. The expressions of Kir2.1 and SERCA2 were downregulated, while p-CaMKII, p-RyR2, and cleaved caspase-3 were upregulated. Iso-induced electrophysiological abnormalities were also manifested with resting potential (RP) depolarization (P < 0.01), APD prolongation (P < 0.01) in adult cardiomyocytes, and calcium overload in cultured NRVMs (P < 0.01). Zacopride treatment effectively retarded myocardial hypertrophy and fibrosis, preserved the expression of Kir2.1 and some key players in Ca2+ homeostasis, normalized the RP (P < 0.05), and abbreviated APD (P < 0.01), thus lowered cytosolic [Ca2 +]i (P < 0.01 or P < 0.05). IK1channel blocker BaCl2 or chloroquine largely reversed the cardioprotection of zacopride. We conclude that cardiac electrical remodeling is concurrent with structural remodeling. By enhancing cardiac IK1, zacopride prevents Iso-induced electrical remodeling around intracellular Ca2+ overload, thereby attenuates cardiac structural disorder and dysfunction. Early electrical interventions may provide protection on cardiac remodeling.

Project description:Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

Project description:BackgroundAndersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel.Methods and resultsWe identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations.ConclusionThe extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias.

Dataset Information

The IK1/Kir2.1 channel agonist zacopride prevents and cures acute ischemic arrhythmias in the rat.

Publications

The IK1/Kir2.1 channel agonist zacopride prevents and cures acute ischemic arrhythmias in the rat.

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