The Anti-ischemic Arrhythmia Effect Mediated By κ-opioid Receptor Is Via The Inhibition Of ET/ETR Signaling Pathway

BackgroundIschemic heart disease exhibits the highest mortality around the world. The incidencerate increases with the aging of population. Arrhythmia induced by myocardial ischemia,particularly ventricular fibrillation, is the main cause of sudden cardiac death. Thus, thepathogenesis and treatment strategies of ischemia-induced arrhythmia need to be clarifiedurgently.Endothelin (ET), an endogenous vascular active peptide of21amino acids, is the mostpotent vasoconstrictor. ET distributes not only in the endothelium, but also widely intissues and cells. It is an important factor to regulate cardiovascular function and plays akey role in maintaining basal vascular tone and homeostasis. ET is implicated in cardiovascular diseases, including hypertension, heart failure, acute myocardial infarction,atherosclerosis and cerebral angiospasm, etc. Exogenous ET-1has a direct effect onmyocardial ischemia and arrhythmia, while the increased synthesis and release ofendogenous ET-1are closely related to the ischemic arrhythmia. It is reported that ET-1can active c-Src tyrosine protein kinase through the activation of its receptor, and furtherinduce functional alteration of Cx43(the most significant connexin in cardiomyocytes).Then, Cx43dysfunction leads to uncoupling and reentry, and finally induces ventriculararrhythmia.Our previous study shows that the selective κ-opioid receptor agonist significantlyinhibits arrhythmia induced by myocardial ischemia/reperfusion through the activation ofκ-opioid receptor. Our recent experimental results exhibite that κ-opioid receptor agonistcan dramatically down-regulate the level of plasma ET-1, which indicates that theanti-arrhythmic effect mediated by κ-opioid receptor may be related to the inhibiton of ETand ET receptor system. This study aims to investigate the role of ET and ET receptorsystem in the anti-arrhythmic effect mediated by κ-opioid receptor, which gives newinsights into the mechanism of the anti-arrhythmic effect mediated by κ-opioid receptorand provides experimental supports for the clinical application of opioids in the preventionand treatment of ischemic arrhythmia.AimsThe study was designed to explore whether the effect of anti-ischemic arrhythmiamediated by κ-opioid receptor is via the regulation of κ-opioid receptor on endothelin (ET)and ET receptor (ETR) system, and downstream signal pathway, namly, c-Src tyrosineprotein kinase-Cx43.MethodsMale Sprague-Dawley (SD) rats (250±30g) were randomly divided into differentgroups. Myocardial ischemia reperfusion model was established in rats. ABP, LVP,±LVdp/dtmaxand ECG were examined, the arrhythmia score were determined. Thecorresponding tissue samples were retained according to the following study.ELISA was used to investigate the level of plasma ET-1. Real-time PCR was used to investigate the mRNA level of ET-1and ET receptor A(ETRA).Western blotting was used to investigate the protein level of ETRA, c-Src and Cx43.Immunofluorescence was used to investigate the expression and distribution of Cx43.ResultsThe effect of κ-opioid receptor stimulation on arrhythmia induced by ET-1innormal rats and rats subjected to myocardial ischemiaET-1triggered serious ventricular arrhythmia in normal rats and aggravated ventriculararrhythmia in rats subjected to myocardial ischemia. Prior administration of U50488H (aselective κ-opioid receptor agonist) dramatically inhibited ventricular arrhythmia inducedby ET-1, which were partly abolished by nor-BNI. The results above indicated thatκ-opioid receptor participated in suppressing the incidence of arrhythmia induced by ET-1.The effect of κ-opioid receptor stimulation on ET-1and ETRAlevelET-1expression was elevated during myocardial ischemia, while the activation ofκ-opioid receptor significantly inhibited ET-1expression both in normal and myocardialischemia rats and downregulated ET-1mRNA expression, which were abolished bynor-BNI. After the rats’ hearts were subjected to I/R for2h, ETRAexpression incardiomyocytes was elevated, which was also inhibited dramatically by U50488H. Theresults above indicated that the effect of κ-opioid receptor agonist U50488H on arrhythmiainduced by ischemia and ET-1might be related to the inhibition of the expression of ET-1and ETRA.The effect of κ-opioid receptor stimulation on c-Src tyrosine protein kinaseexpression levelBoth ET-1and myocardial ischemia triggered the elevation of phosphorylated c-Src(p-Src) tyrosine protein kinase expression separately. In addition, ET-1triggered a furtherincrease of p-Src tyrosine protein kinase in myocardial ischemia, and κ-opioid receptoractivation inhibited the elevation of p-Src tyrosine protein kinase expression mentionedabove. The effect of κ-opioid receptor on the change of Cx43During myocardial ischemia, the expression of Cx43and p-Cx43decreased and thedistribution of them relocated, ET-1triggered a further decrease of Cx43, but the p-Cx43dramatically increased and relocated, which were inhibited by the prior activation ofκ-opioid receptor.ConclusionsThe present study, for the first time, suggests that the anti-ischemic arrhythmia effectmediated by κ-opioid receptor is via the inhibition of ET and ETRAsystem through theactivation of κ-opioid receptor, and further inhibits c-Src tyrosine protein kinasephosphorylation and regulates expression and distribution of Cx43.