Remodeling Of The Inward Rectifier Potassium Channel After Myocardial Infarction And The Intervention Mechanisms Of Valsartan

Research backgroundDespite the continuing progress in contemporary social medical treatment level and our gradually deeper understanding of a healthy lifestyle, myocardial infarction (MI) is still the most common clinical cardiovascular diseases and becomes a major threat to the public health in our country. About 540 thousand people died of sudden cardiac death in our country every year. Nearly 90% of sudden death due to malignant arrhythmia after acute MI. Previous studies have found that ventricular arrhythmias (VAs) and sudden cardiac death are associated with potassium ion channel remodeling, especially the inward rectifier potassium channel (Kir). Kir is the main component of the normal action potential, maintains the resting membrane potential, and participates in the action potentials during the early and terminal repolarization. The most abundant Kir in myocardial cells is the IK1 channel, mediated by heart type Kir2.1 protein, which is encoded by KCNJ2 gene. One of the main characteristics of electrical remodeling in ventricular myocytes after MI is decreased IK1 current density, resulting in the increase of the incidence or susceptibility of VAs. Although there are a variety of antiarrhythmic drugs in current clinical application, these mature antiarrhythmic drugs show proarrhythmic effects, especially in the application of patients with prolonged QT interval. MI patients with long QT are predicted of high risk of malignant ventricular arrhythmia or even sudden death. Therefore, the application of the existing antiarrhythmic drugs in MI patients is limited. Most MI patients in clinical are associated with hypertension or other risk factors. Interestingly, renin-angiotensin-aldosterone system (RAAS) antagonist can significantly reduce the incidence of malignant arrhythmia. But the mechanism is not yet clear. Therefore, if we could explore the target and mechanism of RAAS antagonists to play a role in antiarrhythmic effect, we would contribute to find new antiarrhythmic drugs, and improve the prognosis of MI.Part I. Remodeling of the inward rectifier potassium channel after myocardial infarction in rats and the intervention effects of valsartanObjectiveTest the expression level of inward rectifier potassium channel (Kir2.1) in myocytes of the infarcted border and non infarcted left ventricular free wall (LVEF) after acute MI in rats, as well as the effect of valsartan on Kir2.1. And evaluate the intervention of valsartan in heart rate, blood pressure, ventricular arrhythmia susceptibility and hemodynamics in MI rats.MethodsRats underwent permanent ligation of the anterior descending branch of the left coronary artery to get acute MI. Evaluate the MI model. The rats were randomly divided into sham operation group (Control group), sham operation and valsartan group (Control+Valsartan group), myocardial infarction group (MI group), myocardial infarction and valsartan group (MI+Valsartan group). Small animal telemetry and tail-cuff plethysmography were used to record the heart rate, blood pressure and electrocardiogram for 7 days after MI. qPCR and Western blot were used to detect KCNJ2 mRNA levels and Kir2.1 protein levels in the infarcted border and non infarcted LVEF of the four groups.ResultsElectrocardiographic monitoring and postoperative Masson’s staining proved of successful MI model.7 days after MI, the heart rate and blood pressure of rats in the MI group were higher than Control group and Control+Valsartan group, while MI+Valsartan group decreased. Extended QTc and QTcd after MI are also tend to be normal because of valsartan intervention. In addition, valsartan effectively improve cardiac ejection fraction and other hemodynamic parameters after MI. At the same time, KCNJ2 mRNA and Kir2.1 protein levels were lower in the MI group compared with Control group and Control+Valsartan group, while the remodeling of the ion channel was improved in MI+Valsartan group.ConclusionsIn MI rats, IK1 channel expression level was significantly reduced. Valsartan can reverse this phenomenon, accompanied by reduced arrhythmia susceptibility. In the myocardium of normal rats, there is no significant regulation effect of valsartan on IK1 channel.Part Ⅱ. Research on the mechanisms of valsartan attenuating IK1 after myocardial infarctionA:Valsartan ameliorates IK1 in rats with myocardial infarction via the NF-KB-miR-16 pathwayObjectiveMicroRNAs have an important role in regulating arrhythmogenesis. MicroRNA-16 (miR-16) is predicted to target KCNJ2. The regulation of miR-16 is primarily due to NF-κB. Whether valsartan could downregulate miR-16 via the inhibition of NF-κB after MI and whether miR-16 targets KCNJ2 remain unclear.MethodsMI rats received valsartan or saline for 7 days. The protein levels of NF-κB p65, inhibitor κBα (IκBα) and Kir2.1 were detected by Western blot analysis. The mRNA levels of Kir2.1 and miR-16 were examined by quantitative real-time PCR. Whole cell patch-clamp techniques were applied to record IK1. Luciferase assay to verify whether KCNJ2 is the target gene of miR-16. CHIP to verify the DNA binding activity of NF-kB.ResultsMiR-16 expression was higher in the infarct border, and was accompanied by a depressed IK1/KIR2.1 level. Additionally, miR-16 overexpression suppressed KCNJ2/KIR2.1 expression. In contrast, miR-16 inhibition, or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-16 target. In the MI rats, compared to saline treatment, valsartan reduced NF-κB p65 and miR-16 expression, and increased IκBα and Kir2.1 expression. In vitro, angiotensin Ⅱ increased miR-16 expression and valsartan inhibited it. Over-expressing miR-16 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. NF-κB activition directly upregulates miR-16 expression.ConclusionsMiR-16 controls KCNJ2 expression, and valsartan ameliorates Kir2.1 after MI partly depending on the NF-KB-miR-16 pathway.B:Valsartan upregulates Kir2.1 in rats suffering from myocardial infarction via casein kinase 2ObjectiveThe regulation on IK1 of protein kinases like PKC mainly depends on PIP2. And few reports focused on the downstream regulatory mechanism. Recent studies found that casein kinase 2 (CK2) binds and phosphorylates Spl, a transcription factor of KCNJ2 that encodes Kir2.1. Whether valsartan represses CK2 activation to ameliorate IK1 remodeling following MI remains unclear.MethodsWistar rats suffering from MI received whether valsartan or saline for 7 days. The protein levels of CK2 and Kir2.1 were each detected via a Western blot analysis. The mRNA levels of CK2 and Kir2.1 were each examined via quantitative real-time PCR. Whole cell patch-clamp techniques were applied to record IK1. EMSA was used in vivo and in vitro to detect Spl DNA binding activity.ResultsCK2 expression was higher at the infarct border; and was accompanied by a depressed Kir2.1 protein level. Additionally, CK2 overexpression suppressed KCNJ2/Kir2.1 expression. By contrast, CK2 inhibition enhanced KCNJ2/Kir2.1 expression, establishing that CK2 regulates KCNJ2 expression. Among the rats suffering from MI, valsartan reduced CK2 expression and increased Kir2.1 expression compared with the rats that received saline treatment. In vitro, hypoxia increased CK2 expression and valsartan inhibited CK2 expression. The over-expression of CK2 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. EMSA showed that CK2 inhibited Spl DNA binding activity. After inhibition of CK2 by TBB, Spl DNA binding activity increased. The Spl DNA binding activity decreased in myocardial tissue of rats after MI, and increased after the intervention of valsartan.ConclusionsAT1 receptor antagonist valsartan reduces CK2 activation, increases Kir2.1 expression and thereby ameliorates IK1 remodeling after MI in the rat model.C:Valsartan attenuates Kir2.1 by down-regulating the Thl immune response in rats following myocardial infarctionObjectiveMI results in decreased inward-rectifier K+ current (IK1), which is mediated primarily by the Kir2.1 protein and is accompanied by upregulated T cells. IFN-y, secreted predominantly by Thl cells, causes a decrease in IK1 in microglia. Whether Thl cells can induce IK1/Kir2.1 remodeling following MI and whether valsartan can ameliorate this phenomenon remain unclear.MethodsRats experiencing MI received either valsartan or saline for 7 days. Th1-enriched lymphocytes and myocytes were co-cultured with or without valsartan treatment. Thl cells were monitored via flow cytometry. The protein levels of Kir2.1 were detected via Western blot analyses. The mRNA levels of T-bet、GATA-3、IFN-γ and IL-10 were each examined via quantitative real-time PCR.IK1 was recorded via whole-cell patch clamping. The plasma levels of IFN-y, IL-2 and TNF-a were detected by ELISA.ResultsThl cell number and cytokine expression levels were higher following MI, and the Kir2.1 protein level was decreased. In MI rats, valsartan reduced Thl cell number and cytokine expression levels and increased Kir2.1 expression and the IK1 current compared with the rats that received saline treatment; these results are consistent with the effect of valsartan in co-cultured lymphocytes and myocytes. In vitro, IFN-y overexpression suppressed the IK1 current, whereas IL-2 and TNF-a had no significant effect on the current, establishing that Thl cell regulation of IK1/Kir2.1 expression is mainly dependent on IFN-y.ConclusionsValsartan ameliorates IK1/Kir2.1 remodeling by downregulating the Thl immune response following MI.