GSK-3β Inhibitor Increases The Expression Of Inward Rectifier Potassium Channel Kir2.1 To Improve Electrical Remodeling In Rats With Myocardial Infarction

Background and Objective Cardiac remodeling is the main underlying mechanism in the development of various cardiovascular diseases and a major cause of death globally.Although the prevalence and morbidity rate has declined in the past few years due to advancement in treatment method and preventions while it stills remains responsible for the higher number of death rate.Cardiac remodeling includes structural remodeling and electrical remodeling on the basis of physiological and pathological changes occur in the heart.Electrical remodeling is the main reason for malignant arrhythmia and sudden cardiac death.Elucidating the cellular and molecular mechanisms of myocardial remodeling electrical remodeling is of great significance for discovering potential therapeutic targets and for controlling the occurrence of malignant ventricular arrhythmias.Glycogen synthase kinase-3β（GSK-3β）,a serine/threonine protein kinase,has been involved in many aspects of myocardial remodeling.GSK-3βis an important cardiotropic negative regulator,which plays an essential role in regulating cardiomyocyte apoptosis and in promoting vascular proliferation in vascular tissues.Thus,it has a certain ability to be used as a therapeutic target for improving myocardial viability after ischemia.In recent years,GSK-3βhas been studied on a variety of ion channels in the autonomic nervous system.However,its effect on ion channels in cardiomyocytes and electrical remodeling has not been elucidated.The present study was designed the observe the effect of GSK-3βantagonist SB216763 in myocardial infarction（MI）and in the hypoxic model of rat cardiomyocyte H9C2 cell line followed by the analysis of the cardiac function and QT interval in myocardial remodeling while targeted ion channels screening further verified in vitro by these interventions.Method1.Establish myocardial infarction model:The acute myocardial infarction model was established by ligation of the left anterior descending coronary artery（LAD）.The whole process of ECG monitoring was performed.After the operation,the ST segment was significantly elevated,and thus the model was successfully established.2.Grouping and administration:Male 8-week-old SD rats were randomly divided into 3 groups（10 mice in each group for 2 days group,11 mice in each group for 7days group）,Sham,MI and MI+SB group.One hour before surgery,GSK-3βinhibitor SB216763,administered by tail vein injection,(0.6 mg·kg^-1·d^-1)respectively.In 2 days,SB216763 was given every 24 hours while in 7 days the drug was given every 24 hours,and the animal was sacrificed after 7 days of continuous administration.The Sham group was only operated without ligation,and the MI+SB group was treated along with the MI group.3.To observe the effect of SB216763 on myocardial infarction in rats:H&E staining,Masson staining,electrocardiogram,echocardiography,serum myocardial enzyme method were used to detect the effect of SB216763 on rat heart tissue morphology,fibrosis,cardiac function and QT interval.The qPCR method was used to screen the ion channels affecting the QT interval,and the selected ion channels were verified by qPCR and Western blot for analyzing mRNA and protein levels repectively.4.To observe the effect of SB216763 on hypoxia H9C2 cell activity and potassium channel expression:The cell hypoxia model was established by using the Research Science AW400TG cell workstation.Grouping and administration:H9C2 was divided into 7 groups:NC（normal）,hypoxia group（6h,12h,24h）,SB216763 pretreatment group（SB/6h,SB/12h,SB/24h）respectively.The pretreatment group was pretreated with 10μM GSK-3βinhibitor SB216763 for 1h and then treated with hypoxia.The effect of SB216763 on the protein expression level of Kir2.1 was detected according to the selected 12 h time point.Results1.Protective effect of GSK-3βinhibitor SB216763 on myocardial morphology and fibrosis after myocardial infarction in ratsThe results of HE staining showed that vacuolization and inflammatory infiltration occurred in the MI group,and the degree of its expression was more obvious in 7 days day than that in the 2 days.The corresponding MI+SB group in the same period could reduce the malignant degree of cell damage.Masson staining after surgery showed that the collagen fibrosis in the MI group in 2 and 7 days after surgery was significantly higher than that in the Sham group（P<0.001,P<0.01）,and the corresponding MI+SB group was higher than MI.The groups were all relieved（P<0.01,P<0.05）.2.GSK-3βinhibitor SB216763 can alleviate the damage of cardiac function after myocardial infarction in ratsCompared with the Sham group,the MI group was impaired in cardiac function.In 2 days after myocardial infarction,and LVEF and LVFS were significantly decreased（LVEF:P<0.01,LVFS:P<0.01）.Compared with the MI group,the MI+SB group improved.Adverse effects of LVEF and LVFS（LVEF:P<0.05,LVFS:P<0.01）.Compared with the Sham group,the MI group suffered from impaired cardiac function in 7 days after myocardial infarction（LVEF:P<0.001,LVFS:P<0.01）.Compared with the MI group,the MI+SB group improved the adverse outcomes of LVEF and LVFS.（LVEF:P<0.05,LVFS:P<0.05）.3.GSK-3βinhibitor SB216763 reduces serum LDH and cTn-I levels after myocardial infarction in ratsIn the 2 days animal serum was compared with the Sham group,the myocardial enzymes in the MI group had a significant increase in LDH level except for cTn-I（LDH:P<0.001,cTn-I:P>0.05）.Compared with the MI group,the LDH was significantly lower in the SB group except for cTn-I（LDH:P<0.001,cTn-I:P>0.05）.In the 7 days myocardial enzymes LDH and cTn-I were significantly increased in the MI group compared with the Sham group（LDH:P<0.001,cTn-I:P<0.001）,compared with the MI group,the SB group LDH cTn-I was significantly reduced（LDH:P<0.001,cTn-I:P<0.001）.4.GSK-3βinhibitor SB216763 shortens QT interval of electrocardiogram after myocardial infarction in ratsElectrocardiograph II lead was used to measure the ECG results of the three groups of rats before and after operation and after 2 and 7 days of the operation,the heart rate（HR）,QT interval and QTc were calculated.There was no significant difference in HR,QT and QTc between the groups before operation.The abnormal waveform of the MI group and the ST-end elevation showed that the myocardial infarction model was successful.The results of 2 and 7 days after operation showed that HR,QT and QTc were significantly increased in MI group compared with Sham group,and QT and QTc were significantly lower in SB group than that of MI group differently.5.GSK-3βinhibitor SB216763 has an up-regulation effect on the decrease of potassium channel Ki2.1 mRNA（KCNJ2）and protein expression.SCN 5A（Nav1.5）,KCND2（Kv4.2）,CACNA1C（Cav1.2）,KCNQ1（K_V7.1）,hERG（K_V11.1）and KCNJ2（Kir2.1）mRNA expression levels were detected in the ischemic region of myocardial tissue in 2 and 7 days after myocardial infarction in rats.The results showed that the mRNA expression levels of SCN 5A（Nav1.5）,KCND2（Kv4.2）,KCNQ1（K_V7.1）,hERG（K_V11.1）,KCNJ2（Kir2.1）were decreased after myocardial infarction,only the MI+SB group of KCNJ2 up-regulated the down-regulation of the gene after myocardial infarction in rats（P<0.05）.Western blot results showed that the protein expression of Kir2.1 in MI group was significantly lower compared with Sham group（P<0.01）,and the expression of Kir2.1 was higher in MI+SB group than that in MI group（P<0.05）.6.Effects of GSK-3βinhibitor SB216763 on H9C2 cell activity10μM of GSK-3βinhibitor SB216763 was applied to H9C2 cells for different time.The results showed that SB216763 at this concentration was cultured in different time points of H9C2 cells in 6h,12h,24h and 48h compared with NC group.Cellular activity has no effect.7.GSK-3βinhibitor SB216763 reduces hypoxia-induced troponin release in H9C2 cellsAfter cell group treatment,the supernatant of the cells was collected for cTn-I detection.Compared with NC,the level of cTn-I in H9C2 cells in 6h,12h and 24h after hypoxia was significantly increased（P<0.01,P<0.01,P<0.001）,the inhibitor group significantly reduced that of cTn-I in 12 h（P<0.01）.8.GSK-3βinhibitor SB216763 can attenuate the decrease of potassium channel Kir2.1 in hypoxic H9C2 cellsCompared with NC,Kir2.1 protein expression level was significantly down-regulated in hypoxic H9C2 cells for 12h（P<0.01）,and Kir2.1 protein expression was up-regulated after SB216763 pretreatment（P<0.05）.Conclusions1.GSK-3βinhibitor SB216763 can improve cardiac function damage after myocardial infarction in rats.2.GSK-3βinhibitor SB216763 upregulate the mRNA and protein expression levels of potassium channels KCNJ2/Kir2.1 in rats after myocardial infarction.3.GSK-3βinhibitor SB216763 has an upregulated effect on the expression of potassium channel Kir2.1 induced by hypoxia for 12 h in H9C2.