The Role And Mechanism Of Kir2.1 In Myocardial Fibrosis Induced By Diabetic Cardiomyopathy

Objective:Diabetes mellitus（Diabetes）is a metabolic disease affecting people’s health worldwide,of which diabetic cardiomyopathy（DCM）is one of the major complications of diabetes^[1-2],with myocardial fibrosis as its main pathological feature.Cardiac fibroblasts（CFs）are the main effector cells in myocardial fibrosis,and sustained elevation of blood glucose can lead to a high activation of CFs^[3].Various ion channels are distributed on the membrane surface of cardiac fibroblasts^[4-5],among which the main role of the inwardly rectifying potassium channel Kir2.1 is to maintain the resting potential of the cell membrane,which is associated with a range of diseases:including diabetic retinopathy,myocardial infarction,and myocardial ischemia-reperfusion arrhythmias.However,the role and mechanism of Kir2.1 on myocardial fibrosis in rats with diabetic cardiomyopathy are not well studied.Methods:In vivo experiments:6 weeks male SD rats were randomly divided into four groups of 12rats each:normal group（Control group）,diabetic cardiomyopathy group（DCM group）,diabetic cardiomyopathy+zacopride group（DCM+Zacopride group）,and zacopride group（Zacopride group）.During the course of 12 weeks of modeling,the changes of blood glucose and body weight of rats in each group were recorded.The morphological changes of the hearts of the rats in each group were observed as well as the structural changes of the ventricles of the rats in each group by echocardiography,the severity of myocardial inflammation infiltration and myocardial fibrosis in each group by histological staining,and the changes of fibrosis-related protein expression by immunohistochemistry and Western blot assay.In vitro experiments:（1）primary isolation of cultured SD mammary rat cardiac fibroblasts by mixed enzyme digestion,and identification of isolated cells using Vimentin（fibroblast marker）andα-SMA（myofibroblast marker）;（2）intervention of CFs with high glucose（HG,25 mmol/L）for 24 h to establish a DCM myocardial fibrosis model,using Western blot and immunofluorescence were used to detect the protein expression changes of Kir2.1 and fibrosis-related proteinsα-SMA,CollagenⅠand CollagenⅢin CFs,and whole-cell membrane slice clamp technique was used to detect the inward current changes on the membrane surface of cardiac fibroblasts.（3）Zacopride（1μmol/L）was pretreated on DCM myocardial fibrosis model for 30 min.changes in protein expression of Kir2.1,α-SMA,CollagenⅠ,CollagenⅢin CFs were detected by Western blot and immunofluorescence,and intracellular calcium ion was detected in each group using calcium probe in the mechanistic study.The changes of TGF-β1,Smad 2/3,p-Smad 2/3pathway protein expression were detected by western blot.RESULTS:In vivo experiments showed that,compared with the DCM group,administration of the Kir2.1-selective agonist Zacopride resulted in significantly lower left ventricular systolic internal diameters（LVIDs）,left ventricular diastolic internal diameters（LVIDd）,ejection fraction（LVEF）,and left ventricular short-axis shortening（LVEF）.diameter（LVIDd）were significantly reduced（P<0.05）,and left ventricular ejection fraction（LVEF）and left ventricular shortening shortening（LVFS）were significantly increased（P<0.05）,the expression of myocardial interstitial fibrosis protein decreased,and morphological observations showed a significant reduction in cell fracture and necrosis and a decrease in the area of collagen fiber deposition,suggesting that Zacopride could improve the DCM-induced ventricular structure and the degree of fibrosis.The results of the isolation experiments showed that high glucose-induced myocardial fibrosis model was accompanied by reduced Kir2.1 inward current and decreased protein expression,and the expression of fibrosis-related proteinsα-SMA,CollagenⅠand CollagenⅢcould be inhibited by pretreatment with Zacopride;in addition,further studies revealed that radical Zacopride could reduce intracellular calcium ion concentration and In addition,it was further found that kinetic Zacopride could reduce intracellular calcium concentration and inhibit the protein expression of TGF-β1,p-Smad 2/3（P<0.05）,thus reducing the high glucose-induced myocardial fibrosis.Conclusion:Activation of Kir2.1 inhibits myocardial fibrosis in diabetic cardiomyopathy rats,and the mechanism may be related to inhibiting Ca²⁺overload and TGF-β1/Smads signaling pathway,and down-regulating the expression ofα-SMA,Collagen I and Collagen Ⅲ.