The Role Of Myocardial Deletion Of SNX16 In Cardiac Fibrosis Induced By Isoproterenol

Purpose:When epinephrine is elevated,it binds to β1 receptors of myocardial cells to enhance heart contractility,speed up heart rate,increase cardiac output and myocardial oxygen consumption,increase blood pressure in the body,and its long-term effects might lead to myocardial hypertrophy and cardiac fibrosis.Cardiomyocytes and fibroblasts are the two main components of heart,which maintain the functions of heart together.In the case of long-term sympathetic nerve excitement,hypertension etc,cardiac fibroblasts differentiate into myofibroblasts and secrete excessive collagen fibers,resulting in cardiac fibrosis.Transforming growth factor(TGF-β)plays an important role in mediating fibroblasts differentiation and the development of cardiac fibrosis.Isoproterenol(ISO)is an adrenergic beta receptor agonist.Repeated subcutaneous injections of ISO have been used to induce cardiac fibrosis in mice.Sorting Nexin 16(Sorting Nexin 16,SNX16)belongs to the Sorting Nexin family.As a sorting Nexin,SNX16 is involved in cell endocytosis,protein sorting,cell signal transduction,membrane transport,membrane remodeling and organelles transporting etc.Whether SNX16 is involved in cardiac fibrosis,its role in this process has not been reported.This study aims to explore the effect of myocardial cell specific knockout of SNX16 on cardiac fibrosis and its molecular mechanism.Methods:1.Rat cardiomyocytes(H9C2),were stimulated by different concentrations of ISO and the expression of TGF-β was detected.2.SNX16 F/F mice were obtained with CRISPR/Cas-9 technology,and bred with Cre mice in which Cre recombinase were driven by cardiomyocyte light chain2 v specifically to obtain cardiomyocyte-specific knockout SNX16 mice(F/F+,with Cre)mice and control mice(F/F-without Cre).3.Constructing an animal model of cardiac fibrosis: F/F-and F/F+ male mice at8-12 weeks were divided into two groups: experimental group and control group.The experimental group was injected subcutaneously with ISO(20mg/kg/day)for 14 days to induce cardiac fibrosis in mice,and the control group was given the same amount of normal saline.4.Echcardiography and electrocardiogram detection were performed to evaluate the cardiac function of the mice and determine whether the mouse cardiac fibrosis model is successfully constructed.5.The heart tissues were collected,weighed and fixed to perform Mason staining.6.Cardiac fibrosis markers and TGF-β,an important cytokine for fibrosis,were measured whether the cardiac fibrosis model is successfully constructed.7.Rat cardiomyocytes H9C2 were transfected with SNX16 plasmid.Then,the cells were subjected to ISO stimulation for 24 hours,the cells and supernatants were collected to detect the contents of intracellular and secreted TGF-β in supernatants of cardiomyocytes And WB was overexpression of SNX16 protein.8.Immunofluorescent microscopy was used to observe the intracellular expression and distribution of TGF-β in overexpressing SNX16 cardiomyocytes under ISO stimulation.9.Primary cells from newborn mice were isolated by multiple steps with enzymatic digestion,then separation of primary cardiomyocytes and primary fibroblasts by their differential adherence properties.The cardiomyocytes were stimulated with 10 μM ISO for 24 h(the control group was stimulated with 1/1000Saline),and then the cells were harvested for later analysis and the supernatants were collected.Primary fibroblasts were administrated with the media from cardiomyocytes for 24 h,and the supernatants and cells were also collected.The expressions of fibrosis marker Collagen Ⅲ,α-SMA and TGF-β were determined by WB or RT-PCR,respectively,in primary fibroblasts or cardiac myocytes.The contents of TGF-β in the supernatants of cardiomyocytes were also measured in WB.Results:1.After 24 hours of exposure to ISO 10μM,the expression of TGF-β in cardiomyocytes were increased significantly.2.Myocardial specific knockout of SNX16 was confirmed at both of mRNA and protein levels of SNX16 in mice.There were no significant differences in the growth and development and heart function between SNX16 CKO and the control mice.3.In the ISO-induced cardiac fibrosis mouse model,myocardial specific knockout of SNX16 significantly improved heart functions,attenuated ISO-mediated left ventricular mass increasing,R wave amplitude extension of ECG,inner wall thickening.4.Mason staining results showed that myocardial specific knockout of SNX16 significantly reduced ISO-mediated fibrosis in the left ventricle.5.The specific knockout of SNX16 in cardiomyocytes significantly reduced the contents of ISO-mediated TGF-β in the hearts.6.Overexpression of SNX16 can significantly increase the ISO-mediated secretion of TGF-β in rat cardiomyocytes H9C2.7.The specific knockout of SNX16 in cardiomyocytes has no significant effect on ISO-mediated TGF-β transcription.However,the contents of TGF-β in cardiomyocytes were much higher than the control group,and the contents of TGF-βin the secretory supernatants were much less than the control group.The results indicated that knocking out SNX16 may reduce ISO-mediated TGF-β secretion.8.ISO-induced cardiomyocyte secretion supernatants significantly increased the transcription and expression of Collagen Ⅲ in fibroblasts,while the secretion from cardiomyocytes were hat specifically knocked out SNX16 significantly alleviated this fibroblast phenotype.Conclusion:The results of this study showed that myocardial cell specific knockout of SNX16 significantly reduced ISO-induced cardiac fibrosis.Loss of SNX16 in cardiomyocytes may reduce ISO-mediated TGF-β secretion from intracellular to extracellular,thereby affecting the conversion of fibroblasts to myofibroblasts,and alleviating cardiac fibrosis.