The Role Of Fkbp51 Gene In Cardiac Development And Cardiac Fibrosis And The Exploration Of Molecular Mechanisms

[Objectives]Cardiac fibrosis refers to an abnormal thickening of the heart valves due to inappropriate proliferation of cardiac fibroblasts but more commonly refers to the excess deposition of extracellular matrix in the cardiac muscle.Fibrotic cardiac muscle is stiffer and less compliant and is seen in the progression to heart failure.The description below focuses on a specific mechanism of valvular pathology but there are other causes of valve pathology and fibrosis of the cardiac muscle.Glucocorticoids(GCs)belong to a class of steroid hormones secreted by the adrenal cortex,which are not only through the glucocorticoid receptor(GR)to regulate the biosynthesis and metabolism of sugar,fat and protein,but also play an important role in the cardiovascular system balance;As a co-chaperone protein of the glucocorticoid receptor,the FKBP51 participates in the glucocorticoid receptormediated signaling process.In this thesis,using isoproterenol(ISO)induced cardiac fibrosis model,we identified the role of FKBP51 in heart development and cardiac fibrosis.[Methods]The role of Fkbp51 gene in cardiac fibrosis was analyzed by establishing cardiac fibrosis model 1)Establishment of mice cardiac fibrosis model:The mice were divided into four groups:WT-control and KO-control which slowly released normal saline by slow-released pump for three weeks;WT-ISO and KO-ISO,which slowly released isoproterenol(ISO)by slow-released pump for three weeks;2)Cardiac function analysis:Using the ultrasound and ECG,mice cardiac function of ISO treated or without treated was analyzed;3)Cardiac histotology and pathology analysis:The weight of the mice was weighed by an electronic balance,and observed the size and morphology of the experimental mice heart.The hearts of four groups of mice were routinely paraffin-embedded and sliced,HE staining and Masson staining were used to investigate the histology changes of WT and KO mice;4)Cardiac fibrosis related genes exprsson differenct was analyzed by real-time PCR;5)Immunohistochemistry and immunofluorescence were used to detect thechanges of protein levels in cardiac fibrosis related genes;6)Serum index detection.Inflammatory factors and cytokine validation of 4 groups were analyzed;7)The changes of fibrosis marker protein,TGF-?pathway-related protein and m-TOR pathway-related protein in heart were detected by immunoblottingin four groupsof mice.[Results]1)After 3 weeks ISO treatment,WT-ISO group clearly showed cardiac fibrosis,but there was no obvious fibrosis in KO-ISO group;2)Through ultrasound and ECG analyses,we found that the mean amplitude of the main wave of Fkbp51 KO mice was significantly higher than that of WT mice,and the heart rate was significantly slower,indicating that there may be atrioventricular block in KO mice,and KO mice's ventricular muscle more thickness than WT mice.After the ISO treatment,the ejection fraction,the left ventricular posterior wall thickness and the shortening of the left ventricular short axis of the WT-ISO mice were decreased by ultrasound study,indicating the cardiac function was significantly decreased in WT-ISO.However,the KO-ISO group mice did not change significantly.After the ISO treatment,ECG results show thatthe main wave,heart rate,conduction and cardiac contractility of WT-ISO mice was significantly increased compared to KO-ISO;3)It was found that after the ISO treatment group,the heart of the WT mice became significantly larger and heavier,while the Fkbp51 KO had no obvious change.4)Cardiac pathology analysis found that,after ISO treatment,WT mouse heart hypertrophy,heart wall thinning,appear a large number of fibrosis;and Fkbp51 knockout mice did not significant change in the heart wall,and no appear fibrosis.5)The expression of Fkbp51 gene was significantly increased following ISO treatment,and the expression of Collagen ? and Ang? in Fkbp51 knockout mice was significantly lower than that in wild-type mice,and the cytokine TGF-? and TGF-? R ?,TGF-? R? also significantly increased 6)Immunofluorescence test,Collagen I protein was found to be less in the heart of Fkbp51 knockout mice than in the wild-type mice after ISO treatment.Immunohistochemical experiments were verified on ?-SMA and TIMP1,it was found that the expression in Fkbp51 knockout mice was significantly higher than that in wild type mice after Fkbp51 knockout and ISO treatment.7)After ISO treatment,the fibrosis related markers,including fibroblast growth factor,mouse superoxide dismutase,angiotensin ? and mouse L-lactate dehydrogenasemouse tumor necrosis factor alpha and small gamma interferon showed significant changes in WT-ISO group,while KO mice no significant change;8)AfterFkbp51knockoutthe expression of a-SMA protein,P-STAT3 protein and mTOR protein showed significantly increased.The expression of FKBP51 protein in WT mice was increased after isoproterenol treatment,and the expression of Collagen ? protein and GR protein also increaseafter isoproterenol treatment.In addition,the expression of Smad protein in KO mice was significantly higher than that in WT mice,indicating that TGF-? pathway was closely related to this process.The expression of mTOR-related protein in KO mice was increased after Fkbp51knockout and after isoproterenol treatment,indicating that mTOR protein was closely related to this process.[Conclusions]FKBP51 protein plays an important role in regulating mouse heart development and isoproterenol-induced mouse cardiac fibrosis.Objective Useisoproterenol and left anterior descending coronary artery ligation to construct wild-type mouse(WT)and Fkbp51 gene knockout(KO)mice cardiac fibrosis model,analysis the chang of heart gene expression profiles from cardiac fibrosis model,research the role of Fkbp51 gene in cardiac development and cardiac fibrosis.Methods The second generation of high-throughput gene sequencing technique was used to sequester the heart mRNAof the fibrotic model mice constructed by two methods of isoproterenol and left anterior descending coronary artery ligation.The sequenced data were analyzed by DEGseq,After limiting the differences,the difference gene of the mouse heart was screened,by comparing the data between groups acquire gene volcanic map,and further explore the different between group and make the heat map,then use online tools DAVID to analysis GO and KEGG pathway from different expression gene,and the gene were annotatedusing Genecards.Results(1)The deletion of Fkbp51 mainly leads to protein digestion and uptake of organelles associated with ribosomes,causing cardiac developmental damage through specific functional changes in ribosomes.(2)After isoproterenol treatment,WT mice showed fibrosis,while KO mice had no fibrosis.WT mice after treated PI3K-Akt signaling pathway was significantly changed,and KO mice there is a significant change in the ribosome.(3)After ligation of the left anterior descending artery,both WT and KO mice had fibrosis,but KO mice had a higher mortality rate.WT mice mainly affect the metabolism of enzyme activity,KO mice mainly affect the enzyme activity and ionic activity,and to some extent affect the infection-related pathways.(4)KO group after treatment,there is a significant change is still ribosomes,and Fkbp51 gene knockout after the change,the gene knockout may be the reasons of the effect on heart development and ISO treatment's.(5)After LAD treatment,WT and KO mice compared to the main changes are enzyme activity changes,but KO mice there is a high mortality,may be due to oxidoreductase changes and related diseases susceptibility.Conclusions In this study,ISO treatment and left anterior descending ligation of Fkbp51 KO and WT mice construct cardiac fibrosis model were used to construct RANseq data.It was found that the role of Fkbp51 in cardiac development and cardiac fibrosis was a polygene,Multi-channel,multi-signal pathway interaction process,which provided a theoretical basis for further study on the molecular mechanism of Fkbp51 gene in cardiac development and cardiac fibrosis,further for theory of bedding of Fkbp51play a role in heart disease.