The Effect And Mechanism Of SIRT3 Deficiency On Cardiac Remodeling In Uremic Mice

Objective:1.To observe the effects of SIRT3 gene knockout on cardiac remodeling and cardiac function in uremic mice;2.To explore the mechanism of SIRT3 in the pathogenesis of uremic cardiomyopathy in mice.Methods:1.Genotypic identification of mice: DNA extracted from the tail of 4-week-old mice and genotypic identification was carried out according to the position of the bands in agarose gel electrophoresis.some mouse hearts were taken to further verify the expression of SIRT3 by Western Blot.2.Experimental groups: 10-week-old male wild type(SIRT3-WT,C57BL/6background)and SIRT3 gene knockout(SIRT3KO,C57BL/6 background)mice weighing about 23±2g were randomly divided into two groups: partial nephrectomy(5/6nx)and sham operation(sham).They were divided into four groups: SIRT3-WT sham operation group(WT-sham),SIRT3-KO sham operation group(KO-sham)SIRT3-WT model group(WT-5/6nx)and SIRT3-KO model group(KO-5/6nx).3.Six months after operation,the changes of heart rate and blood pressure were measured by non-invasive tail sleeve method,and the changes of cardiac structure and function were detected by echocardiography.4.After fully anesthetized mice,the venous blood of right ventricle was taken,and the serum was separated and the renal function indexes such as serum creatinine(CR)and urea nitrogen(UR)were detected.5.The body weight(Body Weight),heart weight(Heart Weight),lung weight(Lung Weight)and tibia length(Tibia Length)were recorded,and heart weight/body weight(HW/BW),heart weight/ tibia length(HW/TL),lung weight/ body weight(LW/BW)and lung weight/ tibia length(LW/TL)were calculated.6.Cardiac pathological sections were stained with hematoxylin-eosin staining(HE),wheat germ agglutinin staining(WGA)and Masson trichrome staining(Masson)to detect cardiac remodeling changes such as myocardial hypertrophy and myocardial interstitial fibrosis.Image J was used to calculate the cross-sectional area of the heart and the percentage of collagen.7.The expression of SIRT3,Collagen I,GSK3 β,p-GSK3 β,β-Catenin,Active β-Catenin(N-p β-Catenin)was detected by Western Blot,and the gray value of the protein was semi-quantitatively analyzed by Image J.8.The m RNA expression level of SIRT3 gene and the m RNA expression level of pathological hypertrophy marker genes Nppa and Nppb were detected by Real-time PCR.9.Statistical methods: all the results of this study were analyzed by SPSS25.0software,and the measurement data were expressed by mean ±standard error.The two groups of data were compared by independent sample T test,the rest of the data were analyzed by one-way analysis of variance(One-way ANOVE),and multiple comparisons between groups were conducted by Bonferroni.Results:1.The results of agarose gel electrophoresis showed that wild type mice had a band at 1114bp(R1F1)and 611bp(R3F1),while SIRT3 knockout mice had only one band at 837bp(R1F1).The results of Western Blot detection of SIRT3 expression in the heart of wild type mice showed that SIRT3 was normally expressed in the heart of wild type mice,while SIRT3 protein was not expressed in the heart of SIRT3 knockout mice,indicating that SIRT3 knockout mice were successfully constructed.2.The serum urea nitrogen and creatinine of uremic mice induced by 5/6nx were significantly increased,and the uremic mouse model was successfully established,and SIRT3 knockout did not affect the levels of serum creatinine and urea nitrogen in uremic mice.3.SIRT3 knockout had no effect on heart rate in uremic mice,but significantly increased SBP,DBP and MBP in uremic mice.4.Echocardiography showed that the cardiac structure of uremic mice changed,such as LVAWd,LVPWd and LV mass increased significantly,while EF and FS,which reflect cardiac contractile function,decreased in uremic mice,and SIRT3 knockout could promote these changes.5.HW,HW/BW,HW/TL,cross-sectional area of cardiomyocytes and the expression of Nppa m RNA and Nppb m RNA were significantly increased in uremic mice,and SIRT3 gene knockout could promote these changes,which indicated that SIRT3 gene knockout could aggravate cardiomyopathic hypertrophy in uremic mice;6.The expression of Collagen I in the heart of uremic mice was significantly increased,and Masson staining indicated that the percentage of fiber volume was significantly increased,and SIRT3 gene knockout aggravated this change,indicating that SIRT3 deletion can promote cardiac fibrosis in uremic mice.7.LW,LW/BW and LW/TL did not increase significantly in uremic mice,but SIRT3 gene knockout significantly increased LW,LW/BW and LW/TL,indicating that SIRT3 deletion can promote the progression of left heart failure in uremic mice;8.The relative expressions of p-GSK3 β / GSK3 β and Active β-Catenin/ β-Catenin in uremic mice were significantly increased,and the deletion of SIRT3 further promoted this change,indicating that SIRT3 knockout promote cardiac remodeling in uremic mice may be related to GSK3 β / β-Catenin pathway.Conclusion:SIRT3 gene knockout can promote hemodynamic changes in uremic mice,aggravate cardiac remodeling such as cardiomyopathic hypertrophy and myocardial interstitial fibrosis,and impair cardiac contractile function.These changes may be related to the regulation of GSK3 β / β-Catenin pathway.