Effect Of Shc4 Knockout On Aortic Valve Disease In Mice

Objective(s):Valvular heart disease(VHD)can lead to decreased physical function and quality of life,and in severe cases can reduce survival time,placing a heavy physical,mental and financial burden on patients and their families.The pathology is mainly characterized by abnormal extracellular matrix(ECM).Excessive production of ECM protein can lead to mucoid degeneration of the valve,and then lead to functional changes in valve stenosis and/or regurgitate.The prevalence rates of aortic stenosis and aortic insufficiency were 47.20% and18.00%,respectively.The heart is the earliest internal organ formed during embryonic development,and its development and differentiation are complicated regulated by many genes.Based on our previous study in cardiac tissues from patients with valvular heart disease,we found that SHC4 expression was significantly down-regulated in patients with valvular heart disease.Src homology and collagen 4(SHC4)were finally identified as target proteins by biogenic means and validation experiments.SHC4 is an important intracellular cohesion protein.Therefore,this project was designed for the purpose of constructing a mouse model of Shc4 gene knockout.To explore the relationship between SHC4 gene and heart valve development.Methods:1.HPA and NIH databases were used to search whether SHC4 was expressed in cardiac tissues and the expression of SHC4 in each tissue.Systemic Shc4 gene knockout mouse model was constructed by CRISPR-Cas9 technology,and PCR genotype identification was completed by extracting genomic DNA from mouse tail.Sanger sequencing of PCR products was used to verify whether Shc4 sequence was knocked out.They were divided into three groups according to genotype,The wild type Shc4+/+ mice were used as control group.Heterozygous Shc4+/-mice were used in experimental group 1,and homozygous Shc4-/-mice were used in experimental group 2.The expression of Shc4 c DNA was detected by PCR.The expression of Shc4 protein was detected by Western blot.2.The cardiovascular phenotype and cardiac function of Shc4 knockout mice were examined by echocardiography.3.Aortic valve tissue from a mouse heart was collected and analyzed by histological staining,and the morphology and collagen content of aortic valves were detected by Masson staining,the protein polysaccharide content of aortic valve was detected by Movat’s staining,the lipid content of aortic valve was detected by oil red O staining,and the calcification of aortic valve was detected by alizarin red staining.4.Mouse heart aortic valve tissues were collected,RNA was extracted,and transcriptome analysis was performed on three groups mouse heart aortic valve tissues by high-throughput sequencing technology,so as to explore the influence of Shc4 on the expression of genes related to heart development.Results:1.Through searching NIH and HPA databases,it was found that SHC4 was expressed in heart tissue;The genotypes of Shc4+/+,Shc4+/-and Shc4-/-mice were identified by PCR.Sanger sequencing showed that the 5-6 exon sequence of Shc4 gene was correctly knocked out,and the expression of Shc4 m RNA was decreased by RT-q PCR(Shc4+/-,p =0.0182;Shc4-/-,p = 0.0038),PCR results confirmed that the Shc4 c DNA sequence knockout was correct,and Western blot showed no significant differences in the expression of Shc4 protein.Successful construction of Shc4 knockout mice was preliminarily confirmed.2.Compared with wild-type mice,aortic stenosis and/or regurgitation were found in24-week-old Shc4+/-and Shc4-/-mice,and the peak pressure difference of the aortic valve was significantly increased(Shc4+/-,p = 0.0335;Shc4-/-,p = 0.0036),the contractile neck width of aortic valve regurgitation was different(Shc4+/-,p = 0.0009;Shc4-/-,p < 0.0001),the cardiac function of some mice decreased with prolonged survival time.3.Histological analysis showed that aortic lobe thickened(p = 0.0006)and proteoglycan content increased(p = 0.0379)in 24-week-old Shc4-/-mice compared with wild-type mice.Compared with mice of the same genotype with no echocardiographic lesions,mice with aortic lesions detected by echocardiography had thickened aortic lobes(p < 0.0001),increased collagen deposition(p = 0.0380)and proteoglycan content(p = 0.0004).4.Transcriptome analysis showed that the expression of genes related to extracellular matrix,such as SPP1,COMP,and CHAD,was significantly affected in the cardiac aortic valve tissues of the experimental groups of mice(Shc4+/-and Shc4-/-).Conclusion(s):1.Shc4 knockout caused aortic stenosis and/or regurgitation in mice hearts,and collagen deposition and thickening of the aortic valve leaflets.2.Shc4 knockout may affect the gene expression associated with extracellular matrix,such as SPP1,COMP and CHAD.