Hypertrophic Cardiomyopathy Phenotype In Knock-in Mice Of Sarcomere Gene Mutations

Background:Hypertrophic cardiomyopathy（HCM）is the most common inherited cardiovascular disease,the leading cause of sudden death in young people.HCM is mainly caused by mutations in genes encoding cardiac sarcomere proteins via an autosomaldominant manner.The establishment of HCM mouse models by knock-in of sarcomere mutations into mouse is necessary for investigation of HCM molecular pathogenesis and intervention strategy.The lack of such models in China has greatly hindered the study of HCM.In this study,the relative hot-spot and malignant mutations in Chinese HCM patients,the R92W mutation in TNNT2 gene and the R453C mutation in MYH7 gene,were chosen to construct their knock-in mouse model and to investigate their effect on cardiac structure and function and to evaluate whether it can mimic the phenotype of human HCM.Objectives:Knock in mouse models with R92W mutation in TNNT2 gene and R453C mutation in MYH7 gene were established respectively to observe the effect of mutations on cardiac structure and function,characterize the evolution of the disease,and lay the foundation for in vivo research and intervention of HCM in animal models.Methods and results:Knock-in mouse of TNNT2 gene R92W mutation and MYH7 gene R453C mutation was constructed through CRISPR/Cas9 genome edition.Heterozygous crosses to prepare homozygous mice and draw survival curves.Three-week old TNNT2^R92W/R92W mice have severe right ventricle dilation,significant hypertrophy of the ventricular septum,increased heart weight/body weight ratio accompanied by cardiac fibrosis,disordered muscle fiber arrangement,and increased cross-sectional area of ventricular myocytes,70%of which died within 25 days.HCM caused by sarcomere mutation is heterozygous dominant disease,the heterozygous(TNNT2^R92W/+)and their wildtype littermates（WT）of 8,12 and 22 months old underwent echocardiography monitoring for the cardiac structure and cardiac functions,respectively.Heart weight/body weight ratio,hematoxylin-eosin（H&E）staining,Masson staining,wheat germ agglutinin（WGA）staining and quantitative PCR（qPCR）detection of expression of molecular markers of cardiac hypertrophy were performed in 22 months old mice.Compared with their wild type littermates,TNNT2^R92W/+ mice developed cardiac h ypertrophy at 8 months,12 months,and 22 months,accompanied by a significant increase in ejection fraction,indicating increased contractile function.TNNT2^R92W/+ mice developed muscle fiber disorder,interstitial fibrosis and increased cross-sectional area of cardiomyocytes,and the expression of atrial natriuretic peptide（ANP）and brain natriuretic peptide（BNP）did not increase significantly.Similarly,the heterozygous(MYH7^R453C/+)and their wildtype littermates（WT）of 4,8 and 16 weeks old underwent echocardiography monitoring for the cardiac structure and cardiac functions,respectively.The heart of MYH7^R453C/+ mice was generally normal.Compared with their wildtype littermates,no myocardial hypertrophy was detected untill 4 months of age.Conclusion:TNNT2^R92W/+ mice could almostly replicate the phenotypes of human HCM;MYH7^R453C/+mice have not shown obvious HCM phenotypes until 4 months old,so the disease process still needs to be identified in older mice clear.Both homozygous mutants died at infancy or before birth.It was found that the right ventricle of TNNT2^R92W/R92Wmice was severely affected for the first time,and the mechanism needs to be further explored.