Disease Modeling And Mechanism Investigation Of Congenital Heart Disease With Patient Specific IPSC

Background:Cardiovascular diseases are the most serious threat to the human health.As a type of cardiovascular diseases,congenital heart disease(CHD)frequently occurs in newborns with an incidence of 1%in China.Mutations presented in cardiac key regulators could often lead to CHD.Induced pluripotent stem cell(iPSC),which is generated from somatic cells,shares the same genetic information with the donor and resembles the features of embryonic stem cell(ESC),including the self-renewal and differentiation potential.Patient-specific heart disease model using iPSC can be established for the molecular investigation,drug screening and personalized medicine.GATA binding protein 4(GATA4)serves as a key cardiac transcription factor involved in cardiogenesis,such as cardiomyocyte differentiation and its proliferation.In this study,patient-specific iPSC with GATA4T280M mutation(iPSC-GATA4T280M)was established to reveal the underlying mechanisms of CHD.Methods:Patient-specific iPSC and genome-editted ESC carrying GATA4T280M heterozygous mutation were esatblished.The generated cardiomyocytes were subjected to analyze the cell proliferation.For mechansim investigation,data acquired from ChIP-Seq and RNA-Seq was applied to analyze the target gene of GATA4 in cardiomyocytes.Results:Our data indicated that both iPSC-GATA4T280M and wildtype iPSC hold the same capacity of self-renewal and differentiation potential.The decreased cell proliferation of cardiomyocytes derived from iPSC-GATA4T280M was observed,and the phenotype was further confirmed in cardiomyocytes derived from ESC with GATA4T280M(ESC-GATA4T280M,H7G4).Interestingly,the nuclear localization of GATA4T280M protein was the same to wildtype GATA4 protein,but its transcriptional activity on its targets was dramatically decreased as confirmed by luciferase assays.Through bioinformatics analysis with ChIP-Seq and RNA-Seq data,we found that four highly-enriched candidates might be involved in regulating CHD,but only FGF16 had been proved to participate in heart development and cell proliferation.The expression of FGF16 was decreased in cardiomyocytes derived from iPSC-GATA4T280M when compared to the cardiomyocytes derived from wildtype iPSC.GATA4 protein could regulate FGF16 directly,and the decreased cell proliferation of iPSC-GATA4T280M derived-cardiomyocytes was restored by FGF16 overexpression.ChIP-PCR and luciferase analysis demonstrated that wildtype GATA4 protein could directly bind to the promoter of FGF16 and positively regulate its expression,but not GATA4T280M protein.Conclusion:Both the patient-specific iPSC-GATA4T280M and genome-edited ESC-GATA4T280M had the pluripotency,and GATA4T280M cardiomyocytes showed decreased cell proliferation.The decreased cell proliferation of GATA4T280M cardiomyocytes could lead to the CHD.In term of regulatory mechanism,GATA4T280M protein failed to regulate the expression of FGF16,thus led to the decreased cell proliferation in GATA4T280M cardiomyocytes.The direct relationship between GATA4T280M mutation and CHD in human cardiomyocyte model was firstly proved in this study.The patient-specific iPSC disease model provided an important platform for prenatal diagnosis of CHD and drug screening to treat the CHD with GATA4T280M mutation.