| Congenital heart disease(CHD)refers to abnormal embryonic development during embryonic development due to genetic and/or environmental interactions.It is the most common congenital malformation in neonates and can easily cause fetal intrauterine growth retardation,pregnancy miscarriage,stillbirth,and fetal death after birth,causing heavy economic pressure and psychological burden on families and society.Although countless scholars have spent a lot of time and energy,the cause of congenital heart disease in the fetus has not yet been fully elucidated.As an emerging small bioactive molecule substance,peptides can cross the placental barrier and enter the blood circulation to affect fetal development,which has attracted our attention.In the part I of this study,we selected a common type of congenital heart disease-Tetralogy of Fallot(TOF),and constructed a comparative peptidomic analysis of maternal serum between normal and TOF fetuses to screen out differential peptides that may be involved in the pathological process of tetralogy of Fallot.In the part II,an abnormally over-expressed peptide PDTTN-1 with possibly cardiovascular activity selected from the results of part I was synthesized and its potential role in cardiac development was also explored in both cell and animal experiments for providing theoretical basis for early diagnosis,treatment and prevention of congenital heart disease.Part I Peptidomic Analysis of Maternal Serum in fetal Tetralogy of Fallot Objective:To screen out bioactive peptides that may be involved in the cardiac development by comparing differentially expressed peptides between maternal serum of tetralogy of Fallot and normal controls,which will provide the basis for prediagnosis of the disease and subsequent functional studies.Methods:Serum samples were collected from the peripheral blood of pregnant women at 23-26 weeks of gestation and divided into two groups: the TOF group(n =3)and normal controls(n =3).The TOF fetuses were identified by echocardiography.We construct a comparative peptidomic profiling of maternal serum between normal fetuses and fetuses suffering from TOF and preliminarily screen out peptides with significantly differentially expression(fold change >2.0)and the difference is statistically significant(P <0.05).Further,biologically active peptides that may be closely related to cardiac development were predicted by analyzing these differentially expressed peptides through a series of bioinformatics.Results:Totally,2672 non-redundant peptides in the maternal serum of normal and TOF fetuses were identified and quantified.Of these,278 peptides originating from 227 precursor proteins were significantly differentially expressed(fold change >2.0),including 94 overexpressed peptides and 184 under-expressed peptides.Bioinformatics analysis showed that 12 differentially expressed peptides were derived from precursor proteins associated with cardiovascular diseases and located in their functional domains.Among them,7 peptides were derived from Talin1,and one abnormally overexpressed differential peptide was derived from Titin with a fold change of 3.349.We focused on these two proteins and their differentially expressed peptides,and found that the fold change of Talin1-derived peptide 2390-2404(SQGLISAARMVAAAT)and Titin-derived peptide 21722-21733(RIFVPIKGRPAP)was more than two.Besides,these two peptides were located in the ILWEQ domain and the IG domain respectively,which were closely related to cardiac development.Conclusions:The expression pattern of peptides in maternal serum of fetal tetralogy of Fallot has changed significantly compared with normal controls.Two of the differentially expressed peptides may be involved in the pathological process of TOF and may be used as novel biomarkers for prenatal diagnosis of TOF.Part ? The role of peptide PDTTN-1 in cardiac development-a preliminary studyObjective: To explore the potential role of PDTTN-1 in embryonic cardiac development.Methods: In previous study,PDTTN-1 was identified abnormally over-expressed in maternal serum of fetuses with TOF.In the present study,bioinformatic databases are firstly searched to collect its characteristics and sequence conservation.Then,we evaluated the toxicity of PDTTN-1 and its effect on the differentiation of P19 embryonal carcinoma cells by detecting changes in cell viability and expression of cardiomyocyte markers.Besides,PDTTN-1 was injected into the zebrafish embryo by microinjection.The effects of PDTTN-1 on zebrafish embryo heart development were assessed by comparing changes in morphology,heart rate,and atrial and ventricular specific gene expression.Results: With bioinformatics analysis,PDTTN-1 was located in the IG domain,which were involved in a variety of functions,including cell-cell recognition,cell-surface receptors,muscle structure and the immune system.And,PDTTN-1 was highly conservative in the heart of mouse,zebrafish and rat.In addition,the corresponding gene TTN of PDTTN-1 was highly expressed in heart.The results of studies on P19 illustrated that PDTTN-1 had no or little toxicity effect on cell.Compared with blank controls,PDTTN-1 can enter into cells and affect the formation of embryoid bodies and the expression of myocardial marker genes(NKx2.5,c Tn T,GATA4)during the progress of P19 differentiation.Results of studies on zebrafish shown that mortality of zebrafish embryo gradually increased in a dose-dependent manner with the increase of PDTTN-1 injection concentration.Compared with the group of wild type and negative control,Embryos and juveniles of zebrafish showed different degrees of pericardial edema,changes in size and shape of heart and abnormal atrial and ventricular structures after microinjection of PDTTN-1 with appropriate concentration.In addition,heart rate counts showed a significant decrease,and the results of in situ hybridization showed a decrease in atrial and ventricular specific gene expression,consistent with the results of real-time quantitative PCR.Conclusions: PDTTN-1 could not only hinder the differentiation of P19 embryonal carcinoma cells to cardiomyocyte-like cells,but also cause abnormal development of zebrafish embryo hearts.Therefore,it may be involved in the pathological process of abnormal development of human embryonic hearts. |
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