The Effect Of Triclosan On Embryonic Heart Cardiogenesis And DNA Methylation Regulation

Among all birth defects,congenital heart disease?CHD?is the most prevalent and remains the leading cause of death.In most cases the causes of CHD remain unknown,while a growing number of studies have indicated the potential role of environmental agents as risk factors in CHD occurrence.Triclosan?TCS?,as a highly effective spectral antibacterial agent,is widely used in personal care products,has lipophilicity,persistence and accumulation,and can directly penetrate the placental barrier,affecting the normal development of the embryonic system.Our previous study found that TCS could affect the differentiation of mouse embryonic stem cells into cardiomyocytes and zebrafish heart development.The above results all indicate that TCS can have adverse effects on heart development,but the mechanism is still unclear.In the present study,the human embryonic stem cell?h ESC?-derived cardiomyocytes are used as a model of the early stages of heart development to determine the developmental toxicity of TCS.Morphology of cardiomyocytes,the percentage of beating EBs,genes and proteins related to cardiac development are examined and compared between TCS-treated cells and the control cells.To explore the mechanism involved in TCS' cardiac toxicity,we investigated the DNA methylation and histone acetylation patterns between the treatment and control groups.This may provide greater insight into the epigenetic mechanism of TCS on cardiogenesis and help to identify biological markers for risk assessment of EDCs exposure causing CHD.In addition,h ESC-derived cardiomyocytes will be used as an excellent model system for screening for developmental toxicity of environmental chemicals.It may provide evidence for the prevention of CHD and make contributions to improve the quality of the population and children's health.Objectiveinvestigating the effect of TCS on cardiomyocyte differentiation induced by h ESC and exploring the epigenetic mechanism of TCS affecting cardiomyocyte differentiation process from the perspective of methylation.Methods1.Base on orientated differentiation from human embryonic stem cells?h ESCs?named H9 to cardiomyocytes in vitro model,1?M dose of TCS and 0.1% dose of DMSO were added into differentiation culture medium as experimental group and control group,then induced cardiomyocytes were detected.2.After induction,the spontaneous beating myocardial cell mass were observed under an optical microscope on the 12 th day,the beat frequency of the cardiomyocytes was recorded.Additionally,immunofluorescence method was performed on spontaneous pulsatile cell mass to detect the cardiomyocyte-specific factor TNNT2 and myocardial microstructure was observed.3.At the end of the exposure,pulsed and unstimulated clumps were obtained by mechanical dissection and were labeled as control and TCS experimental groups,respectively.Total RNA was extracted and the m RNA expression levels of cardiac genetics,DNA methylation-related enzymes and DNA demethylationrelated genes were detected by real-time quantitative RT-PCR?q RT-PCR?followed by RNA-Seq sequencing.Western Blot technique was employed to detect the related protein expression levels?NKX2.5 and TET2?in control and TCS experimental groups4.RNA-seq and DNA methylation chip technique were used to screen out the Cp G sites with abnormal DNA methylation pattern,and we used bisulfite sequencing method to examine the DNA methylation pattern of abnormally expressed m RNA gene.Result1.Human embryonic stem cells were successfully differentiated into cardiomyocytes:During the experiment,we used the microscope to observe the changes of cell morphology in the plate daily,the gradual convergence of the cells can be observed.And on the 12^th day of induction,cardiomyocyte mass with rhythmic spontaneous beating was observed,which means that the stem cells were successfully differentiated into cardiomyocytes.2.TCS exposure inhibited the directional differentiation of human embryonic stem cells into cardiomyocytes:In the process of time,the TCS reduced the cardiomyocyte beating rate significantly on day 12 th,and the differentiation rate of cardiomyocytes in TCS-treated group was decreased,while the cardiomyocytes exhibited normal cardiac structure,demonstrated by immunofluorescent staining of specific factor TNNT2 and ?-actinin3.TCS exposure influences the expression of genetic markers,DNA methylation-related genes and related protein expression level during h ESC differentiation into cardiomyocytes in the stage of myocardial differentiation: In differentiated cardiomyocytes,TCS-treated group showed lower levels of gene expression than those in the DMSO control group?P <0.05?,and the methylation-related gene m RNA expressions in the TCS group were higher than the DMSO control group.Demethylation-related gene TET family?TET1,TET2,TET3?m RNA expressions in TCS group were lower than control group.Additionally,Western blot analysis showed that the expression of Nkx2.5 in TCS group was significantly lower than that in control group,and the expression level of TET2 in TCS group was significantly higher than that in control group.All above results suggested that TCS might play an important role in inhibiting myocardial differentiation by suppressing cardiac related gene expression,and DNA methylation and demethylation play an important role in TCS inhibition of myocardial differentiation.4.By m RNA-seq and DNA methylation chip technique,we found that the methylation status of TNNT2 and GATA4 genes were inversely associated with their m RNA expression patterns,above results suggested that methylation play an important role in inhibiting the directional differentiation of human embryonic stem cells into cardiomyocytes.ConclusionTCS can inhibit the differentiation of human embryonic stem cells into cardiomyocytes by inhibiting the expression of cardiomyocyte-specific genes and proteins,resulting in a decrease in the rate of cardiomyocyte differentiation and the frequency of autonomous beats,thereby affecting its differentiation efficiency.At the same time,the methylation levels in the promoter region of cardiomyocyte-specific genes were increased,suggesting that DNA methylation may play a regulatory role in the process of TCS inhibiting the directional differentiation of cardiomyocytes.This method provides a basis for assessing the developmental toxicity of myocardium from environmental chemicals using stem cell model,provides a basis for the prevention and treatment of CHD,and contributes to the improvement of population quality and the promotion of children's health.