Study On Mechanism Of Trichloroethylene-induced Differentiated Inhibition From Human Embryonic Stem Cells To Cardiomyocytes

Objective:To investigate effects of trichloroethylene(TCE) toxicity on differentiation from human embryonic stem cells to cardiomyocytes and its mechanisms.Methods:Base on orientated differentiation from human embryonic stem cells(hESCs)named H9 to cardiomyocytes in vitro model, different doses of TCE(100 ppb, 1 ppm and 10 ppm, in respectively treated group and control group with DMSO) were added into differentiation culture medium. Embryoid body formation and cells in the different developmental stages after TCE induction were examined with observing several parameters such as cell viability by MTT assay, autorhythmic beating frequency from the embryoid body, cell differentiation ratio by flow cytometry analysis, specific gene expression from cardiomyocytes by quantitative PCR, related proteins expressions within each differentiated phase by immunofluorescence method and impact of TCE on genes expressions in cardiomyocytes associated with intracellular calcium ion channel by microarray way.Results:1. Successful orientated differentiation from human embryonic stem cells to cardiomyocytesIn the process of time, cells were in order experienced pluripotent stem cell period,embryoid body(EBs) suspension period and adherent EBs formation period with cell autorhythmic beating and morphological changes of hESCs could be observed. Using stage-specific markers for RT-PCR testing, the results showed the success of cardiomyocyte differentiation.2. Cell toxicity of TCEMTT assay displayed that the cytoactive degrees of both undifferentiated hESCs and terminally differentiated cardiomyocytes were not significantly influenced by concentration of the TCE used in all experiment(n = 12, P> 0.05), which indicate that experimental doses of TCE used do not affect cell viability.3. TCE inhibited directed differentiation from hESCs to cardiomyocytesIn the process of time for cells culture in vitro, the number of beating EBs was increased and time of autorhythmic beating from EBs was delayed compared with the control group. Flow cytometry showed that, with increasing concentrations of TCE, the positive rate of cardiac-specific marker named cTnt was continuously decreased, which suggest that inhibition of cardiomyocyte differentiation induced by TCE has a significant dose-dependent manner.Collected cells culture at D21 consecutively treated by TCE with different dose in each group, the expression of cTnt in the cell was detected by immunofluorescence, and the results showed that the affected area of cells increased significantly.4. TCE suppressed the expressions of cardiomyocytes differentiation stage makersExpression levels of pluripotent genes such as Nanog, Tert, Sox2, and mesoderm formation related gene as Gata4 and T were not significantly affected by TCE with different dose(P> 0.05). However, TCE could significantly decrease expression levels of specific markers, cTnt Mhy-7, in cardiomyocytes at maturation period in a dose-dependent manner(P< 0.05), which indicate that TCE obviously affect changes in cardiomyocytes differentiation from precursors phase to maturation phase.5. Effects of TCE with different doses on expression levels of myocardial calcium channel related geneQuantitative PCR testing showed that calcium channel-related genes as Serca2 and Cav1.2 expression levels were significantly reduced(P< 0.05), which mean that TCE greatly interfere expression of cardiomyocytes calcium channel related gene.The cardiomyocytes with autorhythmic beating response were analyzed by microarray(gene chip) and result told that expression levels of cardiomyocytes calcium channel related genes in autorhythmic beating cells were generally decreased by TCE treatment.Conclusion:Trichloroethylene remarkably inhibit differentiated transition from myocardial progenitor cells to matured cardiomyocytes, decrease the differentiated proportion of matured cardiomyocytes and interfere expressions of cardiomyocytes calcium channel related genes, which results in cardiomyocytes differentiation and development more difficult, and bring about a poor autorhythmic beating activities.