Hck Genes And Embryonic Heart Development And Nfatc1 Gene And The Cardiac Developmental Defects Relations

Part I The Study of Relationship between Hck Gene and Embryonic Heart DevelopmentObjective To investigate the spatiotemporal expression of Hck (hemopoietic cell kinase) gene in the developing rattus heart, the effect of Hck over-expression in the cardiomyocyte differentiation of P19 cells, and the effect of over-expression Hck to the functions of the cells, and to analyze the relationship between Hck gene expression and the heart development. Methods 1) Rattus hearts in embryonic days 12, 15, 19 were included in this study. The mRNA level of Hck gene expression was detected by the methods of RT-PCR and in situ hybridization. 2) Hck-pCDNA3.1 vector was constructed and transfected into P19 cells, a stable cell line was also selected and further induced to differentiate into cardiomyocytes by DMSO. At differentiation day 10(D10), beating cardiomyocytes were checked under microscopy and cytoplasmic proteins were extracted and Western-blot were performed to detect the expression of cTnI. 3) GFP targeted p56Hck plasmid and its constitutively active form were constructed and transiently transfected into HeLa cells, F-actin staining and Indirect immunofluorescence for microtubules were then performed. Phagokinetic track motility assay and In vitro invasion assays were also investigated after transiently transfection respectively. Results 1) In the developing rattus hearts, Hck gene was mainly expressed in the interstitial tissue, no expression in the cardiomyocytes, and it was up-regulated with the embryonic aging. 2) On the D10, the cell clusters or the areas of beating cells have no changes between Hck over-expression P19cells and control cells transfected with empty vectors, nor was cTnI expression between the two groups (P>0.05). 3) Ectopically expressing a constitutively active form of 56Hck will lead to membrane protrusion and F-actin reorganization in HeLa cells. Both 56Hck and its constitutive active form will lead to redistribution of microtubules and enhancement of cell motility and cell invasion. Conclusion Hck was expressed in the interstitial tissue of the developing rattus hearts, no expression in the cardiomyocytes. With the development of rat hearts, the expresion of Hck gene in the level of mRNA is dynamic. Hck have no effects on the cardiomyocyte differentiation of P19 cells. Hck may play a role in the migratory and invasive abilities of cardiac interstitial cells. The deficiency of Hck may contribute to congenital heart defect by the ways of influencing the function of cardiac interstitial cells. Part II The Study of Relationship between NFATc1 Gene and Congenital Heart DefectObjective to explore the basis of defects in heart valves and septum observed in mice lacking NFATc1 and the transcriptional targets of NFATc1 within the endocardium. Method 1) embryos of NFATc1 knock out mice and wide type mice were selected by genotyping and from E9.5 to E13.5. embryos were fixed, embedded, then made into serial slides. Immunohistochemistry was further performed to detect the expression of apoptosis-related cysteine peptidase: caspase 3, cell proliferating mark: PCNA,KI67, and FGFR1, RGS10. 2) Primary culture of embryonic (E11.5) endocardial cells (ECC), then the Cacinurin-NFATc1 pathway in ECC was inhibited by CSA or FK506, or activated by PMA+ionomycin so as to determinate the effects of Cacinurin-NFATc1 pathway in the expression of FGFR1 and RGS10. 3) The binding sites of NFATc1 in the intron of FGFR1 were predicted and cloned. A luciferase report gene system was constructed and then co-transfected with the vector included with NFATc1 into MEF cells. Luciferase activity was measured when cell culture medium was supplemented with ionomycin or not. Result 1) the advent of apoptosis in the developing heart of NFATc1 knock out mice was just the same as that of wide type mice. In both KO mice and WT mice of E12.5, apoptotic cells began to appear in the cushion area, especially in the valves and membranous parts of interventricular septum, reached to peak at E13.5, however, more apoptotic cells in KO mice than that of WT ones.2) Endocardial cells in the aortic valves and pulmonary valves had been showed as impaired proliferating in the critical time of valve formation.3) FGFRl expression was down-regulated at the two phases during heart development in NFATc1 KO mice. At E10.5, the first wave of low FGFR1 expression appeared in aortic sac and cushion areas; later, atE13.5, the FGFR1 expression was repressed in the endocardial cells of valves. We also identified an NFATc1 binding site in the intron of FGFR1. Finally, FGFR1 would be down-regulated when Cacinurin-NFATc1 pathway was inhibited and would be up-regulated when Cacinurin-NFATc1 pathway was activated. 4) The expression of RGS10 was up-regulated in the endocardial cells of NFATc1 KO mice; More RGS10 proteins were in the ECCs lacking of NFATc1 and vice versa. RGS10 mRNA would be up-regulated when Cacinurin-NFATc1 pathway was inhibited and would be down-regulated when Cacinurin-NFATc1 pathway was activated. Conclusion Firstly, the increasing apoptosis in cushion areas and reduced proliferating in endocardial cells of aortic valves and pulmonary valves is the basis of septum defects and valve malformation in NFATc1 mice. Secondly, in NFATc1 KO mice, the down-regulation of FGFR1 in the developing heart may contribute to the apoptosis in cushion areas. Last, NFATc1 activates FGFR1 and represses RGA10 in vertebrate heart valve morphogenesis.