| Incidence rate of congenital heart disease (CHD) in normal crowd is about 1% (0.6%,1.2%). China has about 10 million CHD patients and the medical cost has reached 12 billion. It is estimated that the CHD will still be the first human killer until 2030. Congenital heart valve disease, accounted for 20-25% of CHD, is a large class of the disease. The disease costs a huge amount of money every year, for example, there are about 82000 patients with valve replacement each year in the United States. Therefore, the research on the development of heart and valve as well as its molecular regulation mechanism is of great significance for the prevention and treatment of the disease.Our previous study showed that the zebrafish gene HPRG1 may be involved in the early morphogenesis of the heart and probably affects the BMP and TGF-β signaling pathways that play an important role in heart development by interacting with Smad3, Smad4, Smad7 and Smad8. In this paper zebrafish model was used for further study of the functions of HPRG1 in vertebrate animal heart development and its role in the formation of heart valve.To figure out the function of HPRG1 in cardiac development, we utilized the wild type and transgentic zebrafish with specific marker GFP (nppa:GFP) as models. HPRG1 specific morpholino antisense nucleic acid was used to knockdown the HPRG1 protein expression. The results showed that the low level expression of HPRG1 lead to heart malformations, for example, AV structure became narrow and linear, cardiocoelom seemed larger and so on. When over-expressing HPRG1 mRNA, abnormalities types were observed in zebrafish embryos, such as widened atrioventricular orifice and larger cardiocoelom etc. Combined with (Semi-automated) Heart Beat Analysis Program (M-mode Heart physiological analysis software), the analysis showed that the cardiac rhythm of HPRG1 knocked-down zebrafish slowed down, accompanied by the hange of volume of ventricular atrium. Compared with wild type, the cell number HPRG1 knocked-down hearts in zebrafish appeared to be decreasedand the atrioventricular orifices became smaller. These results suggest that HPRG1 is involved in the regulation of early heart development and plays a role in zebrafish embryos during the cardiac development. According to the experimental results of RT-PCR, knock-down of HPRG1 protein expression results in down-regulation of heart genes marker like Nkx2.5, Gata4, myh6, SRF, cardiac actin, and B-catin, of which Nkx2.5, myh6 and B-catin down-regulation was obvious. In addition, the experimental results with Western blot demonstrated that knock-down of HPRG1 expression down-regulated the expression of Nkx2.5, Gata4, and cardiac catin, but the express of ISL1 was up-regulated. The knocked-down of HPRG1 protein expression can affect several the expression of the heart marker genes that affect the zebrafish early embryonic cardiac development with deformity embryos. The results of overall zebrafish slices with HE immunohistochemistry and antibody immune dyeing indicated that zebrafish heart valves appeared partially missing when the protein expression of HPRG1 was knocked down. Moreover, HPRG1 protein can co-express with the valve endothelial marker Zn-5. These results suggest that HPRG1 may be involved in the formation of zebrafish heart valve.In order to further study the role of HPRG1 in zebrafish early embryonic heart development, the antibody staining of the early embryo was performed. The results showed that HPRG1 protein was expressed in all of the developmental stages analyzed, and in the 128 cell stage, its high expression began to appear in a single cell. The early zebrafish embryonic cells were isolated, and the results with embryonic cell antibody dying showed that HPRG1 protein had a high expression in some cells and could be co-expressed with the genes labeling mesoderm cells such as Nkx2.5, MF20, S46, Gata4and ISL1. |
PDF Full Text Request