The Roleof Cypher And Enhinmice's Early Heart Developmentand Its Molecular Mechanism

BackgroundThe sarcomere Z-line is a high density protein complex. It plays as an essential part of sarcomere that maintains cytoskeleton integrity, cell signal transduction and muscle contraction functions. ENH and Cypher are subfamily proteins of Enigma family, which locate in Z-line structure. The expression products of Cypher and ENH are homologous. The expression products of Cypher and ENH also play important roles in maintaining Z-line structure integrity, cell signal transduction and muscle contraction functions. But the loss or mutation in Cypher and ENH at the same time will affect mice heart development or not is still unknown.In this experiment, we try to establish a Cypher and ENH double knockout mice model to investigate the changes during mice embryonic heart development. By observing changes in mice embryonic heart development, we can get characteristics of morphology, pathology and related protein changes in the double knockout mice model. It can help us to understand biological mechanism in double knockout mice embryonic heart. We also provide experimental evidence for the study of related gene mutation patients. MethodsEstablish and identify gene knockout mice:in order to knockout mice ENH gene,a fragment containing ENH exon-3sequence was inserted into the3rd intron of mice.A fragment sequence containing (3-galactosidase (LacZ) was inserted into mice after Cypher gene translation initiation site ATG to interrupt mice Cypher gene expression. We can then get the double knockout mice by crossing two lines of Cypher knockout line and ENH knockout line. Fresh samples of mice tail tip and fetal membrane were used to identify genotype of knockout mice by PCR technology.Investigation on survival rate of mice embryos:we took out mice embryos on different time point to investigate survival rate in different genotype mice embryos.Morphology of mice embryonic heart:we took out fresh mice embryos then compared and took pictures for whole embryos. All of the fresh mice embryos samples were fixed in paraffin block randomly. We performed H.E staining on paraffin sections to observe mice embryonic heart morphology and take photos.Observation of mice heart ultrastructure:fresh mice embryos samples were fixed. Transmission electron microscope (TEM) was used to observe ultrastructure of mice embryonic heart on ultrathin sections. Test mice embryonic heart protein level and locations:fresh mice embryos samples were fixed in frozen blocks. By using section and whole mount immune staining technology, we test particular protein location changes in mice embryonic heart. Then we used confocal microscope and fluorescence microscope to observe and take photos from staining samples. Western blotting protocol was also applied for fresh mice embryos to test protein level changes. We also use yeast two hybridization and proximity ligation assay to test interactions between protein.Test RNA level in adult mice's heart:RNA level in6weeks old adult mice's heart were tested by dot blotting; we also did the semi-quantitative analysis on RNA level.ResultsThe loss of Cypher and ENH would cause early death, thinning of ventricular wall and heart dilation in mice embryos. Meanwhile, the Z-line could not assemble properly; sarcomere also had maturation problems in double knockout mice's heart. We also investigate the expression and location of Z-line related proteinin mice embryos where a-Actinin and thin filaments were disrupted.We further identified Cypher can interacted with a-Actinin, when LIM domain of Cypher was essential for interaction between Cypher and intracellular cytoplasmic region of Integrin. However, the expression and location of other protein in Z-lineand thick filaments seemed normally.ConclusionBoth ENH and Cypher were important for mice's embryonic heart development. These two genes might play a redundancy role for each other. We found that neither Cypher nor ENH knock-out would not cause early lethality when Cypher and ENH double knock-out would cause mice's early embryo dead (E10.5). What is more, those Cypher and ENH double knock-out embryos showed development retardation, cardiac dilation and heart ventricular wall thinning. During the development of mice's cardiacmyogenesis, Cypher/ENH could interact with a-Actinin to maintain crosslink and assembly in thin filament and Z-line structure. Meanwhile, both CypherLandCypherS took part in early cardiac development of mice embryo.CypherLamd ENH double knock-out mice embryo died in early development stage (E12.5); but CypherS and ENH double knock-out mice embryo could survive through adult. It showed that the role of CypherS in mice's early development can be replaced by CypherL which contained LIM domain. This phenomenon suggests LIM domain of Cypher binding to cytoplasmic region of Integrin might took part into intra-cellular signal transduction that could regulated cardiac myogenesis.