role of extracellular matrix signalling on cardio-pulmonary circulation in mouse embroys

Rho A，而不是Rho B，已被证明促进新生大鼠心肌细胞肥大，然而，Rho蛋白是否或如何促进心肌发育仍不清楚。我们首先在体内和体外检测了Rho A和Rho B在小鼠胚胎心肌细胞中的免疫定位。然后，为了进行功能分析，用以下几种载体对胚胎第13天(ED)的心肌细胞进行了转基因实验：(1)成分活性突变体，HA标记的V14 Rho A或V14 Rho B；(2)显性负性突变体，HA标记的N17 Rho A或N17 Rho B；(3)GEP标记的C3外酶，它全面抑制Rho的功能。转染后1~2天，用免疫组织化学方法检测F-肌动蛋白、肌瘤α-肌动蛋白或肌动蛋白的定位。Rho A在所有胚胎天数(ED8.5~17)均有表达，而Rho B在ED14之前表达。除胞浆弥漫染色外，两种Rho蛋白均以两种方式免疫定位于肌原纤维上：沿Z线或沿M线。转染V14Rho A或Rho B的培养心肌细胞在基底侧形成致密的前肌原纤维。结构性激活的Rho蛋白对顶端横纹肌原纤维的形成没有显著影响。GEP标记的C3可引起心肌细胞的各种形态改变以及肌原纤维网络或束状结构的破坏。显性负性Rho诱导的表型与C3相似，但程度要小得多。Rho A和Rho B不仅在发育中的心肌细胞中以时空调节的方式表达，而且免疫定位在肌原纤维上。这些分子联合起来促进前肌原纤维的形成，可能有助于肌节结构的稳定。

Rho A, but not Rho B, has been shown to promote hypertrophy in rat neonate cardiomyocytes, however, whether or how Rho proteins contribute to myocardial development is still unclear. We first examined the immunolocalization of Rho A and Rho B in mouse embryo cardiomyocytes in vivo and in vitro. Then, for analysis of function, transfection experiments were performed in embryonic day (ED) 13 cardiomyocytes using the following plasmids: (1)constitutively active form mutant, HA-tagged V14 Rho A or V14 Rho B; (2)dominant negative form mutant, HA-tagged N17 Rho A or N17 Rho B; (3)GEP-tagged C3 exoenzyme which inhibits overall Rho function. One or two days after transfection, we evaluated the localization of F-actin, sarcomeric alpha-actinin or titin by using immunohistochemical technique. Rho A was consistently expressed in the myocardium at all embryonic days examined (between ED 8.5 and 17), whereas Rho B was expressed before ED 14. In addition to diffuse staining in the cytoplasm, both Rho proteins were immunolocalized on myofibrils in two patterns : along Z line or along M line. The cultured cardiomyocytes transfected with V14 Rho A or Rho B formed thick and condensed premyofibrils on the basal side. Formation of striated myofibrils on the apical side was not significantly influenced by constitutively activate Rho proteins. GEP-tagged C3 induced various morphological changes of cardiomyocytes as well as disruption of myofibril network or bundling. Transfection with dominant negative Rho induced similar phenotype to that induced by C3 but to a much lesser extent. Not only Rho A, but also Rho B, is expressed in developing cardiomyocytes in a spatiotemporally regulated manner with immunolocalization on myofibrils. These molecules combinatorially promote premyofibril formation and possibly contribute to the stabilization of sarcomere structure.