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

细胞外基质信号传导对小鼠胚胎心肺循环的作用

基本信息

批准号：
10671024
负责人：
YASUI Hiroshi
金额：
$ 0.51万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671024/
关键词：
heart development extracellular matrix インテグリンマウス in situ hybridization シグナル伝達

项目摘要

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.

Rho A，而不是Rho B，已被证明能促进大鼠新生儿心肌细胞肥大，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研究了Rho A和Rho B在小鼠胚胎心肌细胞中的免疫定位。然后，为了分析功能，使用以下质粒在胚胎日（ED） 13心肌细胞中进行转染实验：(1)组成型活性突变体，ha标记的V14 Rho A或V14 Rho B；(2)显性阴性突变体，ha标记N17 Rho A或N17 Rho B；(3) gep标记的C3外泌酶，抑制整体Rho功能。转染后1或2天，我们用免疫组织化学技术评估f -肌动蛋白、肌动蛋白或肌动蛋白的定位。Rho A在检查的所有胚胎日心肌中一致表达（在ED 8.5和17之间），而Rho B在ED 14之前表达。除了细胞质中的弥漫性染色外，两种Rho蛋白在肌原纤维上以两种模式免疫定位：沿Z线或沿M线。转染V14 Rho A或Rho B的培养心肌细胞在基侧形成厚而凝聚的肌前原纤维。组成性激活的Rho蛋白对顶端侧横纹肌原纤维的形成没有显著影响。gep标记的C3诱导心肌细胞的各种形态变化以及肌原纤维网络或捆绑的破坏。Rho显性阴性转染诱导的表型与C3诱导的表型相似，但程度要小得多。在心肌细胞发育过程中，Rho A和Rho B均以时空调控的方式表达，并在肌原纤维上进行免疫定位。这些分子共同促进肌原纤维前形成，并可能有助于肌节结构的稳定。