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

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10671024
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Embryonic/Neonatal medicine
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: YASUI Hiroshi Kyoto Prefectural University of Medicine, Department of Medicine, assistant, 医学部, 助手 (60210241)
• Co-Investigator(Kenkyū-buntansha): MORISHIMA Masae Tokyo Women's Medical University of Medicine, Department of Medicine, assistant, 医学部, 助手 (00241068)
• Project Period (FY): 1998 – 1999
• Keywords: heart / development / extracellular matrix

Research Abstract

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.