The initiation of cardiac differentiation has been a topic of vigorous investigation, and many transcription factors have been described as regulators of the genesis of cardiomyocytes from mesodermal stem cells and the subsequent activation of genes responsible for cardiac contractility and morphogenesis. However, the mechanisms regulating transcriptional activity of cardiac transcription factors, especially at the post-translational level, are largely unknown in early cardiogenesis. Our preliminary results point to a role for Rho kinase in inhibiting cardiac cell differentiation and in regulating cardiac morphogenesis. We observed that both p160ROCK and ROKalpha, two members of the Rho kinase family, are expressed in early mouse embryos before the onset of cardiac differentiation, and p160ROCK is highly enriched in the developing heart. Treatment of early stage chick embryos with a specific pharmacological Rho kinase inhibitor (Y27632) induced precocious expression of cardiac alpha-actin (an early marker of cardiomyocyte differentiation), cardia bifida, an open neural tube and abnormal left-right asymmetry. In cell culture, we observed that Rho kinase phosphorylates SRF, a critical transcription factor in mesoderm specification and cardiac differentiation, and selectively inhibits SRF transcriptional activity on the cardiac alpha-actin promoter. It is thus important to investigate the role of Rho kinase in mammalian cardiac development by a direct genetic approach. We have successfully generated p160ROCK deficient mice for loss-of-function studies.
The Specific Aims of this proposal are: 1) to determine the spatial-temporal regulation of Rho kinase expression and activity during cardiomyocyte differentiation; 2) to demonstrate the role of Rho kinase in mammalian cardiac development through Rho kinase knockout and conditional knockout mouse models; 3) to determine if Rho kinase selectively represses SRF-dependent cardiac gene expression in undifferentiated cardiac cells through direct phosphorylation of SRF. The proposed study will elucidate fundamental roles of Rho kinase in mammalian cardiogenesis and embryogenesis, and gain insight into the mechanisms by which Rho kinase regulates cardiomyocyte differentiation. Understanding the mechanisms of cardiomyocyte differentiation not only has fundamental importance for understanding heart development, but also has important implications for the possibility of cardiac repair through genetic manipulation of embryonic stem cells.
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