Rho Kinase in Mammalian Cardiac Development

描述（由申请人提供）：心脏分化的起始一直是积极研究的主题，并且许多转录因子已被描述为从中胚层干细胞发生心肌细胞以及随后激活负责心脏收缩性和形态发生的基因的调节剂。然而，在早期心脏发生中，调节心脏转录因子转录活性的机制，特别是在翻译后水平，在很大程度上是未知的。我们的初步研究结果表明Rho激酶在抑制心肌细胞分化和调节心脏形态发生中的作用。我们观察到，p160 ROCK和ROK α，两个成员的Rho激酶家族，表达在早期小鼠胚胎的心脏分化开始之前，和p160 ROCK是高度富集在发育中的心脏。用特异性药理学Rho激酶抑制剂（Y27632）处理早期鸡胚诱导了心脏α-肌动蛋白（心肌细胞分化的早期标志物）的早熟表达、心裂、开放的神经管和异常的左右不对称。在细胞培养中，我们观察到Rho激酶磷酸化SRF，中胚层规范和心脏分化的关键转录因子，并选择性地抑制心脏α-肌动蛋白启动子上的SRF转录活性。因此，通过直接遗传学方法研究Rho激酶在哺乳动物心脏发育中的作用具有重要意义。我们已经成功地产生了p160 ROCK缺陷小鼠的功能丧失的研究。本研究的具体目的是：1）确定Rho激酶在心肌细胞分化过程中表达和活性的时空调控; 2）通过Rho激酶基因敲除和条件性敲除小鼠模型来证明Rho激酶在哺乳动物心脏发育中的作用; 3）确定Rho激酶是否选择性地抑制SRF-通过直接磷酸化SRF在未分化的心肌细胞中依赖心脏基因表达。该研究将阐明Rho激酶在哺乳动物心脏发生和胚胎发生中的基本作用，并深入了解Rho激酶调节心肌细胞分化的机制。了解心肌细胞分化的机制不仅对了解心脏发育具有根本重要性，而且对通过胚胎干细胞的遗传操作进行心脏修复的可能性具有重要意义。

DESCRIPTION (provided by applicant): 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.