Rho Kinase in Mammalian Cardiac Development

描述(申请人提供)：心脏分化的启动一直是一个积极研究的主题，许多转录因子被描述为调节中胚层干细胞形成心肌细胞以及随后激活负责心脏收缩和形态形成的基因。然而，在早期心脏发生中，调控心脏转录因子转录活性的机制，特别是在翻译后水平上，在很大程度上是未知的。我们的初步结果表明，Rho激酶在抑制心脏细胞分化和调节心脏形态发生方面发挥了作用。我们观察到Rho激酶家族的两个成员p160ROCK和ROKalpha在小鼠心脏分化开始之前的早期胚胎中都有表达，并且p160ROCK在发育中的心脏中高度丰富。用一种特定的药物Rho激酶抑制剂(Y27632)处理早期鸡胚，会导致心脏α-肌动蛋白(心肌细胞分化的早期标志)早熟表达，心裂，神经管开放和异常的左右不对称。在细胞培养中，我们观察到Rho激酶使SRF磷酸化，SRF是中胚层规范和心脏分化的关键转录因子，并选择性地抑制SRF在心脏α-肌动蛋白启动子上的转录活性。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是很重要的。我们已经成功地产生了p160ROCK缺陷小鼠，用于功能丧失研究。该建议的具体目的是：1)确定Rho激酶在心肌细胞分化过程中的时空调节；2)通过Rho激酶基因敲除和条件性基因敲除小鼠模型，展示Rho激酶在哺乳动物心脏发育中的作用；3)确定Rho激酶是否通过直接磷酸化SRF选择性地抑制未分化心肌细胞中依赖SRF的心脏基因的表达。这项研究将阐明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重要意义，而且对通过胚胎干细胞的基因操作进行心脏修复的可能性也具有重要意义。

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.