Regulation of Cardiac Myocyte Differentiation

描述（由申请人提供）：拟议的研究具有广泛的重要性和影响，因为心脏发育缺陷可能导致胚胎发生问题，但也可能导致儿童甚至成年生活中的问题。心肌细胞分裂、形状和囊泡运输的控制是心脏形态发生的关键。虽然单个基因对这些事件的调节似乎不太可能，但我们最近发表的研究表明，Lek1在调节细胞分裂、形状和运输中起着关键作用。对最初应用的主要批评是，我们缺乏支持Lek1在心肌细胞中的功能的数据，并且Lek1功能的抑制与任何发育缺陷或心脏病无关。我们现在证明，1)Lek1调节心肌细胞的这些关键功能，2)Lek1基因的条件破坏导致心壁发育异常，导致扩张性心肌病。这些新数据使我们提出了一个中心假设，即Lek1在心脏形态发生中起着核心作用，该基因的突变导致分化心脏功能受损。三个简单的目标将检验这一假设。第一个目的是确定心肌细胞分裂、生长和/或凋亡的变化是否解释了“小心脏”表型。这将伴随着出生后心脏的生理分析，以确定心肌细胞的正常适应是否被破坏。第二个目标将量化发育中和出生后心脏中囊泡运输中断的程度和时间，使用GLUT4运输作为模型。这一目标的第二部分更具推测性，但分析起来很简单。也就是说，我们将确定插入椎间盘的产生和维持是如何随着Lek1基因突变而改变的。这不仅可以作为囊泡运输和心肌细胞形状的模型，还可以为心脏功能所必需的椎间盘调节提供关键的见解。第三个目标是确定nuc-和cyt-LEK1在心脏发育中的具体作用。这将通过对Lek1-/-心脏的转基因“拯救”来实现。没有其他研究小组拥有阐明这种独特基因在心脏发育和疾病中的功能所需的遗传、分子、免疫化学或细胞生物学工具。

DESCRIPTION (provided by applicant): The proposed studies are of broad importance and impact because developmental defects in the heart can lead to problems in embryogenesis but also problems that are manifested in childhood or even adult life. Control of cardiac myocyte division, shape and vesicular transport is critical for cardiac morphogenesis. While regulation of these events by a single gene may seem improbable, our recently published studies demonstrate that Lek1 plays critical roles in regulation of cell division, shape and transport. The major criticism of the original application was that we lacked data supporting Lek1 function in cardiac myocytes and that inhibition of Lek1 function was not linked to any developmental defect or cardiac disease. We now demonstrate that 1) Lek1 regulates these critical functions in cardiac myocytes and that 2) conditional disruption of the Lek1 gene results in developmental abnormalities of the heart wall leading to dilated cardiomyopathy. These new data lead us to the central hypothesis that Lek1 plays a central role in cardiac morphogenesis and that mutation of this gene leads to impaired function in the differentiated heart. Three straightforward aims will test this hypothesis. The first aim will determine whether changes in cardiac myocyte division, growth and/or apoptosis account for the "small heart" phenotype. This will be accompanied by physiological analyses of the postnatal heart to determine whether the normal adaptation of the myocyte is disrupted. The second aim will quantify the degree and timing of vesicular transport disruption in the developing and postnatal heart using GLUT4 trafficking as a model. The second part of this aim is more speculative but simple in terms of its analysis. Namely, we will determine how generation and maintenance of the intercalated disc is altered with mutation of the Lek1 gene. This will not only serve as a model for vesicular transport and myocyte shape but provide critical insight into regulation of the disc which is essential for cardiac function. The third aim focuses on determining the specific roles of nuc- and cyt-LEK1 in heart development. This will be accomplished using transgenic "rescue" of the Lek1-/- heart. No other group has the genetic, molecular, immunochemical or cell biological tools needed to elucidate the function of this unique gene in cardiac development and disease.