Identification of a Stretch-Activated Channel with a Role in Cardiac Development

描述(申请人提供)：血压和肌肉伸展等力量转化为生物信号是心血管功能的核心，状态改变会导致心力衰竭、高血压和心律失常。在发育过程中，血流变化可能会产生先天性缺陷，如左心发育不良综合征中出现的严重心肌细胞低增殖。然而，关键机械传感器的身份仍然未知。一种潜在的机械转导方法是产生拉伸敏感离子电流。这些在新生儿/成人心血管组织中已经被电生理学观察到，并且可能在压力感中起作用。然而，由于我们对产生它们的通道的身份的无知，这一机制的研究一直受到阻碍，因为电生理学很难适应克隆。此外，尽管在新生儿组织中发现了拉伸敏感电流，但它们在胚胎心脏中的存在仍未被发现。识别这些途径可能会促进我们的知识，并为心血管疾病提供新的治疗靶点。例如，在动物模型中，这些电流的抑制剂对房颤有效，但设计高亲和力的药物将取决于分离通道本身。同样，研究这些通道在心肌细胞增殖中所起的作用可能会被证明是再生医学领域的关键，因为众所周知，胎儿心脏的成熟取决于心跳产生的力量。因此，这项建议的目的是研究拉伸敏感电流是否在胚胎心脏组织的增殖中发挥作用，并克隆拉伸激活的钙通道。为了识别早期心脏发生中的这些电流，将新鲜解剖胚胎组织，并在早期发育的几个时间点进行电生理学检查。我们将在拉伸条件下培养这些细胞，同时在药物上阻断电流，分析随后的心肌细胞增殖。为了克隆这些通道，在缺乏合适的高亲和力通道阻滞剂的情况下，我们将进行独立的基因组和蛋白质组筛选。基因组学方法将使用果蝇-RNAi筛选那些在拉伸反应中抑制钙进入的克隆。这种方法在克隆新的通道和钙信号分子方面取得了巨大的成功。然后我们将通过计算识别哺乳动物的同源基因。蛋白质组学方法将利用这一发现，即当细胞在流动的而不是静态的培养液中培养时，拉伸激活的通道被上调。因此，表面蛋白质将在静态和流动条件下被标记和纯化，那些表面表达随着流动而增加的蛋白质的身份将通过质谱学方法来确定。假定的拉伸激活通道将从这个子集中分离出来。该项目的成功完成将为心脏疾病提供新的治疗靶点，识别参与力量反应的分子，并允许创造新的工具来研究力量在器官发育中的作用。

DESCRIPTION (provided by applicant): The transduction of forces such as blood pressure and muscle stretch into biological signals is central to cardiovascular function, with altered states leading to heart failure, hypertension, and arrhythmias. Within development, alteration of flow can produce congenital defects, such as the severe cardiomyocyte hypo- proliferation seen in hypoplastic left heart syndrome. Yet, the identity of key mechanosensors remains unknown. One potential means of mechanotransduction is the production of stretch-sensitive ionic currents. These have been observed electrophysiologically in neonatal/adult cardiovascular tissue, and perhaps play a role in pressure sensation. Nevertheless, study of this mechanism has been hindered by our ignorance of the identity of the channels creating them, because electrophysiology is difficult to adapt for cloning. Moreover, though stretch-sensitive currents are found in neonatal tissue, their presence in the embryonic heart remains unexplored. Identifying these channels will likely advance our knowledge and provide novel therapeutic targets for cardiovascular disease. For example, inhibitors of these currents are effective against atrial fibrillation in animal models, but designing high-affinty drugs will depend on isolating the channels themselves. Similarly, studying the role these channels play in cardiomyocyte proliferation may prove critical to field of regenerative medicine, as the maturation of the fetal heart is known to depend on the forces created by a heartbeat. Thus, the aims of this proposal are to examine whether stretch-sensitive currents play a role in the proliferation of embryonic cardiac tissue, and to clone a stretch-activated calcium channel. To identify these currents in early cardiogenesis, embryonic tissue will be freshly dissected and examined electrophysiologically at several points in early development. We will culture these cells under conditions of stretch, while blocking currents pharmacologically, assaying for subsequent cardiomyocyte proliferation. For the aim of cloning these channels, we will perform independent genomic and proteomic screens, given the absence of suitable high- affinity channel blockers. The genomic approach will use a Drosophila-RNAi screen for clones that inhibit Ca2+ entry in response to stretch. This approach has had robust success for cloning novel channels and Ca2+ signaling molecules. We will then identify mammalian orthologs computationally. The proteomic approach will take advantage of the finding that stretch-activated channel are upregulated when cells are cultured under flowing, as opposed to static, media. Thus, surface proteins will be labeled and purified under static and flow conditions, and the identity of those whose surface expression increases with flow will be determined by mass spectroscopic methods. Putative stretch-activated channels will be isolated from within this subset. Successful completion of this project will provide novel therapeutic targets for cardiac disease, identify molecules involved in the response to force, and allow the creation of new tools to study the role of force in organ development.