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

描述（由申请人提供）：将血压和肌肉拉伸等力转换为生物信号对心血管功能至关重要，其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，血流的改变会产生先天性缺陷，如左心发育不良综合征中出现的严重心肌细胞增殖不足。然而，关键机械传感器的身份仍然未知。机械转导的一种潜在手段是产生拉伸敏感离子电流。这些已在新生儿/成人心血管组织中观察到电生理学，并且可能在压力感觉中起作用。然而，由于我们不知道产生它们的通道的身份，对这种机制的研究受到了阻碍，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牵张敏感电流，但它们在胚胎心脏中的存在仍未被探索。识别这些通道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，这些电流的抑制剂在动物模型中对房颤有效，但设计高亲和力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的作用可能对再生医学领域至关重要，因为已知胎儿心脏的成熟取决于心跳产生的力量。 因此，这项建议的目的是检查是否拉伸敏感电流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。为了确定这些电流在早期心脏发生，胚胎组织将新鲜解剖和电生理检查在几个点在早期发展。我们将在拉伸条件下培养这些细胞，同时阻断电流，检测随后的心肌细胞增殖。为了克隆这些通道，我们将进行独立的基因组和蛋白质组筛选，考虑到缺乏合适的高亲和力通道阻断剂。基因组方法将使用果蝇-RNAi筛选抑制响应拉伸的Ca 2+进入的克隆。这种方法在克隆新型通道和Ca 2+信号分子方面取得了巨大成功。然后，我们将通过计算确定哺乳动物的直系同源物。蛋白质组学方法将利用这一发现，即当细胞在流动而不是静态培养基下培养时，拉伸激活通道上调。因此，表面蛋白将在静态和流动条件下标记和纯化，并且其表面表达随流动而增加的那些蛋白的身份将通过质谱方法确定。推定的牵张激活通道将从该子集中分离。该项目的成功完成将为心脏病提供新的治疗靶点，确定参与对力的反应的分子，并允许创建新的工具来研究力在器官发育中的作用。 公共卫生相关性：心血管系统感知血压和血流的能力是其正常功能的核心，在高血压、心力衰竭、心律失常和某些先天性心脏缺陷等疾病中发生改变。通过识别感知血压的关键分子，我们希望对这些疾病如何发展获得新的见解，并将这些传感器作为新药设计的目标。

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. PUBLIC HEALTH RELEVANCE: The ability of the cardiovascular system to sense blood pressure and flow is central to its normal function, becoming altered in diseases such as hypertension, heart failure, cardiac arrhythmias, and certain congenital heart defects. By identifying key molecules that sense blood pressure, we hope to gain new insight into how these diseases develop and establish these sensors as targets for the design of novel drugs.