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

鉴定在心脏发育中起作用的牵拉激活通道

基本信息

批准号：
8059366
负责人：
Dipayan Chaudhuri
金额：
$ 5.47万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8059366
关键词：
Ablation Adult Affect Affinity Animal Model Arrhythmia Atrial Fibrillation Biochemical Biological Biological Assay Blood Pressure Blood Vessels Blood flow Calcium Channel Candidate Disease Gene Cardiac Cardiac Myocytes Cardiovascular Diseases Cardiovascular Physiology Cardiovascular system Cataloging Catalogs Cations Cell Culture Techniques Cell Line Cell Surface Proteins Cells Characteristics Cloning Congenital Abnormality Congenital Heart Defects Culture Media Cultured Cells Data Development Disease Drosophila genus Electrophysiology (science)Embryo Embryonic Heart Esthesia Extracellular Matrix Fetal Heart Genetic Genomics Goals Heart Diseases Heart failure Hypertension Hypoplastic Left Heart Syndrome Ion Channel Knowledge Label Mass Spectrum Analysis Mechanics Membrane Membrane Proteins Methods Monitor Mus Muscle Neonatal Organ Orthologous Gene Pharmaceutical Preparations Play Production Property Protein Kinase Proteins Proteomics RNA Interference Regenerative Medicine Resources Role Screening procedure Signal Transduction Signaling Molecule Silicones Stimulus Stretching Surface System Techniques Testing Tissues Up-Regulation cardiogenesis channel blockers design embryo tissue experience inhibitor/antagonist insight interest medical schools model design neonate new therapeutic target novel pressure protein function response sensor success tool

项目摘要

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.

描述（由申请人提供）：将血压和肌肉拉伸等力转化为生物信号对于心血管功能至关重要，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，血流的改变可能会产生先天性缺陷，例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。机械传导的一种潜在方式是产生拉伸敏感离子电流。这些已经在新生儿/成人心血管组织中通过电生理学观察到，并且可能在压力感觉中发挥作用。然而，由于我们对产生这些通道的身份的无知，对该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拉伸敏感电流，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增进我们的知识并为心血管疾病提供新的治疗靶点。例如，这些电流的抑制剂可有效对抗动物模型中的心房颤动，但设计高亲和力药物将取决于通道本身的隔离。同样，研究这些通道在心肌细胞增殖中发挥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。因此，该提案的目的是检查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，将在早期发育的几个点上新鲜解剖胚胎组织并进行电生理学检查。我们将在拉伸条件下培养这些细胞，同时通过药理学阻断电流，检测随后的心肌细胞增殖。为了克隆这些通道，鉴于缺乏合适的高亲和力通道阻断剂，我们将进行独立的基因组和蛋白质组筛选。基因组方法将使用果蝇 RNAi 筛选能够抑制 Ca2+ 响应拉伸而进入的克隆。这种方法在克隆新通道和 Ca2+ 信号分子方面取得了巨大成功。然后我们将通过计算来识别哺乳动物的直向同源物。蛋白质组学方法将利用这一发现：当细胞在流动培养基（而不是静态培养基）下培养时，拉伸激活通道会上调。因此，表面蛋白将在静态和流动条件下被标记和纯化，并且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。推定的拉伸激活通道将从该子集中隔离。该项目的成功完成将为心脏病提供新的治疗靶点，识别参与力反应的分子，并允许创建新工具来研究力在器官发育中的作用。公众健康相关性：心血管系统感知血压和血流的能力对其正常功能至关重要，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会发生改变。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有新的了解，并将这些传感器作为新药物设计的目标。