Cardiac Function and PIP2

心脏功能和 PIP2

基本信息

批准号：
8039956
负责人：
DONALD W HILGEMANN
金额：
$ 38.11万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-15 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8039956
关键词：
Actins Address Adult Ankyrins Apoptosis Arrhythmia Biochemical Biological Biological Assay Calcium Cardiac Cardiac Myocytes Cardiovascular Diseases Cause of Death Cell Culture Techniques Cell Separation Cell surface Cells Cellular Stress Cessation of life Chimeric Proteins Complex Couples Coupling Cultured Cells Cytoplasm Cytoskeleton Data Electric Capacitance Electron Microscopy Endocytosis Event Excision F-Actin Fibroblasts GTP-Binding Proteins Goals Health Ischemia Knock-in Mouse Label Laboratories Lateral Life Liquid substance Mediating Medicine Membrane Membrane Fusion Metabolic Metabolic stress Methods Mitochondria Molecular Monitor Motion Muscle Cells Myocardial Infarction Myosin ATPase Na(+)-K(+)-Exchanging ATPase Oxygen Pathogenesis Pathology Peptide Hydrolases Perfusion Phosphatidylinositol 4,5-Diphosphate Phosphoric Monoester Hydrolases Phosphotransferases Physiologic pulse Play Positioning Attribute Potassium Procedures Process Proteins Quantum Dots Regulation Reperfusion Injury Reperfusion Therapy Resolution Role Side Signal Transduction Staging Surface Tertiary Protein Structure Testing Time United States Work base biological adaptation to stress cell injury cell type clinically significant deprivation extracellular fluorophore improved innovation insight interest nanoGold polymerization programs protein activation public health relevance response sensor tool trafficking

项目摘要

DESCRIPTION (provided by applicant): This proposal focuses on endocytic processes that remove transporters, specifically cardiac Na/Ca exchangers (NCX1), from the surface membrane. Membrane fusion and budding processes are fundamental to all eukaryotic life, and we have developed improved electrophysiological methods to analyze trafficking events at the cell surface, starting with immortalized fibroblasts and proceeding to adult cardiac myocytes. Exploiting unprecedented control of the cytoplasmic milieu with high resolution capacitance recording, we have discovered that cytoplasmic ATP depletion, followed by a Ca transient and ATP replenishment, promotes a massive endocytic response (MEND). We have further determined that NCX1 is internalized during MEND. As NCX1 plays a major role in ischemia-reperfusion damage and related cardiac arrhythmias, removal of NCX1 from the membrane in response to metabolic stress can be of substantial clinical significance. Therefore, we have initiated a detailed analysis of the MEND response. Preliminary Data indicates that MEND is driven by remodeling of actin membrane cytoskeleton with ATP-, Ca- and PIP2- dependent processes all playing essential roles. Further Preliminary Data shows that NCX1 lateral mobility decreases dramatically in steps leading up to MEND, as well as with stabilization of F-actin. Therefore, we will analyze how metabolic state regulates actin cytoskeleton and NCX1-actin cytoskeleton interactions. Additionally, we will identify the Ca sensors underlying MEND, and we will analyze how PIP-kinases involved in MEND are regulated. To address how NCX1 couples to MEND, new NCX1 fusion proteins have been developed for on-line monitoring of NCX1 internalization, pulse-chase tracking of NCX1, and improved analysis of NCX1 mobility. An NCX1 fusion with Dendra2 allows conversion of green transporters to red transporters, followed by tracking of the two transporter species. Halotag fusions on the extracellular side allow sequential NCX1 labeling with different membrane-permeable and -impermeable fluorophores. In the longer term, these fusions will allow the use of quantum dots and Nanogold to study NCX1 trafficking. Overall, the proposed work will generate fundamental insights into a powerful endocytic process that is of wide cell biological interest and is likely to play an important role in cardiac ischemia-reperfusion and related pathologies. PUBLIC HEALTH RELEVANCE: Public Health Relevance Cardiovascular disease is the leading cause of death in the United States. Many deaths in the immediate aftermath of myocardial infarction are caused by cardiac arrhythmias, and in the long-term of cardiac insufficiency malfunction of cardiac excitation-contraction coupling and associated arrhythmias are thought to play an important role. The pathogenesis of arrhythmias is complex and involves numerous molecular entities. The cardiac Na/Ca exchanger, which removes Ca from cardiac myocytes and is the major focus of this study, is thought to play a trigger role in many cases by generating inward membrane current. Also, this transporter is implicated to mediate much cardiac cell damage from ischemia-reperfusion episodes by loading cardiac cells with calcium in response to previous Na loading, thereby causing myocyte hypercontraction and promoting cell death programs to be activated via mitochondrial signaling mechanisms that are set in motion. The experimental program addresses how Na/Ca exchangers may be removed from the cell surface membrane and how this process may be regulated, in particular how it may become inactivated in pathological settings. Endocytic mechanisms have been found to be activated in multiple non-cardiac cell types in response to ischemia and/or oxygen deprivation. We will now explore related mechanisms in cardiac myocytes. To do so, we are taking a highly unique approach by starting from analysis of endocytic mechanisms and their regulation in simple cell culture cells, where Na/Ca exchangers can be expressed, and proceeding to the analysis of the equivalent mechanisms in cardiac myocytes. Our overall goal is a better understanding of the `life-time' and endocytosis of NCX1. This work can be expected to have fundamental implications for cardiac pathologies and ultimately medicine.

描述（由申请人提供）：该提案重点关注从表面膜去除转运蛋白，特别是心脏 Na/Ca 交换器 (NCX1) 的内吞过程。膜融合和出芽过程是所有真核生物的基础，我们开发了改进的电生理学方法来分析细胞表面的运输事件，从永生化成纤维细胞开始，一直到成年心肌细胞。利用高分辨率电容记录对细胞质环境的前所未有的控制，我们发现细胞质 ATP 消耗，随后是 Ca 瞬变和 ATP 补充，促进大量内吞反应 (MEND)。我们进一步确定 NCX1 在 MEND 期间内化。由于 NCX1 在缺血再灌注损伤和相关心律失常中发挥重要作用，因此响应代谢应激而从膜上去除 NCX1 可能具有重要的临床意义。因此，我们开始对 MEND 响应进行详细分析。初步数据表明，MEND 是由肌动蛋白膜细胞骨架重塑驱动的，其中 ATP、Ca 和 PIP2 依赖性过程均发挥着重要作用。进一步的初步数据表明，NCX1 横向移动性在导致 MEND 的步骤中以及随着 F-肌动蛋白的稳定而急剧下降。因此，我们将分析代谢状态如何调节肌动蛋白细胞骨架和NCX1-肌动蛋白细胞骨架相互作用。此外，我们将识别 MEND 背后的 Ca 传感器，并将分析 MEND 中涉及的 PIP 激酶是如何受到调节的。为了解决 NCX1 如何与 MEND 偶联的问题，开发了新的 NCX1 融合蛋白，用于在线监测 NCX1 内化、NCX1 脉冲追踪以及改进的 NCX1 迁移率分析。 NCX1 与 Dendra2 融合可以将绿色转运蛋白转换为红色转运蛋白，然后跟踪这两种转运蛋白。细胞外侧的 Halotag 融合允许使用不同的膜渗透性和不可渗透性荧光团进行连续的 NCX1 标记。从长远来看，这些融合将允许使用量子点和纳米金来研究 NCX1 贩运。总体而言，拟议的工作将为强大的内吞过程提供基础见解，该过程具有广泛的细胞生物学意义，并且可能在心脏缺血再灌注和相关病理学中发挥重要作用。公共卫生相关性：公共卫生相关性心血管疾病是美国的主要原因。心肌梗塞后立即死亡的许多人是由心律失常引起的，并且在长期的心功能不全中，心脏兴奋-收缩耦合和相关心律失常的功能障碍被认为起着重要作用。心律失常的发病机制很复杂，涉及许多分子实体。心脏钠/钙交换器可从心肌细胞中去除钙，是本研究的主要焦点，被认为在许多情况下通过产生内向膜电流发挥触发作用。此外，这种转运蛋白还涉及介导缺血再灌注事件引起的心肌细胞损伤，通过向心肌细胞加载钙来响应先前的钠负荷，从而导致心肌细胞过度收缩并通过线粒体信号机制促进细胞死亡程序被激活。运动。该实验程序解决了如何从细胞表面膜去除钠/钙交换剂以及如何调节该过程，特别是它如何在病理环境中失活。已发现多种非心肌细胞类型响应缺血和/或缺氧而激活内吞机制。我们现在将探讨心肌细胞的相关机制。为此，我们采取了一种非常独特的方法，从分析简单细胞培养细胞中的内吞机制及其调节开始，其中可以表达 Na/Ca 交换剂，然后分析心肌细胞中的等效机制。我们的总体目标是更好地了解 NCX1 的“生命周期”和内吞作用。这项工作预计将对心脏病学乃至医学产生根本性影响。