Cardiac Function and PIP2

心脏功能和 PIP2

• 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
• 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; 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

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）：该提案的重点是从表面膜中去除转运蛋白，特别是心脏NA/CA交换器（NCX1）的内吞过程。膜融合和萌芽过程是所有真核生活的基础，我们已经开发了改进的电生理方法来分析细胞表面的运输事件，从永生的成纤维细胞开始，并继续前往成人心脏心肌细胞。利用高分辨率记录的细胞质环境的前所未有的控制，我们发现细胞质ATP耗竭，然后是CA瞬态和ATP补充，促进了大规模的内粒细胞反应（MEND）。我们进一步确定了NCX1在修补过程中被内在化。由于NCX1在缺血 - 再灌注损害和相关心律不齐中起主要作用，因此从膜上去除NCX1对代谢应激的响应可能具有很大的临床意义。因此，我们已经对修补响应进行了详细的分析。初步数据表明，通过用ATP，CA-和PIP2依赖性过程对肌动蛋白膜细胞骨架进行重塑驱动。进一步的初步数据表明，NCX1的横向迁移率在导致修补的步骤以及F-肌动蛋白的稳定下急剧下降。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，我们将确定修补的CA传感器，并将分析如何调节参与调节的PIP-激酶。为了解决NCX1夫妇如何修补，已经开发了新的NCX1融合蛋白，用于在线监测NCX1内部化，NCX1的脉冲跟踪跟踪以及改善NCX1迁移率的分析。 NCX1与Dendra2的融合允许将绿色转运蛋白转化为红色转运蛋白，然后跟踪两个转运蛋白物种。细胞外侧的挂接融合允许具有不同膜可渗透和可渗透荧光团的顺序NCX1标记。从长远来看，这些融合将允许使用量子点和纳米old研究NCX1运输。总体而言，拟议的工作将产生对强大的内吞过程的基本见解，该过程具有广泛的细胞生物学兴趣，并且很可能在心脏缺血 - 再生灌注和相关病理中起重要作用。公共卫生相关性：公共卫生相关性心血管疾病是美国死亡的主要原因。心肌心律不齐引起的许多死亡发生后，许多死亡都是由心律不齐引起的，并且在心脏激发触发偶联和相关心律失常的长期内，人们认为这起重要作用。心律不齐的发病机理是复杂的，涉及许多分子实体。心脏NA/CA交换器从心肌细胞中去除CA，并且是本研究的主要重点，在许多情况下，通过产生向内的膜电流，在许多情况下都起着触发作用。同样，该转运蛋白与以前的NA负荷响应钙的加载心脏细胞从缺血 - 重新灌注发作中介导了许多心脏细胞损伤，从而导致肌细胞超声促进，并促进通过线粒体信号机制激活的细胞死亡程序，这些细胞死亡程序被设定在运动中。实验程序解决了如何从细胞表面膜中删除NA/CA交换器以及如何调节该过程，特别是在病理环境中如何灭活。已经发现内吞机制在多种非心脏细胞类型中被激活，以响应缺血和/或氧气剥夺。现在，我们将探索心肌细胞中相关的机制。为此，我们通过分析内吞机制的分析及其在简单细胞培养细胞中的调节来采取一种高度独特的方法，可以表达Na/ca交换器，并继续分析心肌肌细胞中的等效机制。我们的总体目标是更好地了解NCX1的“终身”和内吞作用。可以预期，这项工作对心脏病理和最终医学具有根本性的影响。