Cardiac Function and PIP2

描述(由申请人提供)：这项建议侧重于从表面膜上移除转运体，特别是心脏钠/钙交换器(NCX1)的内吞过程。膜融合和萌发过程是所有真核生物生命的基础，我们已经开发出改进的电生理学方法来分析细胞表面的运输事件，从永生化的成纤维细胞到成年心肌细胞。利用高分辨率电容记录对细胞质环境的前所未有的控制，我们发现细胞质中的ATP耗尽，随后是钙瞬变和ATP补充，促进了大规模的内吞反应(MEND)。我们进一步确定NCX1在MEND过程中是内化的。由于NCX1在缺血再灌注损伤和相关的心律失常中起主要作用，在代谢应激下将NCX1从细胞膜上移除具有重要的临床意义。因此，我们已经启动了对修补反应的详细分析。初步数据表明，MEND是由肌动蛋白膜细胞骨架的重塑驱动的，依赖于ATP、Ca和PIP2的过程都起着至关重要的作用。进一步的初步数据显示，随着F-肌动蛋白的稳定，NCX1的侧向迁移率在修复之前的步骤中显著降低。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-肌动蛋白细胞骨架的相互作用。此外，我们还将确定与MEND相关的钙感受器，并分析参与MEND的PIP-激酶是如何调节的。为了解决NCX1如何结合修复的问题，新的NCX1融合蛋白已经被开发出来，用于在线监测NCX1的内化，NCX1的脉冲追踪，以及改进的NCX1迁移率的分析。NCX1与Dendra2的融合可以将绿色转运蛋白转化为红色转运蛋白，然后跟踪这两种转运蛋白。胞外侧的Halotag融合允许用不同的膜透性和不透性荧光团顺序标记NCX1。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的贩运。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意义，并可能在心脏缺血-再灌注和相关病理中发挥重要作用。公共卫生相关性：心血管疾病是美国主要的死亡原因。许多心肌梗死后即刻死亡是由心律失常引起的，而在心功能不全的长期中，心脏兴奋-收缩偶联功能障碍及伴发的心律失常被认为起着重要作用。心律失常的发病机制复杂，涉及众多分子实体。心肌Na/Ca交换器通过产生内向膜电流在许多情况下发挥触发作用，它从心肌细胞中清除钙，是本研究的主要焦点。此外，该转运体通过在先前的钠负荷下向心肌细胞加载钙，从而导致心肌细胞过度收缩，并通过启动的线粒体信号机制激活细胞死亡程序，从而参与了缺血-再灌注过程中的许多心肌细胞损伤。该实验计划解决了如何将钠/钙交换器从细胞表面膜上移除以及如何调节这一过程，特别是如何在病理环境下使其失活。内吞机制已被发现在多种非心脏细胞类型中被激活，以响应缺血和/或缺氧。我们现在将探索心肌细胞中的相关机制。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可表达Na/Ca交换器的内吞机制及其调控开始，并继续分析心肌细胞中的等效机制。我们的总体目标是更好地理解NCX1的“寿命”和内吞作用。这项工作有望对心脏病理学乃至医学产生根本性的影响。

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.