Cardiac Function and PIP2

描述（由申请人提供）：该提案重点关注从表面膜上去除转运蛋白（特别是心脏Na/Ca交换剂（NCX 1））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开始，并进行到成年心肌细胞。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耗尽，随后是Ca瞬变和ATP补充，促进了大规模内吞反应（MEND）。我们进一步确定NCX 1在MEND期间被内化。由于NCX 1在缺血-再灌注损伤和相关心律失常中起主要作用，因此响应于代谢应激从膜去除NCX 1可能具有实质性的临床意义。因此，我们已开始对MEND反应进行详细分析。初步数据表明，MEND是由肌动蛋白膜细胞骨架的重塑驱动的，ATP，Ca和PIP 2依赖性过程都起着重要作用。进一步的初步数据显示，NCX 1横向迁移率在导致MEND的步骤中以及随着F-肌动蛋白的稳定而显著降低。因此，我们将分析代谢状态如何调节肌动蛋白细胞骨架和NCX 1-肌动蛋白细胞骨架相互作用。此外，我们将确定钙传感器潜在的MEND，我们将分析如何参与MEND的PIP激酶的调节。为了解决NCX 1如何与MEND偶联，已经开发了新的NCX 1融合蛋白用于在线监测NCX 1内化、NCX 1的脉冲追踪跟踪和改进的NCX 1迁移率分析。NCX 1与Dendra 2的融合允许将绿色转运蛋白转化为红色转运蛋白，然后追踪两种转运蛋白种类。Halotag融合的细胞外侧允许连续的NCX 1标记不同的膜渗透性和非渗透性的荧光团。从长远来看，这些融合将允许使用量子点和纳米金来研究NCX 1的运输。总的来说，拟议的工作将产生一个强大的内吞过程，是广泛的细胞生物学的兴趣，并可能在心脏缺血再灌注和相关的病理学发挥重要作用的基本见解。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死亡原因。许多心肌梗死后即刻的死亡是由心律失常引起的，而在长期的心功能不全中，心脏兴奋-收缩耦联功能障碍和相关的心律失常被认为起着重要作用。心律失常的发病机制是复杂的，涉及许多分子实体。心脏Na/Ca交换器，从心肌细胞中去除Ca，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通过响应先前的Na负荷而使心肌细胞负载钙来介导缺血-再灌注事件引起的许多心肌细胞损伤，从而引起肌细胞过度收缩并促进细胞死亡程序通过启动的线粒体信号传导机制被激活。该实验程序解决了Na/Ca交换剂如何从细胞表面膜中去除以及如何调节该过程，特别是如何在病理环境中失活。已经发现内吞机制在多种非心脏细胞类型中响应于缺血和/或缺氧而被激活。我们现在将探讨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，从分析内吞机制及其在简单细胞培养细胞中的调节开始，其中Na/Ca交换剂可以表达，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了解NCX 1的“寿命”和内吞作用。这项工作有望对心脏病理学和最终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.