Hexokinases and Cardioprotection

描述（由申请人提供）：缺血和药理学预处理（PC）是心脏免受缺血/再灌注（I/R）损伤的最有力保护；然而，心脏保护的详细分子机制仍在研究中。人们普遍认为线粒体是心脏保护信号机制的最终效应器，多个研究小组已经提出己糖激酶（HK）调节线粒体通透性转变（MPT）。虽然HK与电压依赖性阴离子通道（VDAC）的关联在10多年前就已被阐明，但关于这种相互作用的生理后果的两个基本问题仍未得到解答，因此，阻碍了心脏保护领域的进展。首先，心肌线粒体中HK的分离是细胞死亡的分子触发因素吗？也就是说，HK与线粒体的分离是否先于所有其他细胞死亡事件[例如，MPT、Δφ丢失和细胞色素C （cyto C）释放]？其次，稳定HK-VDAC相互作用并赋予其独特心脏保护特性的未知分子是什么？由于缺乏以下技术，这些问题无法得到明确的答案：(i)与MPT、Δφ损失和活心肌细胞中细胞释放相关的HK空间分布的时间分析，以及（ii）定量HK- vdac复合物的分子成分并破译其化学计量学。鉴于这些挑战，我们的项目量身定制了最先进的活细胞成像和定量蛋白质组学创新，以全面描述hk诱导的心脏保护在生物时间尺度上的动态。我们假设HK是心脏保护的核心调节因子，在多种损伤和预处理模型中都很常见。我们将在活肌细胞中使用实时成像来定义损伤期间分子事件的时间概况（目的1）；我们将使用广泛的生化和遗传工具箱来描述HK与线粒体相互作用的分子范式及其介导心脏保护的生理后果（目的2）；我们将定量定义HK与VDAC相互作用的蛋白质组动力学和分子化学计量学；表征HK- VDAC相互作用组组装中异构体选择性变化；并确定在心脏保护过程中稳定HK与VDAC相互作用所必需的候选蛋白（目的3）；我们将使用HK构建体或其他在Aims 1-3中确定的候选分子的心脏基因递送，来测试一种体内基因治疗策略，以保护成年大鼠免受I/R损伤（Aim 4）。拟议的调查承诺概念、技术和方法上的创新。我们将利用与加州大学洛杉矶分校NHLBI蛋白质组学中心的密切合作，将在细胞/动物模型中获得的知识立即有效地转化为临床研究。这些研究的成功无疑将推动心脏保护领域向前发展。

DESCRIPTION (provided by applicant): Ischemic and pharmacologic preconditioning (PC) constitute the most powerful protection of the heart from ischemia/reperfusion (I/R) injury; however, the detailed molecular mechanisms underlying cardioprotection are still being defined. There is a general consensus that mitochondria are the final effectors of cardioprotective signaling regimes, and hexokinase (HK) has been suggested by multiple groups to regulate the mitochondrial permeability transition (MPT). Though the association of HK with voltage-dependent anion channels (VDAC) was elucidated over 10 years ago, two fundamental questions regarding the physiologic consequences of this interaction have remained unanswered, and consequently, have stalled the progression of the cardioprotection field. First, is the dissociation of HK from cardiac mitochondria a molecular trigger of cell death? That is, does HK dissociation from mitochondria precede all other cell death events [e.g., MPT, Δφ loss, and cytochrome C (cyto C) release]? Second, what are the unknown molecular players that stabilize the HK-VDAC interaction and impart its unique cardioprotective properties? Unequivocal answers to these questions have been unattainable due to the lack of technologies for (i) temporal profiling of the spatial distribution of HK in relation to MPT, Δφ loss, and cyto release in live cardiomyocytes, and (ii) quantifying the molecular constituents of the HK-VDAC complex and deciphering their stoichiometry. In view of these challenges, our program has tailored state-of-the-art live-cell imaging and quantitative proteomic innovations to comprehensively delineate the dynamics of HK-induced cardioprotection on a biological timescale. We hypothesize that HK is a core regulator of cardioprotection, common to multiple models of injury and preconditioning. We will employ real-time imaging in live myocytes to define the temporal profile of the molecular events during injury (Aim 1); we will use an extensive biochemical and genetic toolbox to delineate the molecular paradigm of HK interaction with mitochondria as well as its physiological consequences mediating cardioprotection (Aim 2); we will quantitatively define the proteome dynamics and molecular stoichiometry of HK interaction with VDAC; characterize isoform-selective changes in assembly of the HK- VDAC interactome; and identify candidate proteins essential to stabilize the HK interaction with VDAC during cardioprotection (Aim 3); and we will use cardiac gene delivery of HK constructs or other molecular candidates identified in Aims 1-3, to test an in vivo gene therapy strategy to protect adult rats from I/R injury (Aim 4). The proposed investigations promise conceptual, technological, and methodological innovations. We will leverage close collaborations with the UCLA NHLBI Proteomics Center for immediate and efficient translation of knowledge obtained in cell/animal models to clinical studies. The success of the proposed investigations will undoubtedly propel the field of cardioprotection forward.