Zinc Protection Against Ischemia-Reperfusion Injury in Heart

锌可预防心脏缺血再灌注损伤

• 批准号: 10652915
• 负责人: ZUI PAN
• 金额: $ 44.3万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652915
• 关键词: Acute; Address; Antioxidants; Apoptosis; Area; Award; Biochemistry; CRISPR screen; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Physiology; Cell Death; Cell Survival; Cell membrane; Cells; Clinical Trials; Confocal Microscopy; Data; Doctor of Philosophy; EFRAC; Echocardiography; Formulation; Genes; Genetic; Genetic Transcription; Goals; Growth; Health; Heart; Heart failure; Hispanic; Homeostasis; Human; Hypoxia; Individual; Infarction; Inflammation; Injury; Ionophores; Ischemia; Laboratories; Left ventricular structure; Link; Mammalian Cell; Mediating; Membrane; Metabolism; Metallothionein; Methods; Minority; Molecular; Monitor; Mus; Muscle; Muscle Cells; Myocardial Ischemia; Myocardial dysfunction; Natural regeneration; Necrosis; Nutrient; Osmolar Concentration; Oxidative Stress; Perfusion; Phase; Phlorhizin; Proteins; Quantitative Reverse Transcriptase PCR; Reactive Oxygen Species; Regimen; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporting; Research; Resources; Risk; Role; Sarcolemma; Signal Pathway; Sodium; Stroke; Students; Techniques; Testing; Texas; Therapeutic; Tissues; Toxic effect; Training; United States National Institutes of Health; Validation; Western Blotting; Zinc; Zinc supplementation; bone; candidate validation; cardioprotection; career; cell growth regulation; college; conditioning; doctoral student; dosage; genetic manipulation; genome-wide; graduate student; heart damage; in vivo; inhibitor; innovation; knock-down; member; myoinositol; overexpression; pharmacologic; post stroke; prevent; programs; protective effect; reverse genetics; screening; side effect; temporal measurement; undergraduate student; whole genome; zinc-binding protein

Project Summary This project is aimed to provide NIH R15 REAP research opportunities for our underrepresented graduate students at UT Arlington, a Hispanic-Serving Institute in North Texas. During acute ischemia-reperfusion (I/R), reactive oxygen species (ROS) is generated at the reperfusion phase and results in catastrophic damage to heart. Thus, the administration of antioxidant agents to prevent or ameliorate ROS detrimental effects is an active research area. The nutrient, zinc, has multifaceted antioxidant effects and has been shown to protect against I/R injury in heart. But a safe formulation, dosage, and delivery mode of zinc have not been established. Previously, zinc dosages yielded toxic effects (e.g., halted growth, cell death), and the delivery mode of zinc as an ionophore across plasma membrane produced many side-effects (e.g., membrane damage, cell death, etc.). Our long-term goal is to establish a therapeutic zinc regimen to protect heart against I/R injury. To progress to such human clinical trials, we must initially address two fundamental questions: What entity besides zinc transporters regulates zinc influx in muscle cells? Can enhancement of such intrinsic entity, without using side effect-prone zinc ionophore, protect the cardiomyocytes from I/R injury? Toward addressing the two questions, we performed unbiased genome-scale CRISPR/Cas9-based screening to search for such new entity. Unexpectedly, a gene on the top validated candidate list was SLC5A3, also called SMIT1, a sodium myo-inositol transporter. SLC5A3 has never been linked to zinc metabolism. Our preliminary data showed that knockdown of SLC5A3 negated zinc-induced protection in HL- 1 cardiomyocytes in hypoxia /reoxygenation (H/R) injury. Interestingly, hypoxic post-conditioning (PostC) enhanced HL-1 cell survival upon H/R injury, and its beneficial effect was diminished in SLC5A3-knockdown cells. Moreover, PostC with zinc perfusion protect the heart (in terms of reduced apoptosis, maintaining left ventricle ejection fraction in echocardiography study) from I/R induced cardiac dysfunction in mice. The protective function of zinc in both cultured cardiomyocytes and whole heart was blunted by a SLC5A3 inhibitor phloridzin. Based on these data, we hypothesize that SLC5A3 mediates zinc influx in cardiomyocytes, which can be enhanced to protect heart from I/R injury. Two specific aims are formulated to test this hypothesis: 1) to define the molecular mechanisms by which SLC5A3 mediates zinc influx in cardiomyocytes; 2) to conduct proof-of-concept study by targeting SLC5A3-mediated zinc influx to protect heart from I/R injury. The innovation includes: (i) the first study to show an unrecognized role of SLC5A3 in regulating zinc homeostasis; (ii) the identification of the therapeutic potential of SLC5A3- mediated zinc influx for heart I/R injury. As one of six founding members in Bone-Muscle Research Center at UTA, the PI’s laboratory has required expertise, excellent resources to train PhD students in cardiovascular physiology.

项目概要 该项目旨在为我们代表性不足的毕业生提供 NIH R15 REAP 研究机会 德克萨斯大学阿灵顿分校的学生，这是一所位于德克萨斯州北部的拉美裔服务学院。急性缺血再灌注期间 （I/R），活性氧（ROS）在再灌注阶段产生并导致灾难性的后果 对心脏的伤害。因此，施用抗氧化剂来预防或改善 ROS 有害 效应是一个活跃的研究领域。这种营养素锌具有多方面的抗氧化作用，并且已被 显示可防止心脏缺血再灌注损伤。但锌的安全配方、剂量和给药方式已经 尚未成立。以前，锌剂量会产生毒性作用（例如生长停止、细胞死亡），并且 锌作为离子载体穿过质膜的递送模式产生了许多副作用（例如， 膜损伤、细胞死亡等）。我们的长期目标是建立一种治疗性锌疗法来保护 心脏对抗 I/R 损伤。为了进行此类人体临床试验，我们必须首先解决两个基本问题 问题：除了锌转运蛋白之外，还有什么实体调节肌肉细胞中的锌流入？可以增强 这种内在实体，无需使用容易产生副作用的锌离子载体，即可保护心肌细胞免受 I/R 的影响 受伤？为了解决这两个问题，我们进行了基于 CRISPR/Cas9 的无偏基因组规模的研究 筛选以寻找这样的新实体。出乎意料的是，排名靠前的已验证候选基因是 SLC5A3，也称为 SMIT1，一种钠肌醇转运蛋白。 SLC5A3从未与锌联系在一起 代谢。我们的初步数据表明，敲除 SLC5A3 可以消除 HL- 中锌诱导的保护作用。 1 心肌细胞缺氧/复氧（H/R）损伤。有趣的是，低氧后处理（PostC） H/R 损伤后 HL-1 细胞存活率提高，但 SLC5A3 敲低后其有益作用减弱 细胞。此外，具有锌灌注的 PostC 可保护心脏（在减少细胞凋亡、维持左心 超声心动图研究中的心室射血分数）来自缺血再灌注引起的小鼠心功能障碍。这 SLC5A3 削弱了锌在培养的心肌细胞和整个心脏中的保护功能 抑制剂根皮苷。基于这些数据，我们假设 SLC5A3 介导锌流入 心肌细胞，可以增强其保护心脏免受缺血再灌注损伤。两个具体目标是 制定来检验这一假设：1) 定义 SLC5A3 介导锌的分子机制 流入心肌细胞； 2）通过针对SLC5A3介导的锌流入来进行概念验证研究 保护心脏免受缺血再灌注损伤。创新包括：(i) 第一项研究显示了未被认识到的作用 SLC5A3 调节锌稳态； (ii) SLC5A3-治疗潜力的鉴定 介导的锌流入导致心脏 I/R 损伤。作为骨肌肉研究中心的六位创始成员之一 UTA，PI 的实验室拥有培训心血管博士生所需的专业知识和优质资源 生理。