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.

项目摘要 这个项目的目的是为NIH R15的毕业生提供研究机会 德州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。在急性缺血-再灌注期间 (I/R)，活性氧(ROS)是在再灌流阶段产生的，导致灾难性的 心脏受损。因此，给予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。这种名为锌的营养物质具有多方面的抗氧化作用，一直以来 对心脏I/R损伤有保护作用。但锌的安全配方、剂量和给药方式 没有得到证实。以前，锌剂量会产生毒性效应(例如，生长停滞、细胞死亡)，而 锌作为离子载体通过质膜的输送方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 心脏抗I/R损伤。为了推进这样的人体临床试验，我们首先必须解决两个基本问题 问：除了锌转运体，还有什么实体可以调节肌肉细胞中锌的内流？CAN的增强功能 这种内在实体在不使用易产生副作用的锌离子载体的情况下，保护心肌细胞免受I/R的影响 受伤了吗？为了解决这两个问题，我们进行了基于CRISPR/CAS9的无偏见基因组规模 筛选以搜索这样的新实体。出乎意料的是，在最有效的候选名单上的一个基因是 SLC5A3，也称为SMIT1，是一种钠肌醇转运蛋白。SLC5A3从未与锌有关 新陈代谢。我们的初步数据显示，SLC5A3基因的敲除可以抵消锌对HL-1细胞的保护作用。 1缺氧/复氧(H/R)损伤心肌细胞。有趣的是，低氧后适应(PostC) 增强HL-1细胞在H/R损伤中的存活，并在SLC5A3-基因敲除中的有益作用减弱 细胞。此外，有锌灌流的PostC保护心脏(在减少细胞凋亡方面，维持左 超声心动图研究中的左心室射血分数)对I/R所致心功能障碍小鼠的影响。这个 SLC5A3钝化锌对培养心肌细胞和全心的保护作用 抑制药根霉苷。基于这些数据，我们假设SLC5A3介导锌内流。 心肌细胞，它可以被增强以保护心脏免受I/R损伤。两个具体目标是 用来验证这一假设的公式：1)确定SLC5A3介导锌的分子机制 心肌细胞内流；2)通过靶向SLC5A3介导的锌内流进行概念验证研究 保护心脏免受I/R损伤。这一创新包括：(I)第一项研究表明， SLC5A3在调节锌稳态中的作用；(Ii)SLC5A3治疗潜力的鉴定- 介导锌内流对心脏I/R损伤的影响。作为骨骼肌肉研究中心的六名创始成员之一， UTA，PI的实验室需要专业知识和优秀的资源来培训心血管领域的博士生 生理学。