Mitochondrial dynamics in acute lung injury

急性肺损伤中的线粒体动力学

基本信息

批准号：
9173052
负责人：
Jahar Bhattacharya
金额：
$ 43.96万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9173052
关键词：
Actins Acute Lung Injury Adult Respiratory Distress Syndrome Affect Alveolar Alveolar Macrophages Apoptosis Bioenergetics Bone Marrow Buffers Calcineurin Cardiolipins Cells Cytosol DNA Data Development Diffuse Dynamin Electron Transport Endotoxins Extravasation F-Actin Failure Functional disorder Goals Guanosine Triphosphate Phosphohydrolases Hydrogen Peroxide Impairment In Vitro Inflammasome Injury Inner mitochondrial membrane Lead Length Lung Marrow Measurement Mechanical Stress Mesenchymal Microscopy Mitochondria Mitochondrial Matrix Molecular Morbidity - disease rate Mus Pathogenesis Phosphoric Monoester Hydrolases Physiological Process Production Proteins Regulation Research Role Small Interfering RNA Stromal Cells Testing Therapeutic Time Transfection alveolar epithelium base calcium uniporter cellular imaging cytochrome c depolymerization functional loss lung injury lung repair mitochondrial dysfunction mortality mouse model mutant novel overexpression prevent protective effect public health relevance restoration surfactant two-photon uptake

项目摘要

DESCRIPTION (provided by applicant): Significance. Acute lung injury (ALI) is major cause of mortality and morbidity. Our goal is to define mechanisms that might lead to a cure. We will focus on alveolar mitochondrial mechanisms, which are poorly understood. In ALI, mitochondrial dysfunction might underlie alveolar dysfunction, leading to lung injury. The dysfunction might follow loss of mitochondrial Ca2+ buffering in which Ca2+ diffuses from cytosol to mitochondrial matrix across the mitochondrial Ca2+ uniporter (MCU), preventing proinflammatory increase of the cytosolic Ca2+ (cytCa2+), hence protecting against injury. Better understanding of these mechanisms might lead to restoration of mitochondrial Ca2+ buffering by MCU as a therapy for LPS-ALI. Proposal. We propose the novel hypothesis that mitochondrial Ca2+ (mitCa2+) determine surfactant secretion, because the mitochondrial Ca2+ uptake increases ATP and H2O2 production. In LPS-ALI, sustained cytCa2+ increase causes failure of mitochondrial Ca2+ buffering leading to the activation of the Ca2+-dependent phosphatase, calcineurin, which in turn, dephosphorylates and activates the mitochondrial fission protein, DRP1. The resulting mitochondrial fission leads to abrogation of MCU. Calcineurin also promotes actin depolymerization. Together these effects induce alveolar dysfunction as reflected in loss of surfactant secretion. Specific Aims. The specific aims are to determine the role of the MCU in alveolar surfactant secretion (Aim 1) and to determine the extent to which loss of MCU exacerbates lung injury in LPS-induced ALI (Aim 2). We will establish the role of the MCU as a specific Ca2+ channel, as well as its role in surfactant secretion. We will evaluate molecular mechanisms underlying the loss of mitochondrial Ca2+ buffering in LPS-ALI and we will consider bone-marrow-derived mesenchymal stromal cell (BMSC)-based therapeutic approaches aimed at reinstating Ca2+ buffering by mitochondria damaged alveoli as a strategy for lung repair. Approach. We will achieve these aims by live confocal and two-photon microscopy of isolated perfused mouse lungs and by studies in isolated alveolar type 2 (AT2) cells. Our determinations will include: (1) cytCa2+ and mitCa2+ in intact alveoli and in isolated AT2 cells; (2) regulation of surfactant secretion by single cell imaging through determinations of Ca2+, ATP, H2O2 and the F-actin fence; (3) MCU and Ca2+ buffering in endotoxin ALI through lung and AT2 cell expressions of specific mutants and siRNA; (4) Expression of MCU mutants in BMSCs to test the hypothesis that MCU restitution re-instates mitochondrial Ca2+ buffering; (5) MCU overexpression by mitochondrial transfer as a means to protect against LPS-ALI. Preliminary data. We show (1) MCU regulates cytCa2+ and mitCa2+ and surfactant secretion, (2) endotoxin ALI blocks mitochondrial Ca2+ buffering by downregulating MCU through activation of DRP1, and that (3) while alveolar transfection of a mutant MCU worsens mouse survival in endotoxin ALI, expression of a fill length MCU increases mouse survival. These and other preliminary data on our measurement strategies support feasibility of the project. Impact. This project will provide the first systematic understanding of the role of the MCU as a determinant of alveolar function. The extent to which loss of the MCU exacerbates ALI, and the extent to which MCU reinstatement promotes lung repair will be understood for the first time. Outstandingly new understanding of the pathogenesis of ALI will be achieved.

描述（由申请人提供）：重要性。急性肺损伤（ALI）是死亡率和发病率的主要原因。我们的目标是定义可能导致治愈的机制。我们将专注于肺泡线粒体机制，这些机制知之甚少。在阿里，线粒体功能障碍可能是肺泡功能障碍的基础，导致肺损伤。功能障碍可能是在线粒体Ca2+缓冲的丧失之后的，其中Ca2+从胞质醇到线粒体基质跨线粒体Ca2+ Uniporter（MCU）扩散，从而防止了细胞质CA2+（CytCA2+）的促炎增加，从而保护受伤。更好地理解这些机制可能会导致MCU作为LPS-ALI的疗法恢复线粒体Ca2+缓冲。提议。我们提出了一个新的假设，即线粒体Ca2+（Mitca2+）决定了表面活性剂的分泌，因为线粒体Ca2+摄取会增加ATP和H2O2的产生。在LPS-ALI中，持续的CYTCA2+增加会导致线粒体Ca2+缓冲的失败，从而导致Ca2+依赖性磷酸酶钙调蛋白的激活，而钙调蛋白又会使线粒体裂变蛋白DRP1脱磷酸化并激活线粒体的磷酸化并激活。产生的线粒体裂变导致MCU废除。钙调蛋白还促进肌动蛋白解聚。这些作用共同诱导肺泡功能障碍，反映在表面活性剂分泌的丧失中。具体目标。具体目的是确定MCU在肺泡表面活性剂分泌中的作用（AIM 1），并确定MCU加剧LPS诱导的ALI肺损伤的程度（AIM 2）。我们将确定MCU作为特定CA2+通道的作用，及其在表面活性剂分泌中的作用。我们将评估LPS-ALI中线粒体Ca2+缓冲损失的分子机制，我们将考虑基于骨箭头衍生的间质质子层细胞（BMSC）基于骨骼的治疗方法，该方法旨在通过损害Mitochondria损坏的Lung Repart的Ca2+ buffering的CA2+恢复。。方法。我们将通过分离的灌注小鼠肺的活共聚焦和两光子显微镜以及对分离的牙槽2型（AT2）细胞的研究来实现这些目标。我们的确定将包括：（1）完整肺泡中的cytca2+和mitca2+在孤立的AT2细胞中；（2）通过测定Ca2+，ATP，H2O2和F-肌动蛋白围栏来调节单细胞成像对表面活性剂的分泌；（3）内毒素ALI中的MCU和Ca2+缓冲，通过特定突变体和siRNA的肺和AT2细胞表达；（4）MCU突变体在BMSC中的表达，以检验MCU恢复原状线粒体Ca2+缓冲的假设；（5）通过线粒体转移的MCU过表达，作为预防LPS-ALI的一种手段。初步数据。我们显示（1）MCU调节Cytca2+和Mitca2+和表面活性剂的分泌，（2）内毒素ALI通过激活DRP1的激活来下调MCU，而内毒素ALI阻止了线粒体Ca2+缓冲，而该突变体MCU的肺泡转置在内毒素Ali ali ali ali ali Ali a alveall tription ande（3）中均可阻止。填充长度MCU的表达增加了小鼠的存活。这些以及我们的测量策略的其他初步数据支持该项目的可行性。影响。该项目将对MCU作为肺泡功能的决定因素的作用提供第一个系统的理解。 MCU的损失加剧了ALI的多大程度，以及MCU恢复原状的促进肺修复程度将首次理解。将实现对ALI发病机理的详尽的新理解。