Mechanisms of oxygen off-loading from red blood cells in murine models of human disease

人类疾病小鼠模型中红细胞的氧卸载机制

• 批准号: 10548180
• 负责人: Walter F Boron
• 金额: $ 65.85万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-08 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10548180
• 关键词: 3-Dimensional; AQP1 gene; Address; Affect; Age; Aging; Air; Alveolar; Amino Acid Sequence; Animal Experiments; Biological Assay; Biology; Biophysics; Blood; Blood capillaries; COVID-19; Carbon Dioxide; Cardiovascular system; Cell Membrane Proteins; Cell Shape; Cell Size; Cell membrane; Cells; Collaborations; Complex; Consumption; Data; Data Analyses; Diffusion; Disease; Disease model; Dissociation; Erythrocytes; Exercise; Exhibits; Gases; Genetic; Genetic Polymorphism; Genomics; Genotype; Geometry; Goals; Grant; Health; Heart failure; Hematology; Hemoglobin; Human; Impairment; Indirect Calorimetry; Interdisciplinary Study; Ion Channel; Kinetics; Knock-out; Knockout Mice; Laboratories; Libraries; Life; Lipids; Liquid substance; Lung; Lung diseases; Measures; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Molecular; Mouse Strains; Movement; Mus; Muscle; Mutation; Mutation Analysis; Nature; Oocytes; Oxygen; Oxyhemoglobin; Pathway interactions; Patients; Performance; Permeability; Plasma Cells; Play; Process; Proteins; Proteomics; Protocols documentation; Reaction; Research; Research Personnel; Resistance; Rhesus; Role; Running; Sepsis; Single Nucleotide Polymorphism; Sodium Dithionite; Speed; Structural Biologist; Systems Biology; Testing; Thinking; Thinness; Tissues; Vascular Diseases; Work; cell dimension; cell type; exercise capacity; experimental study; extracellular; falls; follow-up; human disease; human model; hypoperfusion; improved; inhibitor; insertion/deletion mutation; insight; lipidomics; mathematical model; molecular dynamics; mouse model; mutant; novel; premature; public health relevance; tool; treadmill; uptake; water channel

Red blood cells (RBCs) play a vital role in gas transport—carrying O2 from the alveolar air to systemic tissues, and CO2 in the opposite direction. Their task is central to many diseases of major public-health relevance, in- cluding including heart failure, pulmonary disease (including COVID-19), vascular disease, and sepsis (hy- poperfusion). An important component in the movement of these gases within the body is the transport of these gases across of the plasma membrane (PM) of the RBCs. The dogma had been that all gases cross all mem- branes merely by dissolving in and diffusing through membrane lipids. However, challenging this dogma was the discovery of the first CO2 impermeable membranes, and the first evidence that a gas (CO2) moves through a membrane protein (the water channel aquaporin 1, AQP1). In human RBCs, aquaporin-1 (AQP1) and the Rh complex (including RhAG) account for 90% of membrane CO2 permeability. Preliminary data on O2-offloading from RBCs from knockout (KO) suggests that these two channels, together, are responsible for ~55% of O2 permeability (PM,O2). The addition of the membrane-impermeant inhibitor pCMBS to RBCs from the double- knockout (dKO) mouse reduces PM,O2 by ~90%. Aging mice appear to gradually undergo a decrease in PM,O2 that does not occur in dKOs. A surprising preliminary observation is that the knockout (KO) of one or both of these channels reduces maximal O2 uptake rate (V?O2 max) without decreasing—and, in fact, often increasing—running performance. This grant has two aims. Aim 1 is to determine the extent to which channels vs. lipid composition contribute to the rate of O2 offloading (kHbO2). One approach is to study aging wild-type (WT) vs. KO mice. Another is to examine mice with RBCs genetically depleted or replete in AE1, or depleted in MCT1. The third approach is to examine mice of disease models or widely different genetic background. In each case, the investigators will examine hematology, RBC size and shape, proteomics, lipidomics, and genomics. 3D macroscopic mathemati- cal modeling will play a central role in data interpretation. Finally, the investigators will use exercise protocols to to determine V?O2 max, critical speed, exercise economy, and speed of V?O2 kinetics. They will also examine cardio- vascular and muscle parameters. In Aim 2, the goal is to elucidate the molecular mechanism of O2 movement through AQP1, RhAG, and candidate O2 channels (e.g., AE1). The investigators will use an iterative approach, the first step of which involves identifying prioritizing missense single nucleotide polymorphisms (SNPs), as well as other mutations that come forward in Aim 1. The investigators will use a novel neutral buoyance assay to measure O2 uptake into oocytes and thereby assess these mutants channels. Molecular dynamics and molecular biophysics will complete the iteration before choosing additional laboratory mutation for analysis. The proposed research will reorganize thinking about O2 carriage by blood and could lead to therapies to improve exercise in patients with diminished exercise capacity.

红细胞 (RBC) 在气体运输中发挥着至关重要的作用——将氧气从肺泡空气输送到全身组织， 他们的任务是许多与公共卫生相关的疾病的核心，例如： 包括心力衰竭、肺部疾病（包括 COVID-19）、血管疾病和败血症（hy- poperfusion）。这些气体在体内移动的一个重要组成部分是这些气体的运输。 气体穿过红细胞的质膜（PM） 一直以来，人们的教条是所有气体都穿过所有的内存。 膜仅仅通过溶解并扩散通过膜脂然而，挑战这一教条是。 发现第一个 CO2 不渗透膜，以及第一个证据表明气体 (CO2) 穿过 膜蛋白（水通道水通道蛋白 1，AQP1） 在人类红细胞中，水通道蛋白-1 (AQP1) 和 Rh。 复合物（包括 RhAG）占膜 CO2 渗透性的 90%。 O2 卸载的初步数据。 来自基因敲除 (KO) 的红细胞表明这两个通道共同负责约 55% 的 O2 向红细胞中添加非膜渗透性抑制剂 pCMBS（PM、O2）。 基因敲除 (dKO) 可使小鼠 PM,O2 减少约 90%。 衰老小鼠的 PM,O2 似乎逐渐减少。 一个令人惊讶的初步观察结果是，其中一个或两个都被敲除（KO）。 通道会降低最大 O2 摄取率 (V?O2 max)，但不会减少（事实上，经常增加）跑步 此项资助有两个目的：确定通道与脂质成分的程度。 一种方法是研究衰老的野生型 (WT) 小鼠与 KO 小鼠。 第三种方法是检查红细胞中 AE1 基因缺失或充足，或 MCT1 缺失的小鼠。 在每种情况下，研究人员都会检查疾病模型或广泛不同遗传背景的小鼠。 检查血液学、红细胞大小和形状、蛋白质组学、脂质组学和基因组学。 最后，研究人员将使用运动方案来解释数据。 以确定 V?O2 最大、临界速度、运动经济性和 V?O2 动力学速度。他们还将检查有氧运动。 目标 2 的目标是阐明 O2 运动的分子机制。 通过 AQP1、RhAG 和候选 O2 通道（例如 AE1），研究人员将使用迭代方法， 第一步还包括确定错义单核苷酸多态性 (SNP) 的优先级 正如目标 1 中出现的其他突变一样。研究人员将使用一种新颖的中性浮力测定来 测量卵母细胞的 O2 摄取，从而评估这些突变体通道。 生物物理学将在选择其他实验室突变进行分析之前完成迭代。 研究将重新思考血液中氧气的运输，并可能带来改善运动的疗法 运动能力下降的患者。