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: 10343967
• 负责人: Walter F Boron
• 金额: $ 66.46万
• 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/10343967
• 关键词: 3-Dimensional; AQP1 gene; Address; Affect; Age; Aging; Air; Alveolar; Amino Acid Sequence; Animals; 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; Diffuse; 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; Lead; 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; 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）在气体运输中起着至关重要的作用-将O2从肺泡空气运送到全身组织， 而二氧化碳则相反他们的任务对许多具有重大公共卫生意义的疾病至关重要， 包括心力衰竭、肺部疾病（包括COVID-19）、血管疾病和败血症（hy- 灌注不足）。这些气体在体内运动的一个重要组成部分是这些气体的运输。 气体穿过RBC的质膜（PM）。教条是所有的气体都能穿过所有的空气- 膜仅仅通过溶解和扩散通过膜脂。然而，挑战这一教条的是 发现了第一个不透CO2的膜，并首次证明气体（CO2）通过一个 膜蛋白（水通道水通道蛋白1，AQP 1）。在人红细胞中，水通道蛋白-1（AQP 1）和Rh 复合物（包括RhAG）占膜CO2渗透率的90%。O2卸载的初步数据 来自敲除（KO）的RBC的研究表明，这两个通道共同负责约55%的O2 渗透率（PM，O2）。将膜不渗透性抑制剂pCMBS添加到来自双细胞的RBC中， 敲除（dKO）小鼠减少PM，O2约90%。衰老的小鼠似乎逐渐经历PM，O2的减少， 不会发生在dKO中。一个令人惊讶的初步观察是，这些基因中的一个或两个的敲除（KO）， 通道降低最大O2摄取率（V？O2 max）而不减少-事实上，经常增加-运行 性能这笔赠款有两个目的。目的1是确定在何种程度上通道与脂质组成 有助于O2卸载速率（kHbO 2）。一种方法是研究衰老的野生型（WT）与KO小鼠。另一 是检查红细胞基因缺失或充满AE 1或缺失MCT 1的小鼠。第三种方法 是检查疾病模型或遗传背景差异很大的小鼠。在每种情况下，调查人员将 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。三维宏观建模 CAL建模将在数据解释中发挥核心作用。最后，研究人员将使用运动方案， 确定V？O2 max、临界速度、运动经济性和V？O2动力学他们还将检查心脏- 血管和肌肉参数。在目标2中，目标是阐明O2运动的分子机制 通过AQP 1、RhAG和候选O2通道（例如，AE1）。研究人员将使用迭代方法， 其第一步涉及识别优先错义单核苷酸多态性（SNP），以及 和其他在目标1中出现的突变一样。研究人员将使用一种新的中性免疫测定法， 测量卵母细胞的O2摄取，从而评估这些突变体通道。分子动力学和分子 生物物理学将在选择额外的实验室突变进行分析之前完成迭代。拟议 研究将重新组织对血液中氧气运输的思考，并可能导致改善运动的治疗方法， 运动能力下降的患者。