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）在气体传输中起着至关重要的作用 - 将O2从肺泡空气到全身组织， 和二氧化碳朝相反的方向。他们的任务是许多主要公共卫生相关性疾病的核心 包括心力衰竭，肺部疾病（包括COVID-19），血管疾病和败血症（Hy- PoperFusion）。这些气体在体内运动的重要组成部分是这些气体的运输 RBCS的质膜（PM）的气体。教条是所有气体都跨越了所有的纪念 仅通过溶解并通过膜脂质扩散而进行麸皮。但是，挑战这种教条是 发现第一个CO2不可渗透机制，以及第一个证据表明气体（CO2）通过 膜蛋白（水通道水通道1，AQP1）。在人类RBC中，Aquaporin-1（AQP1）和RH 复合物（包括RHAG）占膜CO2渗透性的90％。 O2-Rothloading的初步数据 来自RBC的淘汰（KO）表明，这两个通道共同负责约55％的O2 渗透率（PM，O2）。从双重添加膜 - 覆盖剂抑制剂PCMB从双重的RBC中 敲除（DKO）小鼠将PM降低，O2降低了〜90％。衰老的小鼠似乎逐渐减少PM，O2 在DKO中不发生。令人惊讶的初步观察是，其中一个或两个的淘汰（KO） 频道降低了最大O2摄取率（V？o2 max）而不减少（实际上，通常增加） 表现。该赠款有两个目标。目标1是确定通道与脂质组成的程度 有助于O2卸载率（KHBO2）。一种方法是研究老化野生型（WT）与KO小鼠。其他 是为了检查在AE1中耗尽或替换的RBC的小鼠，或在MCT1中耗尽的小鼠。第三种方法 是检查疾病模型的小鼠或广泛不同的遗传背景。在每种情况下，调查人员都会 检查血液学，RBC的大小和形状，蛋白质组学，脂肪组学和基因组学。 3D宏观数学 CAL建模将在数据解释中发挥核心作用。最后，调查人员将使用练习方案来 确定v？o2最大，临界速度，运动经济和V？o2动力学的速度。他们还将检查心脏 - 血管和肌肉参数。在AIM 2中，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）。调查人员将使用迭代方法， 第一步涉及确定优先级错义单核苷酸多态性（SNP） 正如AIM 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法进行 测量O2摄取卵母细胞，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其他实验室突变进行分析之前完成迭代。提议 研究将重组对血液的O2运输进行重组，并可能导致疗法改善运动 运动能力降低的患者。