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

红细胞(RBC)在将氧气从肺泡空气输送到全身组织中起着至关重要的作用。 和二氧化碳的排放方向相反。他们的任务是许多与重大公共卫生相关的疾病的核心，在- 包括心力衰竭、肺部疾病(包括新冠肺炎)、血管疾病和脓毒症。 灌流)。这些气体在体内运动的一个重要组成部分是这些物质的运输 穿过红细胞质膜(PM)的气体。以前的教条是所有的气体都会穿过所有的金属- 膜仅通过溶解在膜脂中并通过膜脂扩散。然而，挑战这一教条的是 第一个二氧化碳不透膜的发现，以及气体(二氧化碳)通过 膜蛋白(水通道水通道蛋白1，AQP1)。人红细胞中水通道蛋白-1(AQP1)和Rh 复合体(包括RHAG)占膜CO2通透性的90%。O2-排放的初步数据 来自基因敲除(KO)的红细胞表明，这两个通道共同负责大约55%的O2 透气性(PM，O2)。将膜不透膜的缓蚀剂pCMBS添加到红细胞中，使之成为双核细胞。 基因敲除(DKO)小鼠可使PM、O2降低约90%。衰老的小鼠似乎逐渐经历了PM，O2的减少， 不会出现在dKO中。一个令人惊讶的初步观察是，这两个基因中的一个或两个的敲除(KO) 通道会降低最大氧气摄取率(V？O2 max)，但不会减少--事实上，通常会增加--跑步 性能。这笔赠款有两个目的。目标1是确定通道与脂质成分之间的关系 对O2卸载率(KHbO2)有贡献。一种方法是研究老化的野生型(WT)和KO小鼠。另一个 是检查红细胞AE1基因缺失或充满，或MCT1缺失的小鼠。第三种方法 是为了检查疾病模型或遗传背景差异很大的小鼠。在每一起案件中，调查人员都将 检查血液学、红细胞大小和形状、蛋白质组学、脂类组学和基因组学。三维宏观数学-- CAL建模将在数据解释中发挥核心作用。最后，调查人员将使用锻炼方案来 测定VO2最大值、临界速度、运动经济性和VO2动力学速度。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AQP1、RHAG和候选O2信道(例如，AE1)。调查人员将使用迭代方法， 其中的第一步也涉及识别优先顺序的错义单核苷酸多态(SNPs) 作为目标1中出现的其他突变。研究人员将使用一种新的中性浮力测试来 测量卵母细胞对O2的摄取，从而评估这些突变的通道。分子动力学与分子 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。建议数 研究将重组血液携带氧气的思维，并可能导致改善运动的治疗方法 运动能力减弱的患者。

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.