Using calorespirometry to assemble a complete picture of acute hypoxic metabolic depression in fishes

人类活动正在使世界上的水生环境越来越缺氧（低氧）。这威胁到动物的生存，因为氧气需要提供足够的细胞能量（ATP）来满足动物的高代谢需求。然而，许多动物，尤其是鱼类，已经适应了已经经历过缺氧的环境，我们可以看看这些物种的耐受策略，以了解什么可能促进或阻碍动物适应不断变化的世界的能力。一种非常有效的耐缺氧策略是代谢抑制（MD），即代谢率的调节降低，从而使ATP的供应和使用低于静息水平。并不是所有的鱼类都能诱发缺氧MD，但那些能诱发的鱼类往往对缺氧具有高度的耐受性。一些导致缺氧MD的下调的细胞atp供应和使用过程已被单独研究；然而，诱导它们下调的上游信号以及它们的调制通过时间和O2水平（PO2）联系的方式仍然未知。这项发现基金将在我之前的工作基础上进行重大研究，以验证血红蛋白（Hb）介导的组织氧供应减少是鱼类缺氧MD的初始信号这一总体假设。具体来说，当水的PO2变得太低，使Hb无法结合并向组织输送足够数量的O2时，这些组织就会诱发MD，包括好氧和厌氧atp供应途径的停止以及atp使用过程的下调。通过4个假设驱动的项目，该工作将首先通过热肺量测定和Hb- o2亲和力的操纵实验来研究Hb在缺氧MD诱导中的作用。其次，它将确定细胞PO2如何影响高分辨率呼吸测量的有氧ATP供应途径。第三，它将研究如何阻止atp供应和使用过程作为时间和PO2使用培养组织细胞的函数联系起来。第四，它将在细胞水平上研究为什么一些物种不能诱导缺氧MD。这项工作的预期结果是更好地理解缺氧MD诱导的信号，其细胞机制是如何顺序联系的，以及使用和不使用缺氧MD的物种之间的细胞机制差异。目前，这些都是未知的。该计划将培养5名研究生（4名硕士，1名博士）和6名本科生，为他们提供广泛应用的研究和分析技能。此外，它将提高我们对动物对水生缺氧反应的理解，这种现象正在全球范围内（包括加拿大）增加，并威胁着无数的鱼类种群。最近的研究表明，低氧MD可能是一种耐受低氧的快速进化策略。了解其近因和最终机制有助于建立预测模型，以确定潜在的脆弱物种，并采取保护措施保护它们。

Human practices are rendering the world's aquatic environments increasingly hypoxic (low O2). This threatens the resident animals because O2 is required to supply sufficient quantities of cellular energy (ATP) to meet the animals' high metabolic demands. However, many animals, especially fishes, are adapted to environments that already experience hypoxia, and we can look to the tolerance strategies of these species to understand what might facilitate or hinder an animal's ability to adapt to a changing world. A highly effective strategy for tolerating hypoxia is metabolic depression (MD), a regulated reduction of metabolic rate - and thus the supply and use of ATP - below resting levels. Not all fishes can induce hypoxic MD, but those that can tend to be highly tolerant of hypoxia. Some of the downregulated cellular ATP-supply and -use processes that contribute to hypoxic MD have been investigated in isolation; however, the upstream signal that induces their downregulation and the manner by which their modulation is linked via time and O2 level (PO2) remain unknown. This Discovery Grant will build significantly upon my previous work to test the overarching hypothesis that diminished hemoglobin (Hb)-mediated O2 supply to the tissues serves as the initial signal for hypoxic MD in fish. Specifically, when water PO2 becomes too low for Hb to bind and deliver sufficient quantities of O2 to the tissues, those tissues induce MD, including the arrest of aerobic and anaerobic ATP-supply pathways and the downregulation of ATP-use processes. Through 4 hypothesis-driven projects, the work will first investigate the role of Hb in hypoxic MD induction through experiments involving calorespirometry and the manipulation of Hb-O2 affinity. Second, it will determine how cellular PO2 affects aerobic ATP supply pathways using high resolution respirometry. Third, it will investigate how the arrest of ATP-supply and -use processes are linked as a function of time and PO2 using cultured tissue cells. And fourth, it will investigate at the cellular level why some species are unable to induce hypoxic MD. The expected outcome of this work is a better understanding of what signals hypoxic MD induction, how its cellular mechanisms are linked sequentially, and what cellular mechanisms differ between species that do and do not use hypoxic MD. Currently, these are unknowns. The proposed work will train 5 graduate (4 MSc, 1 PhD) and 6 undergraduate students, providing them with widely applicable research and analytical skills. Additionally, it will improve our understanding of animal responses to aquatic hypoxia, a phenomenon that is increasing globally (incl. Canada) and threatens innumerable fish populations. Recent work indicates that hypoxic MD may be a rapidly evolved strategy for tolerating hypoxia. Understanding its proximate and ultimate mechanisms may benefit predictive models for identifying potentially vulnerable species and conservation efforts for protecting them.