Data from: Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation

Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.

本研究采用拉曼显微光谱法（Raman microspectroscopy），监测分离的成年大鼠心肌细胞在缺氧及复氧过程中，肌红蛋白（myoglobin）与细胞色素c（cytochrome c）的细胞内氧化还原状态。肌红蛋白具备亚硝酸盐还原酶活性，可在缺氧条件下于细胞内生成一氧化氮（nitric oxide），该过程与线粒体呼吸抑制密切相关。本研究证实，缺氧后的复氧步骤会使氧合肌红蛋白的水平高于常氧条件下的初始观测水平；同时通过拉曼显微光谱法，还可观测到离体心肌细胞内还原型细胞色素c的水平在缺氧及复氧过程中有所升高。细胞对复氧的响应差异显著，取决于此前构建细胞悬液缺氧环境的方法：若使用化学试剂连二亚硫酸钠（sodium dithionite），则可在去除溶解氧的同时还原细胞色素；若采用氮气鼓泡法，则仅能置换溶解氧。上述结果对细胞缺氧实验模拟的评估具有重要参考价值。本研究采用的光谱技术未来将作为分析手段，用于监测细胞预处理或缺血组织再灌注过程中，向心肌细胞供给的不同水平氧气与营养物质所产生的效应。