Abstract
Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.
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Acknowledgments
The authors sincerely thank Professor Imamura (Kyoto University) for giving us the pcDNA-mitGo-ATeam2.
Funding
This work was supported by the National Natural Science Foundation of China grant No. 81571844.
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Every author made a valuable contribution to the preparation of this manuscript. ZBY and YYW conceived and designed the study. RZZ, XBW, SJ, and NYR performed the experiments. RZZ and XBW analyzed the data. RZZ and BJ produced the figures. RZZ wrote the manuscript. ZBY and YYW critically revised the manuscript. All authors read and approved the final version of the manuscript.
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All animal experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Ethics Committee for the animal care and use of Fourth Military Medical University.
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Zhao, RZ., Wang, XB., Jiang, S. et al. Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia. Pflugers Arch - Eur J Physiol 472, 1619–1630 (2020). https://doi.org/10.1007/s00424-020-02463-5
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DOI: https://doi.org/10.1007/s00424-020-02463-5