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Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice

Abstract

Background

Prolonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function.

Methods

Here we investigated the role of a very short exposure to hyperoxia (95% O2, 5% CO2) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice.

Results

In epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14.

Conclusion

A short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease.

Impact

  • Many studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease.

  • We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells.

  • This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism.

  • We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification.

  • This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction.

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Fig. 1: Four hours of hyperoxia is sufficient to cause dysregulation of oxphos, ETC, and energy production.
Fig. 2: Four hours of hyperoxia causes a persistent reduction in fuel utilization.
Fig. 3: Four hours of hyperoxia does not cause oxidative stress or depolarization of mitochondria.
Fig. 4: Four hours of hyperoxia causes an increase in expression of fusion proteins and mitochondrial mass, which does not persist in recovery.
Fig. 5: Four hours of hyperoxia causes mitochondria to form elongated interconnected networks, which does not persist in recovery.
Fig. 6: Neonatal short-term hyperoxic exposure impairs mouse lung growth.

References

  1. 1.

    Nardiello, C. et al. Standardisation of oxygen exposure in the development of mouse models for bronchopulmonary dysplasia. Dis. Model Mech. 10, 185–196 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Saugstad, O. D. Is oxygen more toxic than currently believed? Pediatrics 108, 1203–1205 (2001).

    CAS  Article  Google Scholar 

  3. 3.

    Vento, M. et al. Preterm resuscitation with low oxygen causes less oxidative stress, inflammation, and chronic lung disease. Pediatrics 124, e439–e449 (2009).

    Article  Google Scholar 

  4. 4.

    Khaw, K. S. & Ngan Kee, W. D. Fetal effects of maternal supplementary oxygen during caesarean section. Curr. Opin. Anaesthesiol. 17, 309–313 (2004).

    Article  Google Scholar 

  5. 5.

    White, L. N. et al. Achievement of saturation targets in preterm infants <32 weeks’ gestational age in the delivery room. Arch. Dis. Child. Fetal Neonatal Ed. 102, F423–f427 (2017).

    Article  Google Scholar 

  6. 6.

    Wang, Y. et al. Pulmonary alveolar type I cell population consists of two distinct subtypes that differ in cell fate. Proc. Natl Acad. Sci. USA 115, 2407 (2018).

    CAS  Article  Google Scholar 

  7. 7.

    Ray, N. B. et al. Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia. Nat. Med. 16, 1120–1127 (2010).

    CAS  Article  Google Scholar 

  8. 8.

    Budinger, G. R. et al. Epithelial cell death is an important contributor to oxidant-mediated acute lung injury. Am. J. Respir. Crit. Care Med. 183, 1043–1054 (2011).

    Article  Google Scholar 

  9. 9.

    Nabhan, A. N., Brownfield, D. G., Harbury, P. B., Krasnow, M. A. & Desai, T. J. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science 359, 1118–1123 (2018).

    CAS  Article  Google Scholar 

  10. 10.

    Mishra, P. & Chan, D. C. Mitochondrial dynamics and inheritance during cell division, development and disease. Nat. Rev. Mol. Cell Biol. 15, 634–646 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Mishra, P., Carelli, V., Manfredi, G. & Chan, DavidC. Proteolytic cleavage of Opa1 stimulates mitochondrial inner membrane fusion and couples fusion to oxidative phosphorylation. Cell Metab. 19, 630–641 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Mizumura, K. et al. Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD. J. Clin. Investig. 124, 3987–4003 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Lottes, R. G., Newton, D. A., Spyropoulos, D. D. & Baatz, J. E. Alveolar type II cells maintain bioenergetic homeostasis in hypoxia through metabolic and molecular adaptation. Am. J. Physiol. Lung Cell Mol. Physiol. 306, L947–L955 (2014).

    CAS  Article  Google Scholar 

  14. 14.

    Das, K. C. Hyperoxia decreases glycolytic capacity, glycolytic reserve and oxidative phosphorylation in MLE-12 cells and inhibits complex I and II function, but not complex IV in isolated mouse lung mitochondria. PLoS ONE 8, e73358 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Yao, H. et al. Fatty acid oxidation protects against hyperoxia-induced endothelial cell apoptosis and lung injury in neonatal mice. Am. J. Respir. Cell Mol. Biol. 60, 667–677 (2019).

    CAS  Article  Google Scholar 

  16. 16.

    Cooney, T. P. & Thurlbeck, W. M. The radial alveolar count method of Emery and Mithal: a reappraisal 1-postnatal lung growth. Thorax 37, 572–579 (1982).

    CAS  Article  Google Scholar 

  17. 17.

    Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76, 4350–4354 (1979).

    CAS  Article  Google Scholar 

  18. 18.

    Tong, W. H. & Rouault, T. A. Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab. 3, 199–210 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    McGrath-Morrow, S. A., Cho, C., Soutiere, S., Mitzner, W. & Tuder, R. The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice. Am. J. Respir. Cell Mol. Biol. 30, 635–640 (2004).

    CAS  Article  Google Scholar 

  20. 20.

    McGrath-Morrow, S. A. & Stahl, J. Apoptosis in neonatal murine lung exposed to hyperoxia. Am. J. Respir. Cell Mol. Biol. 25, 150–155 (2001).

    CAS  Article  Google Scholar 

  21. 21.

    O’Reilly, M. & Thebaud, B. Animal models of bronchopulmonary dysplasia. The term rat models. Am. J. Physiol. Lung Cell. Mol. Physiol. 307, L948–L958 (2014).

    Article  Google Scholar 

  22. 22.

    Farrow, K. N. et al. Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells. Antioxid. Redox Signal. 17, 460–470 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Farrow, K. N. et al. Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn. Respir. Physiol. Neurobiol. 174, 272–281 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    Ratner, V., Sosunov, S. A., Niatsetskaya, Z. V., Utkina-Sosunova, I. V. & Ten, V. S. Mechanical ventilation causes pulmonary mitochondrial dysfunction and delayed alveolarization in neonatal mice. Am. J. Respir. Cell Mol. Biol. 49, 943–950 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Waxman, A. B. & Kolliputi, N. IL-6 protects against hyperoxia-induced mitochondrial damage via Bcl-2-induced Bak interactions with mitofusins. Am. J. Respir. Cell Mol. Biol. 41, 385–396 (2009).

    CAS  Article  Google Scholar 

  26. 26.

    Gardner, P. R., Raineri, I., Epstein, L. B. & White, C. W. Superoxide radical and iron modulate aconitase activity in mammalian cells. J. Biol. Chem. 270, 13399–13405 (1995).

    CAS  Article  Google Scholar 

  27. 27.

    Ma, C. et al. Hyperoxia causes mitochondrial fragmentation in pulmonary endothelial cells by increasing expression of pro-fission proteins. Arterioscler. Thromb. Vasc. Biol. 38, 622–635 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Allen, C. B. & White, C. W. Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP. Am. J. Physiol. 274, L159–L164 (1998).

    CAS  PubMed  Google Scholar 

  29. 29.

    Pantopoulos, K. & Hentze, M. W. Rapid responses to oxidative stress mediated by iron regulatory protein. EMBO J. 14, 2917–2924 (1995).

    CAS  Article  Google Scholar 

  30. 30.

    Gardner, P. R., Nguyen, D. D. & White, C. W. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc. Natl Acad. Sci. USA 91, 12248–12252 (1994).

    CAS  Article  Google Scholar 

  31. 31.

    Morton, R. L., Iklé, D. & White, C. W. Loss of lung mitochondrial aconitase activity due to hyperoxia in bronchopulmonary dysplasia in primates. Am. J. Physiol. Lung Cell. Mol. Physiol. 274, L127–L133 (1998).

    CAS  Article  Google Scholar 

  32. 32.

    Wang, Z. W. et al. Mitochondrial fission mediated cigarette smoke-induced pulmonary endothelial injury. Am. J. Respir. Cell Mol. Biol. https://doi.org/10.1165/rcmb.2020-0008OC (2020).

  33. 33.

    Yao, C.-H. et al. Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation. Elife 8, e41351 (2019).

    Article  Google Scholar 

  34. 34.

    Gomes, L. C., Di Benedetto, G. & Scorrano, L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat. Cell Biol. 13, 589–598 (2011).

    CAS  Article  Google Scholar 

  35. 35.

    Molina, A. J. A. et al. Mitochondrial networking protects beta-cells from nutrient-induced apoptosis. Diabetes 58, 2303–2315 (2009).

    CAS  Article  Google Scholar 

  36. 36.

    Ballweg, K., Mutze, K., Königshoff, M., Eickelberg, O. & Meiners, S. Cigarette smoke extract affects mitochondrial function in alveolar epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 307, L895–L907 (2014).

    CAS  Article  Google Scholar 

  37. 37.

    Meiners, S. & Ballweg, K. Proteostasis in pediatric pulmonary pathology. Mol. Cell. Pediatr. 1, 11 (2014).

    Article  Google Scholar 

  38. 38.

    Attaye, I. et al. The effects of hyperoxia on microvascular endothelial cell proliferation and production of vaso-active substances. Intensive Care Med. Exp. 5, 22 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Ronald Mason for the generous gift of DMPO antibody. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health under award number HL139080 (to D.G.), NIH T32 HL134625 (to D.G.), the Institutional Development Award (IDeA) from the NIGMS of NIH under grant #P20GM103652 (to H.Y.), the Falk Medical Research Trust Catalyst Award (to H.Y.), and the Warren Alpert Foundation at Brown University (to P.A.D.).

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Conception and design: D.G., P.A.D. Acquisition and analysis of data: all authors. Drafting and revising the paper and final approval of the version to be published: D.G., J.F.C., P.A.D.

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Correspondence to Phyllis A. Dennery.

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Garcia, D., Carr, J.F., Chan, F. et al. Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice. Pediatr Res 90, 58–65 (2021). https://doi.org/10.1038/s41390-020-01224-5

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