Skip to main content
SpringerLink
Log in

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

Experimental data, statistical analysis results, protocols used for cell manipulation, and hypoxia incubator electric schemes that supported this study can be found within the paper, in the supplementary materials or can be provided from the corresponding author on reasonable request.

References

  1. Virani, S. S., Alonso, A., Benjamin, E. J., Bittencourt, M. S., Callaway, C. W. & & Carson, A. P. American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. (2020). Heart disease and stroke statistics-2020 update: A report from the American Heart Association.Circulation, 141(9), e139–e596. https://doi.org/10.1161/CIR.0000000000000757.

    Article  PubMed  Google Scholar 

  2. Flora, G. D., & Nayak, M. K. (2019). A brief review of cardiovascular diseases, associated risk factors and current treatment regimes. Current Pharmaceutical Design, 25(38), 4063–4084. https://doi.org/10.2174/1381612825666190925163827.

    Article  CAS  PubMed  Google Scholar 

  3. Fryar, C. D., Chen, T.-C., & Li, X. (2012). Prevalence of uncontrolled risk factors for cardiovascular disease: United States, 1999-2010. NCHS Data Brief, 103, 1–8

    Google Scholar 

  4. Oprea, A. D., Russell, R. R., Russell, K. S., & Abu-Khalaf, M. (2017). Chemotherapy agents with known cardiovascular side effects and their anesthetic implications. Journal of Cardiothoracic and Vascular Anesthesia, 31(6), 2206–2226. https://doi.org/10.1053/j.jvca.2015.06.020.

    Article  CAS  PubMed  Google Scholar 

  5. Bilo, G., Gatterer, H., Torlasco, C., Villafuerte, F. C., & Parati, G. (2022). Editorial: Hypoxia in cardiovascular disease. Frontiers in Cardiovascular Medicine, 9, 990013. https://doi.org/10.3389/fcvm.2022.990013.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Parati, G., Agostoni, P., Basnyat, B., Bilo, G., Brugger, H., Coca, A., & Torlasco, C. (2018). Clinical recommendations for high altitude exposure of individuals with pre-existing cardiovascular conditions: A joint statement by the European Society of Cardiology, the Council on Hypertension of the European Society of Cardiology, the European Society of Hypertension, the International Society of Mountain Medicine, the Italian Society of Hypertension and the Italian Society of Mountain Medicine. European Heart Journal, 39(17), 1546–1554. https://doi.org/10.1093/eurheartj/ehx720.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Parati, G., Bilo, G., Faini, A., Bilo, B., Revera, M., Giuliano, A., & Mancia, G. (2014). Changes in 24 h ambulatory blood pressure and effects of angiotensin II receptor blockade during acute and prolonged high-altitude exposure: A randomized clinical trial. European Heart Journal, 35(44), 3113–3122. https://doi.org/10.1093/eurheartj/ehu275.

    Article  CAS  PubMed  Google Scholar 

  8. Lucero García Rojas, E. Y., Villanueva, C., & Bond, R. A. (2021). Hypoxia inducible factors as central players in the pathogenesis and pathophysiology of cardiovascular diseases. Frontiers in Cardiovascular Medicine, 8, 709509. https://doi.org/10.3389/fcvm.2021.709509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen, X.-Y., Wang, J.-Q., Cheng, S.-J., Wang, Y., Deng, M.-Y., Yu, T., & Zhou, W.-J. (2021). Diazoxide Post-conditioning Activates the HIF-1/HRE Pathway to Induce Myocardial Protection in Hypoxic/Reoxygenated Cardiomyocytes. Frontiers in Cardiovascular Medicine, 8, 711465. https://doi.org/10.3389/fcvm.2021.711465.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bloise, A. C., Dos Santos, J. A., de Brito, I. V., Bassaneze, V., Gomes, L. F., & Alencar, A. M. (2020). Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples. In Vitro Cellular & Developmental Biology. Animal, 56(8), 604–613. https://doi.org/10.1007/s11626-020-00497-8.

    Article  CAS  Google Scholar 

  11. Palomares, C. V. V., Barreto, Y. B., Bexiga, N. M., Toma, S. H., Julival dos Santos, J., Araki, K., … Bloise, A. C. (2023). Metabolic profiling of murine macrophages exposed to silver nanoparticles at dose and time dependencies. Particle & Particle Systems Characterization, 2200191. https://doi.org/10.1002/ppsc.202200191

  12. Vivas, C. V., dos Santos, J. A., Barreto, Y. B., Toma, S. H., dos Santos, J. J., Stephano, M. A., … Bloise, A. C. (2023). Biochemical response of human endothelial and fibroblast cells to silver nanoparticles. BioNanoScience. https://doi.org/10.1007/s12668-023-01091-4

  13. Campos, L. C. G., Ribeiro-Silva, J. C., Menegon, A. S., Barauna, V. G., Miyakawa, A. A., & Krieger, J. E. (2018). Cyclic stretch-induced Crp3 sensitizes vascular smooth muscle cells to apoptosis during vein arterialization remodeling. Clinical Science. https://doi.org/10.1042/CS20171601

  14. Jensen, L., Neri, E., Bassaneze, V., De Almeida Oliveira, N. C., Dariolli, R., Turaça, L. T., & Krieger, J. E. (2018). Integrated molecular, biochemical, and physiological assessment unravels key extraction method mediated influences on rat neonatal cardiomyocytes. Journal of Cellular Physiology, 233(7), 5420–5430. https://doi.org/10.1002/jcp.26380.

    Article  CAS  PubMed  Google Scholar 

  15. Freshney, R. I. (2010). Culture of animal cells: A manual of basic technique and specialized applications. Hoboken, NJ, USA: John Wiley & Sons, Inc. https://doi.org/10.1002/9780470649367.

    Book  Google Scholar 

  16. Martins-Bach, A. B., Bloise, A. C., Vainzof, M. & & Rahnamaye Rabbani, S. (2012). Metabolic profile of dystrophic mdx mouse muscles analyzed with in vitro magnetic resonance spectroscopy (MRS).Magnetic Resonance Imaging, 30(8), 1167–1176. https://doi.org/10.1016/j.mri.2012.04.003.

    Article  CAS  PubMed  Google Scholar 

  17. Bacchi, P. S., Bloise, A. C., Bustos, S. O., Zimmermann, L., Chammas, R., & Rabbani, S. R. (2014). Metabolism under hypoxia in Tm1 murine melanoma cells is affected by the presence of galectin-3, a metabolomics approach. SpringerPlus, 3, 470. https://doi.org/10.1186/2193-1801-3-470.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lindon, J. C., Holmes, E., & Nicholson, J. K. (2006). Metabonomics techniques and applications to pharmaceutical research & development. Pharmaceutical Research, 23(6), 1075–1088. https://doi.org/10.1007/s11095-006-0025-z.

    Article  CAS  PubMed  Google Scholar 

  19. Madsen, R., Lundstedt, T., & Trygg, J. (2010). Chemometrics in metabolomics–a review in human disease diagnosis. Analytica Chimica Acta, 659(1–2), 23–33. https://doi.org/10.1016/j.aca.2009.11.042.

    Article  CAS  PubMed  Google Scholar 

  20. Worley, B., & Powers, R. (2013). Multivariate analysis in metabolomics. Current Metabolomics, 1(1), 92–107. https://doi.org/10.2174/2213235X11301010092.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wu, Z, Li, D, Meng, J. & Wang, H. (2010). Introduction to SIMCA-P and its application. In: V. Esposito Vinzi, W. W. Chin, J. Henseler & H. Wang, (Eds.) Handbook of partial least squares (pp. 757–774). Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-540-32827-8_33

  22. Xia, J., & Wishart, D. S. (2016). Using metaboanalyst 3.0 for comprehensive metabolomics data analysis. Current Protocols in Bioinformatics, 55, 14.10.1–14.10.91. https://doi.org/10.1002/cpbi.11.

    Article  PubMed  Google Scholar 

  23. Bilal Zorić, A. (2021). Applied statistics: basic principles and application. International Journal of Industrial Electronics and Drives, 7(3), 27–33. https://doi.org/10.18775/ijied.1849-7551-7020.2015.73.2003.

    Article  Google Scholar 

  24. Triba, M. N., Starzec, A., Bouchemal, N., Guenin, E., Perret, G. Y., & Le Moyec, L. (2010). Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells. NMR in Biomedicine, 23(9), 1009–1016. https://doi.org/10.1002/nbm.1516.

    Article  CAS  PubMed  Google Scholar 

  25. Tansey, J. T., Baird, T., Cox, M. M., Fox, K. M., Knight, J., Sears, D., & Bell, E. (2013). Foundational concepts and underlying theories for majors in “biochemistry and molecular biology”. Biochemistry and molecular biology education: a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 41(5), 289–296. https://doi.org/10.1002/bmb.20727.

    Article  CAS  PubMed  Google Scholar 

  26. Zhang, J., Wang, Y. T., Miller, J. H., Day, M. M., Munger, J. C., & Brookes, P. S. (2018). Accumulation of succinate in cardiac ischemia primarily occurs via canonical krebs cycle activity. Cell Reports, 23(9), 2617–2628. https://doi.org/10.1016/j.celrep.2018.04.104.

    Article  CAS  PubMed  Google Scholar 

  27. Prochownik, E. V., & Wang, H. (2021). The metabolic fates of pyruvate in normal and neoplastic cells. Cells, 10(4). https://doi.org/10.3390/cells10040762

  28. Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W., & Walker, J. E. (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 107(39), 16823–16827. https://doi.org/10.1073/pnas.1011099107

  29. Aliu, E., Kanungo, S., & Arnold, G. L. (2018). Amino acid disorders. Annals of translational medicine, 6(24), 471 https://doi.org/10.21037/atm.2018.12.12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Prasun, P. (2019). Disorders of pyruvate metabolism and tricarboxylic acid cycle. In Mitochondrial Medicine (pp. 83–95). Elsevier. https://doi.org/10.1016/B978-0-12-817006-9.00015-0

  31. Kanehisa, M., Furumichi, M., Sato, Y., Ishiguro-Watanabe, M., & Tanabe, M. (2021). KEGG: integrating viruses and cellular organisms. Nucleic Acids Research, 49(D1), D545–D551. https://doi.org/10.1093/nar/gkaa970.

    Article  CAS  PubMed  Google Scholar 

  32. Savla, J. J., Levine, B. D., & Sadek, H. A. (2018). The effect of hypoxia on cardiovascular disease: friend or foe. High Altitude Medicine & Biology, 19(2), 124–130. https://doi.org/10.1089/ham.2018.0044.

    Article  Google Scholar 

  33. Su, Z., Liu, Y., & Zhang, H. (2021). Adaptive cardiac metabolism under chronic hypoxia: mechanism and clinical implications. Frontiers in cell and developmental biology, 9, 625524 https://doi.org/10.3389/fcell.2021.625524.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ross-Ascuitto, N. T., Joyce, J. J., Hasan, A. Z. M. A., & Ascuitto, R. J. (2004). Performance of the chronically hypoxic young rabbit heart. Pediatric Cardiology, 25(4), 397–405. https://doi.org/10.1007/s00246-003-0429-z.

    Article  CAS  PubMed  Google Scholar 

  35. Ferrannini, E., Mark, M., & Mayoux, E. (2016). CV Protection in the EMPA-REG OUTCOME Trial: A “Thrifty Substrate” Hypothesis. Diabetes Care, 39(7), 1108–1114. https://doi.org/10.2337/dc16-0330.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge the Sao Paulo Research Foundation - FAPESP (grants 2011/19678-1; 2013/17368-0; 2014/22102-2; 2014/21646-9; 2018/20910-5), and Medical Sciences Graduate Program-CAPES/PROEX for financial support; Instituto de Pesquisas Tecnologicas (IPT) for providing NMR spectrometer and facilities; Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo School of Medicine for providing cells and scientific assistance; and National Institute of Science and Technology Complex Fluids (INCT-FCX) for daily financial aid.

Author information

Authors and Affiliations

  1. Universidade de Sao Paulo, Instituto de Fisica, Rua do Matao 1371, Sao Paulo, Brazil

    Luis Daniel Montañez Condori, Cristofher Victor Vivas, Yan Borges Barreto, Ligia Ferreira Gomes, Adriano Mesquita Alencar & Antonio Carlos Bloise

Authors
  1. Luis Daniel Montañez Condori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristofher Victor Vivas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Borges Barreto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ligia Ferreira Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Adriano Mesquita Alencar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Antonio Carlos Bloise
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.D.M.C. and A.C.B. contributed to design, coding, calibrations, and the first trials with cells in the HI device. A.C.B. contributed performing all the resonance experiments, biological interpretations, and drafted the entire manuscript. L.D.M.C. contributed to the cell experiments, sample preparation, viability assays, resonance data processing, and statistical analysis. Y.B.B. and C.V.V. made suggestions and corrections to the manuscript. L.F.G. contributed to the revision of biological interpretations of the results and its respective discussions. C.V.V. drew Fig. 6. All authors contributed to the revision of the manuscript and agreed to be fully accountable for ensuring the integrity and accuracy of the work and reading and approving the final manuscript.

Corresponding authors

Correspondence to Ligia Ferreira Gomes or Antonio Carlos Bloise.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

Approval was obtained from the institutional review board of the University of Sao Paulo, School of Medicine, Brazil (#340/12).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Condori, L.D.M., Vivas, C.V., Barreto, Y.B. et al. Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes. Cell Biochem Biophys (2024). https://doi.org/10.1007/s12013-024-01249-1

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12013-024-01249-1

Keywords

Search

Navigation