Cardiovascular Toxicology

, Volume 18, Issue 4, pp 346–355 | Cite as

Necrostatin-1 Protects Against Paraquat-Induced Cardiac Contractile Dysfunction via RIP1-RIP3-MLKL-Dependent Necroptosis Pathway

  • Liping ZhangEmail author
  • Qiming Feng
  • Teng Wang


Paraquat is a highly toxic prooxidant that triggers oxidative stress and multi-organ failure including that of the heart. To date, effective treatment of paraquat toxicity is still not established. Necroptosis, a newly discovered form of programmed cell death, was recently shown to be strongly associated with cardiovascular disease. Receptor interaction proteins 1 (RIP1), receptor interaction proteins 3 (RIP3), and mixed lineage kinase domain like (MLKL) are key proteins in the necroptosis pathway. Necrostatin-1 (Nec-1) is a specific inhibitor of necroptosis which acts by blocking the interaction between RIP1 and RIP3. In the present study, we studied the effect of Nec-1 on paraquat-induced cardiac contractile dysfunction and reactive oxygen species (ROS) production in the heart tissues using a mouse model. Our results revealed impaired contractile function, deranged intracellular Ca2+ handling and echocardiographic abnormalities in mice challenged with paraquat. We further found enhanced expressions of RIP1, RIP3, and MLKL along with overproduction of ROS in mice heart tissues. Nec-1 pre-treatment prevented cardiac contractile dysfunction in paraquat-challenged mice. Furthermore, Nec-1 reduced RIP1–RIP3 interaction, down-regulated the RIP1–RIP3–MLKL signal pathway, and dramatically inhibited the production of ROS. Collectively, these findings suggest that Nec-1 alleviated paraquat-induced myocardial contractile dysfunction through inhibition of necroptosis, an effect which was likely mediated via the RIP1–RIP3–MLKL signaling cascade. Further, ROS appeared to play an important role in this process. Thus, this process may represent a novel therapeutic strategy for the treatment of paraquat-induced cardiac contractile dysfunction.


Paraquat Acute heart injury Necroptosis ROS 


Authors Contribution

LPZ, QMF, and TW performed parts of the experiments; LPZ and TW wrote article and carried out data analysis; LPZ, QMF carried out the study design, supervised and reviewed manuscript. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Chan, Y. C., Chang, S. C., Hsuan, S. L., Chien, M. S., Lee, W. C., Kang, J. J., et al. (2007). Cardiovascular effects of herbicides and formulated adjuvants on isolated rat aorta and heart. Toxicology in Vitro, 21, 595–603.CrossRefPubMedGoogle Scholar
  2. 2.
    Ge, W., Zhang, Y., Han, X., & Ren, J. (2010). Cardiac-specific overexpression of catalase attenuates paraquat-induced myocardial geometric and contractile alteration: Role of ER stress. Free Radical Biology and Medicine, 49, 2068–2077.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Koo, J. R., Kim, J. C., Yoon, J. W., Kim, G. H., Jeon, R. W., Kim, H. J., et al. (2002). Failure of continuous venovenous hemofiltration to prevent death in paraquat poisoning. American Journal of Kidney Diseases, 39, 55–59.CrossRefPubMedGoogle Scholar
  4. 4.
    Li, Q., Yang, X., Sreejayan, N., & Ren, J. (2007). Insulin-like growth factor I deficiency prolongs survival and antagonizes paraquat-induced cardiomyocyte dysfunction: Role of oxidative stress. Rejuvenation Research, 10, 501–512.CrossRefPubMedGoogle Scholar
  5. 5.
    Wang, Q., Yang, L., Hua, Y., Nair, S., Xu, X., & Ren, J. (2014). AMP-activated protein kinase deficiency rescues paraquat-induced cardiac contractile dysfunction through an autophagy-dependent mechanism. Toxicological Sciences, 142, 6–20.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wang, J., Lu, S., Zheng, Q., Hu, N., Yu, W., Li, N., et al. (2016). Cardiac-specific knockout of ETA receptor mitigates paraquat-induced cardiac contractile dysfunction. Cardiovascular Toxicology, 16, 235–243.CrossRefPubMedGoogle Scholar
  7. 7.
    Wang, S., Zhu, X., Xiong, L., & Ren, J. (2017). Ablation of Akt2 prevents paraquat-induced myocardial mitochondrial injury and contractile dysfunction: Role of Nrf2. Toxicology Letters, 269, 1–14.CrossRefPubMedGoogle Scholar
  8. 8.
    Lei, Y., Li, X., Yuan, F., Liu, L., Zhang, J., Yang, Y., et al. (2017). Toll-like receptor 4 ablation rescues against paraquat-triggered myocardial dysfunction: Role of ER stress and apoptosis. Environmental Toxicology, 32, 656–668.CrossRefPubMedGoogle Scholar
  9. 9.
    Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N., et al. (2005). Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nature Chemical Biology, 1, 112–119.CrossRefPubMedGoogle Scholar
  10. 10.
    Davis, C. W., Hawkins, B. J., Ramasamy, S., Irrinki, K. M., Cameron, B. A., Islam, K., et al. (2010). Nitration of the mitochondrial complex I subunit NDUFB8 elicits R 442 IP1- and RIP3-mediated necrosis. Free Radical Biology and Medicine, 48, 306–317.CrossRefPubMedGoogle Scholar
  11. 11.
    Kim, S., Dayani, L., Rosenberg, P. A., & Li, J. (2010). RIP1 kinase mediates arachidonicacid-induced oxidative death of oligodendrocyte precursors. International journal of Physiology, Pathophysiology and Pharmacology, 2, 137–147.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhao, J., Jitkaew, S., Cai, Z., Choksi, S., Li, Q., Luo, J., et al. (2012). Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proceedings of the National Academy of Sciences of the United States of America, 109, 5322–5327.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang, D. W., Shao, J., Lin, J., Zhang, N., Lu, B. J., Lin, S. C., et al. (2009). RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science, 325, 332–336.CrossRefPubMedGoogle Scholar
  14. 14.
    Newton, K. (2015). RIPK1 and RIPK3: Critical regulators of inflammation and cell death. Trends in Cell Biology, 25, 347–353.CrossRefPubMedGoogle Scholar
  15. 15.
    Sun, L., Wang, H., Wang, Z., He, S., Chen, S., Liao, D., et al. (2012). Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell, 148, 213–227.CrossRefPubMedGoogle Scholar
  16. 16.
    Weinlich, R., Oberst, A., Beere, H. M., & Green, D. R. (2017). Necroptosis in development, inflammation and disease. Nature Reviews Molecular Cell Biology, 18, 127–136.CrossRefPubMedGoogle Scholar
  17. 17.
    Kung, G., Konstantinidis, K., & Kitsis, R. N. (2011). Programmed necrosis, not apoptosis, in the heart. Circulation Research, 108, 1017–1036.CrossRefPubMedGoogle Scholar
  18. 18.
    Konstantinidis, K., Whelan, R. S., & Kitsis, R. N. (2012). Mechanisms of cell death in heart disease. Arteriosclerosis, Thrombosis, and Vascular Biology, 32, 1552–1562.CrossRefPubMedGoogle Scholar
  19. 19.
    Zhang, T., Zhang, Y., Cui, M., Jin, L., Wang, Y., Lv, F., et al. (2016). CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis. Nature Medicine, 22, 175–182.CrossRefPubMedGoogle Scholar
  20. 20.
    Luedde, M., Lutz, M., Carter, N., Sosna, J., Jacoby, C., Vucur, M., et al. (2014). RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction. Cardiovascular Research, 103, 206–216.CrossRefPubMedGoogle Scholar
  21. 21.
    Oerlemans, M. I., Liu, J., Arslan, F., den Ouden, K., van Middelaar, B. J., Doevendans, P. A., et al. (2012). Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo. Basic Research in Cardiology, 476(107), 270.CrossRefGoogle Scholar
  22. 22.
    Smith, C. C., Davidson, S. M., Lim, S. Y., Simpkin, J. C., Hothersall, J. S., & Yellon, D. M. (2007). Necrostatin: A potentially novel cardioprotective agent? Cardiovascular Drugs and Therapy, 479(21), 227–233.CrossRefGoogle Scholar
  23. 23.
    Cho, Y. S. (2014). Perspectives on the therapeutic modulation of an alternative cell death, programmed necrosis. International Journal of Molecular Medicine, 33, 1401–1406.CrossRefPubMedGoogle Scholar
  24. 24.
    Koudstaal, S., Oerlemans, M. I., Van der Spoel, T. I., Janssen, A. W., Hoefer, I. E., Doevendans, P. A., et al. (2015). Necrostatin-1 alleviates reperfusion injury following acute myocardial infarction in pigs. European Journal of Clinical Investigation, 45, 150–159.CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang, A., Mao, X., Li, L., Tong, Y., Huang, Y., Lan, Y., et al. (2014). Inhibits Hmgb1-IL-23/IL-17 pathway and attenuates cardiac ischemia reperfusion injury. Transplant International, 27, 1077–1085.CrossRefPubMedGoogle Scholar
  26. 26.
    Bhardwaj, Nitin, & Saxena, Rajiv K. (2014). Elimination of young erythrocytes from blood circulation and altered erythropoietic patterns during paraquat induced anemic phase in mice. PLoS ONE, 9, e99364.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Saad, N. S., Repas, S. J., Floyd, K., Janssen, P. M. L., & Elnakish, M. T. (2017). Recovery following thyroxine treatment withdrawal, but not propylthiouracil, averts in vivo and ex vivo thyroxine-provoked cardiac complications in adult FVB/N mice. BioMed Research International, 2017(2017), 6071031.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Ren, J., Privratsky, J. R., Yang, X., Dong, F., & Carlson, E. C. (2008). Metallothionein alleviates glutathione depletion induced oxidative cardiomyopathy in murine hearts. Critical Care Medicine, 36, 2106–2116.CrossRefPubMedGoogle Scholar
  29. 29.
    Liang, L., Shou, X. L., Zhao, H. K., Ren, G. Q., Wang, J. B., Wang, X. H., et al. (2015). Antioxidant catalase rescues against high fat diet-induced cardiac dysfunction via an IKKbeta-AMPK dependent regulation of autophagy. Biochimica et Biophysica Acta, 1852, 343–352.CrossRefPubMedGoogle Scholar
  30. 30.
    Hui, B., Yao, X., Zhou, Q., Wu, Z., Sheng, P., & Zhang, L. (2014). Pristimerin, a natural anti-tumor triterpenoid, inhibits LPS-induced TNF-α and IL-8 production through down-regulation of ROS-related classical NF-κB pathway in THP-1 cells. International Immunopharmacology, 21, 501–508.CrossRefPubMedGoogle Scholar
  31. 31.
    Kim, J. M., Heo, H. S., Ha, Y. M., Ye, B. H., Lee, E. K., Choi, Y. J., et al. (2015). Mechanism of Ang II involvement in activation of NF-κB through phosphorylation of p65 during aging. AGE, 2012(34), 11–25.Google Scholar
  32. 32.
    Yin, B., Xu, Y., Wei, R. L., He, F., Luo, B. Y., & Wang, J. Y. (2015). Inhibition of receptor-interacting protein 3 upregulation and nuclear translocation involved in Necrostatin-1 protection against hippocampal neuronal programmed necrosis induced by ischemia/reperfusion injury. Brain Research, 1609, 63–71.CrossRefPubMedGoogle Scholar
  33. 33.
    Lim, S. Y., Davidson, S. M., Mocanu, M. M., Yellon, D. M., & Smith, C. C. (2007). The cardioprotective effect of necrostatin requires the cyclophilin-D component of the mitochondrial permeability transition pore. Cardiovascular Drugs and Therapy, 21, 467–469.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Cho, Y. S., Challa, S., Moquin, D., Genga, R., Ray, T. D., Guildford, M., et al. (2009). Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell, 137, 1112–1123.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    He, S., Wang, L., Miao, L., Wang, T., Du, F., Zhao, L., et al. (2009). Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell, 137, 1100–1111.CrossRefPubMedGoogle Scholar
  36. 36.
    Li, J., McQuade, T., Siemer, A. B., Napetschnig, J., Moriwaki, K., Hsiao, Y. S., et al. (2012). The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell, 150, 339–350.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Whelan, R. S., Kaplinskiy, V., & Kitsis, R. N. (2010). Cell death in the pathogenesis of heart disease: mechanisms and significance. Annual Review of Physiology, 72, 19–44.CrossRefPubMedGoogle Scholar
  38. 38.
    Schenk, B., & Fulda, S. (2015). Reactive oxygen species regulate Smac mimetic/TNFalpha-induced necroptotic signaling and cell death. Oncogene, 34, 5796–5806.CrossRefPubMedGoogle Scholar
  39. 39.
    Zhang, Y., Su, S. S., Zhao, S., Yang, Z., Zhong, C. Q., Chen, X., et al. (2017). RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome. Nature Communications, 8, 14329.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., et al. (2012). Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biology and Medicine, 52(1), 1–6.CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Emergency MedicineShanghai Jiao Tong University Affiliated Sixth People’s HospitalShanghaiChina
  2. 2.Shanghai Pudong Newarea Healthcare Hospital for Women and ChildrenShanghaiChina

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