Journal of Anesthesia

, Volume 28, Issue 2, pp 235–241 | Cite as

Combination of necroptosis and apoptosis inhibition enhances cardioprotection against myocardial ischemia–reperfusion injury

  • Shizuka Koshinuma
  • Masami MiyamaeEmail author
  • Kazuhiro Kaneda
  • Junichiro Kotani
  • Vincent M. Figueredo
Original Article



Necroptosis has been proposed as a mode of cell death that is a caspase-independent programmed necrosis. We investigated whether necroptosis is involved in myocardial ischemia–reperfusion injury in isolated guinea pig hearts and, if so, whether simultaneous inhibition of necroptosis and apoptosis confers enhanced cardioprotection.


Isolated perfused guinea pig hearts were subjected to 30 min ischemia and 4 h reperfusion (control = CTL, n = 8). Necrostatin-1 (necroptosis inhibitor, 10 μM), Z-VAD (apoptosis inhibitor, 0.1 μM) and both inhibitors were administered starting 5 min before ischemia and during the initial 30 min of reperfusion (Nec, Z-VAD, Nec + Z-VAD; n = 8 each). Contractile recovery was monitored by left ventricular developed (LVDP) and end-diastolic (LVEDP) pressure. Infarct size was determined by triphenyltetrazolium chloride staining. Tissue samples were obtained after 4 h reperfusion to determine expression of receptor-interacting protein 1 (RIP1) and activated caspase 3 by Western blot analysis.


After reperfusion, Nec + Z-VAD had higher LVDP and lower LVEDP compared with CTL. Infarct size was reduced in Nec and Z-VAD compared with CTL. Combination of necroptosis and apoptosis inhibition further reduced infarct size. Expression of activated caspase 3 was not increased in Z-VAD and Nec + Z-VAD compared with Nec and CTL. Expression of RIP1 was preserved in Z-VAD and Nec + Z-VAD compared with CTL, suggesting RIP1-mediated necrosis is involved in myocardial ischemia–reperfusion injury.


Necroptosis is involved in myocardial ischemia–reperfusion injury, and simultaneous inhibition of necroptosis and apoptosis enhances the cardioprotective effect. These findings may provide a novel, additive strategy for cardioprotection in acute myocardial infarction.


Necroptosis Necrostatin-1 Ischemia–reperfusion Heart 



We thank Dr. Hirofumi Sawai for excellent advice. This study was supported by Osaka Dental University (Osaka, Japan) Research Funds (12-13) (Shizuka Koshinuma) and Grant-in-Aid for Scientific Research (C) 23593008 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Masami Miyamae) (Tokyo, Japan).

Conflict of interest

The authors have no conflicts of interest to report.


  1. 1.
    Gottlieb RA, Engler RL. Apoptosis in myocardial ischemia-reperfusion. Ann N Y Acad Sci. 1999;874:412–26.PubMedCrossRefGoogle Scholar
  2. 2.
    Inamura Y, Miyamae M, Sugioka S, Domae N, Kotani J. Sevoflurane postconditioning prevents activation of caspase 3 and 9 through antiapoptotic signaling after myocardial ischemia-reperfusion. J Anesth. 2010;24:215–24.PubMedCrossRefGoogle Scholar
  3. 3.
    Qiao S, Xie H, Wang C, Wu X, Liu H, Liu C. Delayed anesthetic preconditioning protects against myocardial infarction via activation of nuclear factor-κB and upregulation of autophagy. J Anesth. 2013;27:251–60.PubMedCrossRefGoogle Scholar
  4. 4.
    Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JJ. Apoptosis. Nature (Lond) 1989;338:10.Google Scholar
  5. 5.
    Kitanaka C, Kuchino Y. Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ. 1999;6:508–15.PubMedCrossRefGoogle Scholar
  6. 6.
    Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005;1:112–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Declercq W, Vanden Berghe T, Vandenabeele P. RIP kinases at the crossroads of cell death and survival. Cell. 2009;138:229–32.PubMedCrossRefGoogle Scholar
  8. 8.
    Christofferson DE, Li Y, Hitomi J, Zhou W, Upperman C, Zhu H, Gerber SA, Gygi S, Yuan J. A novel role for RIP1 kinase in mediating TNFα production. Cell Death Dis. 2012;3:e320.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008;4:313–21.PubMedCrossRefGoogle Scholar
  10. 10.
    Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM. Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther. 2007;21:227–33.PubMedCrossRefGoogle Scholar
  11. 11.
    Wang YQ, Wang L, Zhang MY, Wang T, Bao HJ, Liu WL, Dai DK, Zhang L, Chang P, Dong WW, Chen XP, Tao LY. Necrostatin-1 suppresses autophagy and apoptosis in mice traumatic brain injury model. Neurochem Res. 2012;37:1849–58.PubMedCrossRefGoogle Scholar
  12. 12.
    Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, Ganz W. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J. 1981;101:593–600.PubMedCrossRefGoogle Scholar
  13. 13.
    Dorn GW 2nd. Apoptotic and non-apoptotic programmed cardiomyocyte death in ventricular remodelling. Cardiovasc Res. 2009;81:465–73.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Yaoita H, Ogawa K, Maehara K, Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation. 1998;97:276–81.PubMedCrossRefGoogle Scholar
  15. 15.
    Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1:489–95.PubMedCrossRefGoogle Scholar
  16. 16.
    Liu J, van Mil A, Vrijsen K, Zhao J, Gao L, Metz CH, Goumans MJ, Doevendans PA, Sluijter JP. MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1. J Cell Mol Med. 2010;15:1474–82.PubMedCrossRefGoogle Scholar
  17. 17.
    Lim SY, Davidson SM, Mocanu MM, Yellon DM, Smith CC. The cardioprotective effect of necrostatin requires the cyclophilin-D component of the mitochondrial permeability transition pore. Cardiovasc Drugs Ther. 2007;21:467–9.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Garlid KD, Costa AD, Quinlan CL, Pierre SV, Dos Santos P. Cardioprotective signaling to mitochondria. J Mol Cell Cardiol. 2009;46:858–66.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Chavez-Valdez R, Martin LJ, Flock DL, Northington FJ. Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia–ischemia. Neuroscience. 2012;219:192–203.PubMedCrossRefGoogle Scholar
  20. 20.
    Laukens B, Jennewein C, Schenk B, Vanlangenakker N, Schier A, Cristofanon S, Zobel K, Deshayes K, Vucic D, Jeremias I, Bertrand MJ, Vandenabeele P, Fulda S. Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor alpha-induced necroptosis. Neoplasia. 2011;13:971–9.PubMedCentralPubMedGoogle Scholar
  21. 21.
    Mocanu MM, Baxter GF, Yellon DM. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol. 2000;130:197–200.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Schwarz ER, Somoano Y, Hale SL, Kloner RA. What is the required reperfusion period for assessment of myocardial infarct size using triphenyltetrazolium chloride staining in the rat? J Thromb Thrombolysis. 2000;10:181–7.PubMedCrossRefGoogle Scholar
  23. 23.
    Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996;74:86–107.PubMedGoogle Scholar
  24. 24.
    Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S, Ferrari R, Knight R, Latchman D. Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury. Circulation. 2001;104:253–6.PubMedCrossRefGoogle Scholar

Copyright information

© Japanese Society of Anesthesiologists 2013

Authors and Affiliations

  • Shizuka Koshinuma
    • 1
  • Masami Miyamae
    • 2
    Email author
  • Kazuhiro Kaneda
    • 3
  • Junichiro Kotani
    • 3
  • Vincent M. Figueredo
    • 4
  1. 1.Department of Anesthesiology, Graduate School of DentistryOsaka Dental UniversityOsakaJapan
  2. 2.Department of Internal MedicineOsaka Dental UniversityHirakataJapan
  3. 3.Department of AnesthesiologyOsaka Dental UniversityOsakaJapan
  4. 4.Institute for Heart and Vascular Health, Einstein Medical CenterJefferson Medical CollegePhiladelphiaUSA

Personalised recommendations