Journal of Molecular Medicine

, Volume 90, Issue 2, pp 127–138

ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling

  • Ofer Beharier
  • Shani Dror
  • Shiri Levy
  • Joy Kahn
  • Merav Mor
  • Sharon Etzion
  • Daniel Gitler
  • Amos Katz
  • Anthony J. Muslin
  • Arie Moran
  • Yoram Etzion
Original Article

Abstract

Activation of ERK signaling may promote cardioprotection from ischemia–reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

Keywords

Cardiomyocyte survival Reperfusion injury salvage kinase Raf-1 kinase Extracellular signal-regulated kinase 

Supplementary material

109_2011_845_MOESM1_ESM.doc (325 kb)
ESM 1(DOC 325 kb)

References

  1. 1.
    Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury. N Engl J Med 357:1121–1135. doi:10.1056/NEJMra071667 PubMedCrossRefGoogle Scholar
  2. 2.
    Buja LM (2005) Myocardial ischemia and reperfusion injury. Cardiovasc Pathol 14:170PubMedCrossRefGoogle Scholar
  3. 3.
    Hausenloy DJ, Yellon DM (2009) Preconditioning and postconditioning: underlying mechanisms and clinical application. Atherosclerosis 204:334–341PubMedCrossRefGoogle Scholar
  4. 4.
    Hausenloy DJ, Yellon DM (2007) Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection. Heart Fail Rev 12:217–234PubMedCrossRefGoogle Scholar
  5. 5.
    Iliodromitis EK, Papalois A, Gritsopoulos G, Kremastinos DT, Yellon DM, Hausenloy DJ (2009) Remote ischaemic preconditioning and postconditioning and the reperfusion injury salvage kinase pathway. Heart 95:13Google Scholar
  6. 6.
    Philipp S, Critz SD, Cui L, Solodushko V, Cohen MV, Downey JM (2006) Localizing extracellular signal-regulated kinase (ERK) in pharmacological preconditioning’s trigger pathway. Basic Res Cardiol 101:159–167PubMedCrossRefGoogle Scholar
  7. 7.
    Hausenloy DJ, Yellon DM (2009) Cardioprotective growth factors. Cardiovasc Res 83:179–194. doi:10.1093/cvr/cvp062 PubMedCrossRefGoogle Scholar
  8. 8.
    Lips DJ, Bueno OF, Wilkins BJ, Purcell NH, Kaiser RA, Lorenz JN, Voisin L, Saba-El-Leil MK, Meloche S, Pouyssegur J et al (2004) MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo. Circulation 109:1938–1941. doi:10.1161/01.CIR.0000127126.73759.23 PubMedCrossRefGoogle Scholar
  9. 9.
    Bruinsma JJ, Jirakulaporn T, Muslin AJ, Kornfeld K (2002) Zinc ions and cation diffusion facilitator proteins regulate Ras-mediated signaling. Dev Cell 2:567–578PubMedCrossRefGoogle Scholar
  10. 10.
    Jirakulaporn T, Muslin AJ (2004) Cation diffusion facilitator proteins modulate Raf-1 activity. J Biol Chem 279:27807–27815PubMedCrossRefGoogle Scholar
  11. 11.
    Lazarczyk M, Pons C, Mendoza JA, Cassonnet P, Jacob Y, Favre M (2008) Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses. J Exp Med 205:35–42. doi:10.1084/jem.20071311 PubMedCrossRefGoogle Scholar
  12. 12.
    Palmiter R, Findley S (1995) Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 14:639–649PubMedGoogle Scholar
  13. 13.
    Cousins RJ, Liuzzi JP, Lichten LA (2006) Mammalian zinc transport, trafficking, and signals. J Biol Chem 281:24085–24089PubMedCrossRefGoogle Scholar
  14. 14.
    Levy S, Beharier O, Etzion Y, Mor M, Buzaglo L, Shaltiel L, Gheber LA, Kahn J, Muslin AJ, Katz A et al (2009) Molecular basis for zinc transporter 1 action as an endogenous inhibitor of L-type calcium channels. J Biol Chem 284:32434–32443PubMedCrossRefGoogle Scholar
  15. 15.
    Beharier O, Etzion Y, Katz A, Friedman H, Tenbosh N, Zacharish S, Bereza S, Goshen U, Moran A (2007) Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling. Cell Calcium 42:71–82PubMedCrossRefGoogle Scholar
  16. 16.
    Kamalov G, Deshmukh PA, Baburyan NY, Gandhi MS, Johnson PL, Ahokas RA, Bhattacharya SK, Sun Y, Gerling IC, Weber KT (2009) Coupled calcium and zinc dyshomeostasis and oxidative stress in cardiac myocytes and mitochondria of rats with chronic aldosteronism. J Cardiovasc Pharmacol 53:414–423. doi:10.1097/FJC.0b013e3181a15e77 PubMedCrossRefGoogle Scholar
  17. 17.
    White SM, Constantin PE, Claycomb WC (2004) Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol 286:H823–H829PubMedCrossRefGoogle Scholar
  18. 18.
    Seymour EM, Wu SY, Kovach MA, Romano MA, Traynor JR, Claycomb WC, Bolling SF (2003) HL-1 myocytes exhibit PKC and K(ATP) channel-dependent delta opioid preconditioning. J Surg Res 114:187–194. doi:S0022480403002488 PubMedCrossRefGoogle Scholar
  19. 19.
    Facundo HT, de Paula JG, Kowaltowski AJ (2005) Mitochondrial ATP-sensitive K + channels prevent oxidative stress, permeability transition and cell death. J Bioenerg Biomembr 37:75–82. doi:10.1007/s10863-005-4130-1 PubMedCrossRefGoogle Scholar
  20. 20.
    Ruiz-Meana M, Garcia-Dorado D, Miro-Casas E, Abellan A, Soler-Soler J (2006) Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion. Cardiovasc Res 71:715–724. doi:10.1016/j.cardiores.2006.06.019 PubMedCrossRefGoogle Scholar
  21. 21.
    Ljubkovic M, Marinovic J, Fuchs A, Bosnjak ZJ, Bienengraeber M (2006) Targeted expression of Kir6.2 in mitochondria confers protection against hypoxic stress. J Physiol 577:17–29. doi:10.1113/jphysiol.2006.118299 PubMedCrossRefGoogle Scholar
  22. 22.
    Cavalheiro RA, Marin RM, Rocco SA, Cerqueira FM, da Silva CC, Rittner R, Kowaltowski AJ, Vercesi AE, Franchini KG, Castilho RF (2010) Potent cardioprotective effect of the 4-anilinoquinazoline derivative PD153035: involvement of mitochondrial K(ATP) channel activation. PLoS One 5:e10666. doi:10.1371/journal.pone.0010666 PubMedCrossRefGoogle Scholar
  23. 23.
    Yitzhaki S, Huang C, Liu W, Lee Y, Gustafsson AB, Mentzer RM Jr, Gottlieb RA (2009) Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA. Basic Res Cardiol 104:157–167. doi:10.1007/s00395-009-0006-6 PubMedCrossRefGoogle Scholar
  24. 24.
    McMahon RJ, Cousins RJ (1998) Regulation of the zinc transporter ZnT-1 by dietary zinc. PNAS 95:4841–4846PubMedCrossRefGoogle Scholar
  25. 25.
    Heusch G (2009) No risk, no … cardioprotection? A critical perspective. Cardiovasc Res 84:173–175. doi:10.1093/cvr/cvp298 PubMedCrossRefGoogle Scholar
  26. 26.
    Skyschally A, van Caster P, Boengler K, Gres P, Musiolik J, Schilawa D, Schulz R, Heusch G (2009) Ischemic postconditioning in pigs: no causal role for RISK activation. Circ Res 104:15–18. doi:10.1161/circresaha.108.186429 PubMedCrossRefGoogle Scholar
  27. 27.
    Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev 88:581–609. doi:10.1152/physrev.00024.2007 PubMedCrossRefGoogle Scholar
  28. 28.
    Ramaraj R, Forman MB, Jackson EK, Lowenstein CJ, Ibanez B, Cimmino G, Badimon JJ, Korantzopoulos PG, Goudevenos JA, Yellon DM et al (2007) Myocardial reperfusion injury. N Engl J Med 357:2408–2410. doi:10.1056/NEJMc072913 PubMedCrossRefGoogle Scholar
  29. 29.
    Armstrong SC (2004) Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res 61:427–436. doi:10.1016/j.cardiores.2003.09.031 PubMedCrossRefGoogle Scholar
  30. 30.
    Beguin PC, Belaidi E, Godin-Ribuot D, Levy P, Ribuot C (2007) Intermittent hypoxia-induced delayed cardioprotection is mediated by PKC and triggered by p38 MAP kinase and Erk1/2. J Mol Cell Cardiol 42:343–351. doi:10.1016/j.yjmcc.2006.11.008 PubMedCrossRefGoogle Scholar
  31. 31.
    Przyklenk K, Hata K, Kloner RA (1997) Is calcium a mediator of infarct size reduction with preconditioning in canine myocardium? Circulation 96:1305–1312PubMedGoogle Scholar
  32. 32.
    Miyawaki H, Zhou X, Ashraf M (1996) Calcium preconditioning elicits strong protection against ischemic injury via protein kinase C signaling pathway. Circ Res 79:137–146PubMedGoogle Scholar
  33. 33.
    Cain BS, Meldrum DR, Cleveland JC, Meng X, Banerjee A, Harken AH (1999) Clinical L-type Ca(2+) channel blockade prevents ischemic preconditioning of human myocardium. J Mol Cell Cardiol 31:2191–2197PubMedCrossRefGoogle Scholar
  34. 34.
    Schulman D, Latchman DS, Yellon DM (2002) Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway. Am J Physiol Heart Circ Physiol 283:H1481–H1488. doi:10.1152/ajpheart.01089.2001 PubMedGoogle Scholar
  35. 35.
    Brar BK, Jonassen AK, Stephanou A, Santilli G, Railson J, Knight RA, Yellon DM, Latchman DS (2000) Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway. J Biol Chem 275:8508–8514PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Ofer Beharier
    • 1
    • 4
  • Shani Dror
    • 1
    • 4
  • Shiri Levy
    • 1
  • Joy Kahn
    • 1
  • Merav Mor
    • 1
    • 4
  • Sharon Etzion
    • 4
  • Daniel Gitler
    • 1
  • Amos Katz
    • 3
    • 4
  • Anthony J. Muslin
    • 2
  • Arie Moran
    • 1
  • Yoram Etzion
    • 5
  1. 1.Department of PhysiologyFaculty of Health Sciences, Ben-Gurion University of the NegevBeer-ShevaIsrael
  2. 2.Center for Cardiovascular Research, John Milliken Department of MedicineWashington University School of MedicineSt. LouisUSA
  3. 3.Department of CardiologyBarzilai Medical CenterAshkelonIsrael
  4. 4.Cardiac Arrhythmia Research LaboratorySoroka University Medical CenterBeer-ShevaIsrael
  5. 5.Cardiac Arrhythmia Research LaboratoryFaculty of Health sciences, Ben-Gurion University of the Negev & Soroka University Medical CenterBeer-ShevaIsrael

Personalised recommendations