Basic Research in Cardiology

, Volume 101, Issue 2, pp 180–189 | Cite as

Post–conditioning induced cardioprotection requires signaling through a redox–sensitive mechanism, mitochondrial ATP–sensitive K+ channel and protein kinase C activation

  • C. Penna
  • R. Rastaldo
  • D. Mancardi
  • S. Raimondo
  • S. Cappello
  • D. Gattullo
  • G. Losano
  • P. PagliaroEmail author


Post–conditioning (Post–C) induced cardioprotection involves activation of guanylyl–cyclase. In the ischemic preconditioning scenario, the downstream targets of cGMP include mitochondrial ATP–sensitive K+ (mKATP) channels and protein kinase C (PKC), which involve reactive oxygen species (ROS) production. This study tests the hypothesis that mKATP, PKC and ROS are also involved in the Post–C protection. Isolated rat hearts underwent 30 min global ischemia (I) and 120 min reperfusion (R) with or without Post–C (i.e., 5 cycles of 10 s R/I immediately after the 30 min ischemia). In 6 groups (3 with and 3 without Post–C) either mKATP channel blocker, 5– hydroxydecanoate (5–HD), or PKC inhibitor, chelerythrine (CHE) or ROS scavenger, N–acetyl–cysteine (NAC), were given during the entire reperfusion (120 min). In other 6 groups (3 with and 3 without Post–C), 5–HD, CHE or NAC were infused for 117 min only starting after 3 min of reperfusion not to interfere with the early effects of Post–C and/or reperfusion. In an additional group NAC was given during Post–C maneuvers (i.e., 3 min only). Myocardial damage was evaluated using nitro–blue tetrazolium staining and lactate dehydrogenase (LDH) release. Post–C attenuated myocardial infarct size (21 ± 3% vs. 64 ± 5% in control; p < 0.01). Such an effect was abolished by 5–HD or CHE given during either the 120 or 117 min of reperfusion as well as by NAC given during the 120 min or the initial 3 min of reperfusion. However, delayed NAC (i.e., 117 min infusion) did not alter the protective effect of Post– C (infarct size 32 ± 5%; p < 0.01 vs. control, NS vs. Post–C). CHE, 5–HD or NAC given in the absence of Post–C did not alter the effects of I/R. Similar results were obtained in terms of LDH release. Our data show that Post–C induced protection involves an early redox–sensitive mechanism as well as a persistent activation of mKATP and PKC, suggesting that the mKATP/ROS/PKC pathway is involved in post–conditioning.

Key words

ischemia/reperfusion mitochondrial KATP channels postconditioning protein kinase C redox signaling 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Angelos MG, Kutala VK, Torres CA, He G, Stoner JD, Mohammad M, Kuppusamy P (2005) Hypoxic Reperfusion of the Ischemic Heart and Oxygen Radical Generation. Am J Physiol Heart Circ Physiol In Press DOI:10. 1152/ajpheart. 00223. 2005Google Scholar
  2. 2.
    Atmaca G (2004) Antioxidant effects of sulfur–containing amino acids. Yonsei Med J 45:776–488PubMedGoogle Scholar
  3. 3.
    Becker LB (2004) New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res 61:461–470CrossRefPubMedGoogle Scholar
  4. 4.
    Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB (1988) Demonstration of free radical generation in “stunned” myocardium of intact dogs with the use of the spin trap a–phenyl–N–tertbutyl nitrone. J Clin Invest 82:476–485PubMedGoogle Scholar
  5. 5.
    Cao Z, Liu L, Van Winkle DM (2005) Met5–enkephalin–induced cardioprotection occurs via transactivation of EGFR and activation of PI3K. (2005) Am J Physiol Heart Circ Physiol 288:H1955–H1964PubMedGoogle Scholar
  6. 6.
    Carroll R, Gant VA, Yellon DM (2001) Mitochondrial KATP channels protects a human atrial–derived cell line by a mechanism involving free radical generation. Cardiovasc Res 51:691–700CrossRefPubMedGoogle Scholar
  7. 7.
    Chen W, Gabel S, Steenbergen C, Murphy E (1995) A redox–based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res 77:424–429PubMedGoogle Scholar
  8. 8.
    Darling CE, Jiang R, Maynard M, Whittaker P, Vinten–Johansen J, Przyklenk K (2005) ‘Postconditioning’ via Stuttering Reperfusion Limits Myocardial Infarct Size in Rabbit Hearts: Role of ERK 1/2. Am J Physiol Heart Circ Physiol 289:H1618–H1626CrossRefPubMedGoogle Scholar
  9. 9.
    Forbes RA, Steenbergen C, Murphy E (2001) Diazoxide–induced cardioprotection requires signaling through a redox–sensitive mechanism. Circ Res 88:802–809PubMedGoogle Scholar
  10. 10.
    Forman MB, Puett DW, Cates CU, McCroskey DE, Beckman JK, Greene HL, et al. (1988) Glutathione redox pathway and reperfusion injury. Effect of N–acetylcysteine on infarct size and ventricular function. Circulation 78:202–213PubMedGoogle Scholar
  11. 11.
    Gelpi RJ, Morales C, Cohen MV, Downey JM (2002) Xanthine oxidase contributes to preconditioning’s preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end–point for studies of preconditioning. Basic Res Cardiol 97:40–46CrossRefPubMedGoogle Scholar
  12. 12.
    Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS, Sun HY, Guyton RA, Vinten–Johansen J, Zhao ZQ (2004) Myocardial protection with postconditioning is not enhanced by ischemic preconditioning. Ann Thorac Surg 78:961–969CrossRefPubMedGoogle Scholar
  13. 13.
    Hanley PJ, Drose S, Brandt U, Lareau RA, Banerjee AL, Srivastava DK, Banaszak LJ, Barycki JJ, Van Veldhoven PP, Daut J (2005) 5–Hydroxydecanoate is metabolised in mitochondria and creates a ratelimiting bottleneck for beta–oxidation of fatty acids. J Physiol 562:307–318PubMedGoogle Scholar
  14. 14.
    Hausenloy DJ, Tsang A, Yellon DM (2005) The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning. Trends Cardiovasc Med 15:69–75CrossRefPubMedGoogle Scholar
  15. 15.
    Heusch G. Postconditioning: old wine in a new bottle? (2004) J Am Coll Cardiol 44:1111–1112CrossRefPubMedGoogle Scholar
  16. 16.
    Lochner A, Genade S, Moolman JA (2003) Ischemic preconditioning: infarct size is a more reliable endpoint than functional recovery. Basic Res Cardiol 98:337–346CrossRefPubMedGoogle Scholar
  17. 17.
    Kerendi F, Kin H, Halkos ME, Jiang R, Zatta AJ, Zhao ZQ, Guyton RA, Vinten– Johansen J (2005) Remote postconditioning Brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infarct size via endogenous activation of adenosine receptors. Basic Res Cardiol 100:404–412CrossRefPubMedGoogle Scholar
  18. 18.
    Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F et al. (2005) Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res 67:124–133CrossRefPubMedGoogle Scholar
  19. 19.
    Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME et al. (2004) Postconditioning attenuates myocardial ischemia–reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 62:74–85CrossRefPubMedGoogle Scholar
  20. 20.
    Kloner RA, Jennings RB (2001) Consequences of brief ischemia: stunning, preconditioning and their clinical implications. Circulation 104:2981–2989PubMedGoogle Scholar
  21. 21.
    Krenz M, Oldenburg O, Wimpee H, Cohen MV, Garlid KD, Critz SD, Downey JM, Benoit JN (2002) Opening of ATPsensitive potassium channels causes generation of free radicals in vascular smooth muscle cells. Basic Res Cardiol 97:365–373CrossRefPubMedGoogle Scholar
  22. 22.
    Krieg T, Cohen MV, Downey JM (2003) Mitochondria and their role in preconditioning’s trigger phase. Basic Res Cardiol 98:228–234PubMedGoogle Scholar
  23. 23.
    Krieg T, Qin Q, Philipp S, Alexeyev MF, Cohen MV, Downey JM (2004) Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS. Am J Physiol Heart Circ Physiol 287:H2606–H2611CrossRefPubMedGoogle Scholar
  24. 24.
    Ma XL, Gao F, Liu GL, Lopez BL, Christopher TA, Fukuto JM et al. (1999) Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury. Proc Natl Acad Sci USA 96:14617–14622PubMedGoogle Scholar
  25. 25.
    Okawa H, Horimoto H, Mieno S, Nomura Y, Yoshida M, Shinjiro S (2003) Preischemic infusion of alpha–human atrial natriuretic peptide elicits myoprotective effects against ischemia reperfusion in isolated rat hearts. Mol Cell Biochem 248:171–177CrossRefPubMedGoogle Scholar
  26. 26.
    Pabla R, Buda AJ, Flynn DM, Blesse SA, Shin AM, Curtis MJ et al. (1996) Nitric oxide attenuates neutrophil–mediated myocardial contractile dysfunction after ischemia and reperfusion. Circ Res 78:65–72PubMedGoogle Scholar
  27. 27.
    Pagliaro P, Mancardi D, Rastaldo R, Penna C, Gattullo D, Miranda KM et al. (2003) Nitroxyl affords thiol–sensitive myocardial protective effects akin to early preconditioning Free Radic Biol Med 34:33–43CrossRefPubMedGoogle Scholar
  28. 28.
    Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS et al. (2000) Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 87:460–466PubMedGoogle Scholar
  29. 29.
    Patel HH, Gross GJ (2001) Diazoxide induced cardiprotecion: what comes first, KATP channels or reactive oxygen species? Cardiovasc Res 51:633–636CrossRefPubMedGoogle Scholar
  30. 30.
    Penna C, Alloatti G, Cappello S, Gattullo D, Berta G, Mognetti B et. al. (2005a) Platelet–activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3–kinase and protein kinase C activation. Am J Physiol Heart Circ Physiol 288:H2512–H2520CrossRefGoogle Scholar
  31. 31.
    Penna C, Cappello S, Mancardi D, Raimondo S, Rastaldo R, Gattullo D et al. (2005b) Post–Conditioning Reduces Infarct Size in the Isolated Rat Heart: Role of Coronary Flow and Pressure and the Nitric Oxide/cGMP Pathway. Basic Res Cardiol In Press [DOI: 10. 1007/s00395– 005–0543–6]Google Scholar
  32. 32.
    Przyklenk K, Kloner RA (1989) “Reperfusion injury” by oxygen–derived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow. Circ Res 64:86–96PubMedGoogle Scholar
  33. 33.
    Sasaki N, Sato T, Ohler A, O’Rourke B, Marban E (2000) Activation of mitochondrial ATP–dependent potassium channels by nitric oxide. Circulation 101:439–445PubMedGoogle Scholar
  34. 34.
    Sato H, Jordan JE, Zhao ZQ, Sarvotham SS, Vinten–Johansen J (1997) Gradual reperfusion reduces infarct size and endothelial injury but augments neutrophil accumulation. Ann Thorac Surg 64:1099–1107CrossRefPubMedGoogle Scholar
  35. 35.
    Sato T, Marban E (2000) The role of mitochondrial K(ATP) channels in cardioprotection. Basic Res Cardiol 95:285–289CrossRefPubMedGoogle Scholar
  36. 36.
    Serviddio G, Di Venosa N, Federici A, D’Agostino D, Rollo T, Prigigallo F, Altomare E, Fiore T, Vendemiale G (2005) Brief hypoxia before normoxic reperfusion (postconditioning) protects the heart against ischemia–reperfusion injury by preventing mitochondria peroxide production and glutathione depletion. FASEB J 19:354–361CrossRefPubMedGoogle Scholar
  37. 37.
    Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L’Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre–Fouet X, Ovize M (2005) Postconditioning the human heart. Circulation 112:2143–2148CrossRefPubMedGoogle Scholar
  38. 38.
    Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA et al. (2005) Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. Am J Physiol Heart Circ Physiol 288:H1900–H1908PubMedGoogle Scholar
  39. 39.
    Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM (2004) Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3–kinase–Akt pathway. Circ Res 95:230–232CrossRefPubMedGoogle Scholar
  40. 40.
    Tsang A, Hausenloy DJ, Yellon DM (2005) Myocardial postconditioning: reperfusion injury revisited. Am J Physiol Heart Circ Physiol 289:H2–H7CrossRefPubMedGoogle Scholar
  41. 41.
    Valen G, Vaage J (2005) Pre– and postconditioning during cardiac surgery. Basic Res Cardiol 100:179–186CrossRefPubMedGoogle Scholar
  42. 42.
    Vinten–Johansen J, Zhao ZQ, Zatta AJ, Kin H, Halkos ME, Kerendi F (2005) Postconditioning A new link in nature’s armor against myocardial ischemiareperfusion injury. Basic Res Cardiol 100:295–310PubMedGoogle Scholar
  43. 43.
    Wang Y, Takashi E, Xu M, Ayub A, Ashraf M (2001) Downregulation of protein kinase C inhibits activation of mitochondrial K(ATP) channels by diazoxide. Circulation 104:85–90PubMedGoogle Scholar
  44. 44.
    Yang XM, Philipp S, Downey JM, Cohen MV (2005) Postconditioning’s protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3–kinase and guanylyl cyclase activation. Basic Res Cardiol 100:57–63PubMedGoogle Scholar
  45. 45.
    Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV (2004) Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44:1103–1110CrossRefPubMedGoogle Scholar
  46. 46.
    Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA et al. (2003) Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285:H579–588PubMedGoogle Scholar

Copyright information

© Steinkopff-Verlag 2006

Authors and Affiliations

  • C. Penna
    • 1
  • R. Rastaldo
    • 2
  • D. Mancardi
    • 1
  • S. Raimondo
    • 1
  • S. Cappello
    • 2
  • D. Gattullo
    • 1
  • G. Losano
    • 2
  • P. Pagliaro
    • 1
    Email author
  1. 1.Dipartimento di Scienze Cliniche e Biologiche, Università di TorinoOspedale S. Luigi, Regione GonzoleOrbassano (TO)Italy
  2. 2.Sezione di Fisiologia del Dipartimento di NeuroscienzeUniversità di TorinoItaly

Personalised recommendations