Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury

  • Yiru Guo
  • Santosh K. Sanganalmath
  • Wenjian Wu
  • Xiaoping Zhu
  • Yiming Huang
  • Wei Tan
  • Suzanne T. Ildstad
  • Qianhong Li
  • Roberto BolliEmail author
Original Contribution


Although the late phase of ischemic preconditioning is known to be mediated by increased inducible nitric oxide synthase (iNOS) activity, controversy persists regarding the role of iNOS in ischemia/reperfusion (I/R) injury and, specifically, whether this protein is protective or detrimental. We hypothesized that iNOS is protective in myocytes but detrimental in inflammatory cells. To test this hypothesis, we created chimeric mice with iNOS-deficient peripheral blood cells by transplanting iNOS knockout (KO) bone marrow in wild-type (WT) mice after lethal irradiation. 2 months later, the mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. In WT naïve mice (iNOS+/+ naïve; group I, n = 17), infarct size was 56.9 ± 2.8% of the risk region. In iNOS KO naïve mice with whole-body iNOS deletion (iNOS−/− naïve; group II, n = 10), infarct size was comparable to group I (53.4 ± 3.5%). When irradiated WT mice received marrow from WT mice (iNOS+/+ chimera; group III, n = 10), infarct size was slightly reduced versus group I (44.3 ± 3.2%), indicating that irradiation and/or transplantation slightly decrease I/R injury. However, when WT mice received marrow from iNOS KO mice (iNOS−/− chimera; group IV, n = 14), infarct size was profoundly reduced (22.8 ± 2.1%, P < 0.05 vs. group III), indicating that selective deletion of iNOS from peripheral blood cells (with no change in myocardial iNOS content) induces protection against myocardial infarction. Together with our previous work showing the cardioprotective actions of NO donors, iNOS gene therapy, and cardiac-specific overexpression of iNOS, these data support a complex, dual role of iNOS in myocardial infarction (i.e., protective in myocytes but deleterious in blood cells). To our knowledge, this is the first study to identify a critical role of iNOS in peripheral blood cells as a mediator of myocardial I/R injury. The results support heretofore unknown differential actions of iNOS depending on cell source and have important translational implications.


Inducible nitric oxide synthase Myocardial infarction Infarct size Chimeric mice 



This study was supported in part by NIH grants R01 HL55757, HL-70897, HL-76794, and P01HL78825.

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Abu-Soud HM, Hazen SL (2000) Nitric oxide modulates the catalytic activity of myeloperoxidase. J Biol Chem 275:5425–5430PubMedCrossRefGoogle Scholar
  2. 2.
    Baker CS, Dutka DP, Pagano D, Rimoldi O, Pitt M, Hall RJ, Polak JM, Bonser RS, Camici PG (2002) Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium. Basic Res Cardiol 97:409–415. doi: 10.1007/s003950200050 PubMedCrossRefGoogle Scholar
  3. 3.
    Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620–1624PubMedCrossRefGoogle Scholar
  4. 4.
    Bogdan C (2001) Nitric oxide and the immune response. Nat Immunol 2:907–916. doi: 10.1038/ni1001-907 PubMedCrossRefGoogle Scholar
  5. 5.
    Bolli R (2001) Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33:1897–1918. doi: 10.1006/jmcc.2001.1462 PubMedCrossRefGoogle Scholar
  6. 6.
    Bolli R (2000) The late phase of preconditioning. Circ Res 87:972–983PubMedCrossRefGoogle Scholar
  7. 7.
    Bolli R, Becker L, Gross G, Mentzer R Jr, Balshaw D, Lathrop DA (2004) Myocardial protection at a crossroads: the need for translation into clinical therapy. Circ Res 95:125–134. doi: 10.1161/01.RES.0000137171.97172.d7 PubMedCrossRefGoogle Scholar
  8. 8.
    Bolli R, Bhatti ZA, Tang XL, Qiu Y, Zhang Q, Guo Y, Jadoon AK (1997) Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide. Circ Res 81:42–52PubMedCrossRefGoogle Scholar
  9. 9.
    Bolli R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan YT, Jones WK, Takano H, Guo Y, Zhang J (1998) The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol 93:325–338PubMedCrossRefGoogle Scholar
  10. 10.
    Bolli R, Manchikalapudi S, Tang XL, Takano H, Qiu Y, Guo Y, Zhang Q, Jadoon AK (1997) The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res 81:1094–1107PubMedCrossRefGoogle Scholar
  11. 11.
    Das A, Xi L, Kukreja RC (2005) Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling. J Biol Chem 280:12944–12955. doi: 10.1074/jbc.M404706200 PubMedCrossRefGoogle Scholar
  12. 12.
    Feng Q, Lu X, Jones DL, Shen J, Arnold JM (2001) Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation 104:700–704PubMedCrossRefGoogle Scholar
  13. 13.
    Gealekman O, Abassi Z, Rubinstein I, Winaver J, Binah O (2002) Role of myocardial inducible nitric oxide synthase in contractile dysfunction and beta-adrenergic hyporesponsiveness in rats with experimental volume-overload heart failure. Circulation 105:236–243PubMedCrossRefGoogle Scholar
  14. 14.
    Gross GJ, Fryer RM (2000) Mitochondrial K(ATP) channels: triggers or distal effectors of ischemic or pharmacological preconditioning? Circ Res 87:431–433PubMedCrossRefGoogle Scholar
  15. 15.
    Grover GJ, Garlid KD (2000) ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32:677–695. doi: 10.1006/jmcc.2000.1111 PubMedCrossRefGoogle Scholar
  16. 16.
    Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli R (1999) The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96:11507–11512PubMedCrossRefGoogle Scholar
  17. 17.
    Guo Y, Stein AB, Wu WJ, Zhu X, Tan W, Li Q, Bolli R (2005) Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS. Am J Physiol Heart Circ Physiol 289:H2251–H2257. doi: 10.1152/ajpheart.00341.2005 PubMedCrossRefGoogle Scholar
  18. 18.
    Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R (1998) Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol 275:H1375–H1387PubMedGoogle Scholar
  19. 19.
    Hataishi R, Rodrigues AC, Morgan JG, Ichinose F, Derumeaux G, Bloch KD, Picard MH, Scherrer-Crosbie M (2006) Nitric oxide synthase 2 and pressure-overload-induced left ventricular remodelling in mice. Exp Physiol 91:633–639. doi: 10.1113/expphysiol.2005.033068 PubMedCrossRefGoogle Scholar
  20. 20.
    Heger J, Godecke A, Flogel U, Merx MW, Molojavyi A, Kuhn-Velten WN, Schrader J (2002) Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction. Circ Res 90:93–99PubMedCrossRefGoogle Scholar
  21. 21.
    Hendgen-Cotta UB, Merx MW, Shiva S, Schmitz J, Becher S, Klare JP, Steinhoff HJ, Goedecke A, Schrader J, Gladwin MT, Kelm M, Rassaf T (2008) Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury. Proc Natl Acad Sci USA 105:10256–10261. doi: 10.1073/pnas.0801336105 PubMedCrossRefGoogle Scholar
  22. 22.
    Heusch G, Boengler K, Schulz R (2008) Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation 118:1915–1919. doi: 10.1161/CIRCULATIONAHA.108.805242 PubMedCrossRefGoogle Scholar
  23. 23.
    Heusch G, Post H, Michel MC, Kelm M, Schulz R (2000) Endogenous nitric oxide and myocardial adaptation to ischemia. Circ Res 87:146–152PubMedCrossRefGoogle Scholar
  24. 24.
    Ignarro LJ (1991) Signal transduction mechanisms involving nitric oxide. Biochem Pharmacol 41:485–490PubMedCrossRefGoogle Scholar
  25. 25.
    Ischiropoulos H, Zhu L, Beckman JS (1992) Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298:446–451PubMedCrossRefGoogle Scholar
  26. 26.
    Jones SP, Greer JJ, Ware PD, Yang J, Walsh K, Lefer DJ (2005) Deficiency of iNOS does not attenuate severe congestive heart failure in mice. Am J Physiol Heart Circ Physiol 288:H365–H370. doi: 10.1152/ajpheart.00245.2004 PubMedCrossRefGoogle Scholar
  27. 27.
    Kolios G, Valatas V, Ward SG (2004) Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle. Immunology 113:427–437. doi: 10.1111/j.1365-2567.2004.01984.x PubMedCrossRefGoogle Scholar
  28. 28.
    Li H, Colson YL, Ildstad ST (1995) Mixed allogeneic chimerism achieved by lethal and nonlethal conditioning approaches induces donor-specific tolerance to simultaneous islet allografts. Transplantation 60:523–529PubMedCrossRefGoogle Scholar
  29. 29.
    Li H, Kaufman CL, Ildstad ST (1995) Allogeneic chimerism induces donor-specific tolerance to simultaneous islet allografts in nonobese diabetic mice. Surgery 118:192–197 (discussion 197–198)PubMedCrossRefGoogle Scholar
  30. 30.
    Li Q, Guo Y, Ou Q, Cui C, Wu WJ, Tan W, Zhu X, Lanceta LB, Sanganalmath SK, Dawn B, Shinmura K, Rokosh GD, Wang S, Bolli R (2009) Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120:1222–1230. doi: 10.1161/CIRCULATIONAHA.108.778688 PubMedCrossRefGoogle Scholar
  31. 31.
    Li Q, Guo Y, Tan W, Ou Q, Wu WJ, Sturza D, Dawn B, Hunt G, Cui C, Bolli R (2007) Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-kappaB dependent pathway. Circulation 116:1577–1584. doi: 10.1161/CIRCULATIONAHA.107.689810 PubMedCrossRefGoogle Scholar
  32. 32.
    Li Q, Guo Y, Tan W, Stein AB, Dawn B, Wu WJ, Zhu X, Lu X, Xu X, Siddiqui T, Tiwari S, Bolli R (2006) Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences. Am J Physiol Heart Circ Physiol 290:H584–H589. doi: 10.1152/ajpheart.00855.2005 PubMedCrossRefGoogle Scholar
  33. 33.
    Li Q, Guo Y, Wu WJ, Ou Q, Zhu X, Tan W, Yuan F, Chen N, Dawn B, Luo L, O’Brien E, Bolli R (2011) Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences. Basic Res Cardiol 106:1355–1366. doi: 10.1007/s00395-011-0207-7 PubMedCrossRefGoogle Scholar
  34. 34.
    Li Q, Guo Y, Xuan YT, Lowenstein CJ, Stevenson SC, Prabhu SD, Wu WJ, Zhu Y, Bolli R (2003) Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism. Circ Res 92:741–748. doi: 10.1161/01.RES.0000065441.72685.29 PubMedCrossRefGoogle Scholar
  35. 35.
    Liu YH, Carretero OA, Cingolani OH, Liao TD, Sun Y, Xu J, Li LY, Pagano PJ, Yang JJ, Yang XP (2005) Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction. Am J Physiol Heart Circ Physiol 289:H2616–H2623. doi: 10.1152/ajpheart.00546.2005 PubMedCrossRefGoogle Scholar
  36. 36.
    Loke KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, Hintze TH (1999) Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res 84:840–845PubMedCrossRefGoogle Scholar
  37. 37.
    MacMicking J, Xie QW, Nathan C (1997) Nitric oxide and macrophage function. Annu Rev Immunol 15:323–350. doi: 10.1146/annurev.immunol.15.1.323 PubMedCrossRefGoogle Scholar
  38. 38.
    Marfella R, Di Filippo C, Esposito K, Nappo F, Piegari E, Cuzzocrea S, Berrino L, Rossi F, Giugliano D, D’Amico M (2004) Absence of inducible nitric oxide synthase reduces myocardial damage during ischemia reperfusion in streptozotocin-induced hyperglycemic mice. Diabetes 53:454–462PubMedCrossRefGoogle Scholar
  39. 39.
    Martin C, Schulz R, Post H, Boengler K, Kelm M, Kleinbongard P, Gres P, Skyschally A, Konietzka I, Heusch G (2007) Microdialysis-based analysis of interstitial NO in situ: NO synthase-independent NO formation during myocardial ischemia. Cardiovasc Res 74:46–55. doi: 10.1016/j.cardiores.2006.12.020 PubMedCrossRefGoogle Scholar
  40. 40.
    Mohr S, Hallak H, de Boitte A, Lapetina EG, Brune B (1999) Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem 274:9427–9430PubMedCrossRefGoogle Scholar
  41. 41.
    Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, Downey JM (2000) Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals. Circ Res 87:460–466PubMedCrossRefGoogle Scholar
  42. 42.
    Post H, Schulz R, Behrends M, Gres P, Umschlag C, Heusch G (2000) No involvement of endogenous nitric oxide in classical ischemic preconditioning in swine. J Mol Cell Cardiol 32:725–733. doi: 10.1006/jmcc.2000.1117 PubMedCrossRefGoogle Scholar
  43. 43.
    Pryor WA, Squadrito GL (1995) The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol 268:L699–L722PubMedGoogle Scholar
  44. 44.
    Qiu Y, Rizvi A, Tang XL, Manchikalapudi S, Takano H, Jadoon AK, Wu WJ, Bolli R (1997) Nitric oxide triggers late preconditioning against myocardial infarction in conscious rabbits. Am J Physiol 273:H2931–H2936PubMedGoogle Scholar
  45. 45.
    Radi R, Beckman JS, Bush KM, Freeman BA (1991) Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 266:4244–4250PubMedGoogle Scholar
  46. 46.
    Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA (1994) Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 269:26066–26075PubMedGoogle Scholar
  47. 47.
    Sam F, Sawyer DB, Xie Z, Chang DL, Ngoy S, Brenner DA, Siwik DA, Singh K, Apstein CS, Colucci WS (2001) Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction. Circ Res 89:351–356PubMedCrossRefGoogle Scholar
  48. 48.
    Sasaki N, Sato T, Ohler A, O’Rourke B, Marban E (2000) Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 101:439–445PubMedCrossRefGoogle Scholar
  49. 49.
    Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, Zhu Y, Bolli R (2002) Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res 90:602–608PubMedCrossRefGoogle Scholar
  50. 50.
    Szelid Z, Pokreisz P, Liu X, Vermeersch P, Marsboom G, Gillijns H, Pellens M, Verbeken E, Van de Werf F, Collen D, Janssens SP (2010) Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial reperfusion injury. Basic Res Cardiol 105:169–179. doi: 10.1007/s00395-009-0077-4 PubMedCrossRefGoogle Scholar
  51. 51.
    Takano H, Bolli R, Black RG Jr, Kodani E, Tang XL, Yang Z, Bhattacharya S, Auchampach JA (2001) A(1) or A(3) adenosine receptors induce late preconditioning against infarction in conscious rabbits by different mechanisms. Circ Res 88:520–528PubMedCrossRefGoogle Scholar
  52. 52.
    Takano H, Manchikalapudi S, Tang XL, Qiu Y, Rizvi A, Jadoon AK, Zhang Q, Bolli R (1998) Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits. Circulation 98:441–449PubMedCrossRefGoogle Scholar
  53. 53.
    Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R (1998) Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res 83:73–84PubMedCrossRefGoogle Scholar
  54. 54.
    Vegh A, Papp JG, Szekeres L, Parratt JR (1993) Prevention by an inhibitor of the l-arginine-nitric oxide pathway of the antiarrhythmic effects of bradykinin in anaesthetized dogs. Br J Pharmacol 110:18–19PubMedGoogle Scholar
  55. 55.
    West MB, Rokosh G, Obal D, Velayutham M, Xuan YT, Hill BG, Keith RJ, Schrader J, Guo Y, Conklin DJ, Prabhu SD, Zweier JL, Bolli R, Bhatnagar A (2008) Cardiac myocyte-specific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition. Circulation 118:1970–1978. doi: 10.1161/CIRCULATIONAHA.108.791533 PubMedCrossRefGoogle Scholar
  56. 56.
    Ziolo MT, Maier LS, Piacentino V 3rd, Bossuyt J, Houser SR, Bers DM (2004) Myocyte nitric oxide synthase 2 contributes to blunted beta-adrenergic response in failing human hearts by decreasing Ca2+ transients. Circulation 109:1886–1891. doi: 10.1161/01.CIR.0000124231.98250.A8 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Yiru Guo
    • 1
  • Santosh K. Sanganalmath
    • 1
  • Wenjian Wu
    • 1
  • Xiaoping Zhu
    • 1
  • Yiming Huang
    • 2
  • Wei Tan
    • 1
  • Suzanne T. Ildstad
    • 2
  • Qianhong Li
    • 1
  • Roberto Bolli
    • 1
    Email author
  1. 1.Institute of Molecular CardiologyUniversity of LouisvilleLouisvilleUSA
  2. 2.Institute for Cellular TherapeuticsUniversity of LouisvilleLouisvilleUSA

Personalised recommendations