Remote ischemic preconditioning confers late protection against myocardial ischemia–reperfusion injury in mice by upregulating interleukin-10

  • Zheqing P. CaiEmail author
  • Nirmal Parajuli
  • Xiaoxu Zheng
  • Lewis Becker
Original Contribution


Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia–reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of interleukin (IL)-10. Mice were exposed to lower limb RIPC or sham ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120). These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. In IL-10 knockout mice, RIPC cardioprotection was lost, but it was mimicked by exogenous IL-10. Administration of IL-10 to isolated perfused hearts increased phosphorylation of the protein kinase Akt and limited infarct size after I-30/R-120. In wild-type mice, RIPC increased plasma and cardiac IL-10 protein levels and caused activation of Akt and endothelial nitric oxide synthase in the heart at 24 h, which was also blocked by anti-IL-10 receptor antibodies. In the gastrocnemius muscle, RIPC resulted in immediate inactivation of the phosphatase PTEN and activation of Stat3, with increased IL-10 expression 24 h later. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.


Remote ischemic preconditioning Interleukin-10 Reperfusion injury Phosphatase and tensin homologue deleted on chromosome ten Stat3 



Endothelial nitric oxide synthase


Interleukin 10 receptor subunit 1


Infarct size




Left ventricular developed pressure






Phosphatase and tensin homologue deleted on chromosome ten


Myocyte-specific PTEN knockout


Anti-IL-10 receptor antibody


Remote ischemic preconditioning


Signal transducer and activator of transcription 3


Stat3 inhibitor



This work was supported by Public Health Service grants HL88071 (to Z.P. Cai) and HL65608 (to L.C. Becker) from the National Heart, Lung and Blood Institute, National Institutes of Health.

Conflict of interest


Supplementary material

395_2012_277_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 32 kb)
395_2012_277_MOESM2_ESM.doc (34 kb)
Supplementary material 2 (DOC 34 kb)


  1. 1.
    Barrett JS, Wagner JG, Fisher SJ, Wahl RL (1991) Effect of intraperitoneal injection volume and antibody protein dose on the pharmacokinetics of intraperitoneally administered IgG2a kappa murine monoclonal antibody in the rat. Cancer Res 51:3434–3444PubMedGoogle Scholar
  2. 2.
    Birnbaum Y, Hale SL, Kloner RA (1997) Ischemic preconditioning at a distance: reduction of myocardial infarct size by partial reduction of blood supply combined with rapid stimulation of the gastrocnemius muscle in the rabbit. Circulation 96:1641–1646. doi: 10.1161/01.CIR.96.5.1641 PubMedCrossRefGoogle Scholar
  3. 3.
    Bøtker HE, Kharbanda R, Schmidt MR, Bøttcher M, Kaltoft AK, Terkelsen CJ, Munk K, Andersen NH, Hansen TM, Trautner S, Lassen JF, Christiansen EH, Krusell LR, Kristensen SD, Thuesen L, Nielsen SS, Rehling M, Sørensen HT, Redington AN, Nielsen TT (2010) Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet 375:727–734. doi: 10.1016/S0140-6736(09)62001-8 PubMedCrossRefGoogle Scholar
  4. 4.
    Breivik L, Helgeland E, Aarnes EK, Mrdalj J, Jonassen AK (2011) Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion. Basic Res Cardiol 106:135–145. doi: 10.1007/s00395-010-0133-0 PubMedCrossRefGoogle Scholar
  5. 5.
    Cai Z, Semenza GL (2005) PTEN activity is modulated during ischemia and reperfusion: involvement in the induction and decay of preconditioning. Circ Res 97:1351–1359. doi: 10.1161/01.RES.0000195656.52760.30 PubMedCrossRefGoogle Scholar
  6. 6.
    Cai ZP, Shen Z, Van Kaer L, Becker LC (2008) Ischemic preconditioning-induced cardioprotection is lost in mice with immunoproteasome subunit low molecular mass polypeptide-2 deficiency. FASEB J 22:4248–4257. doi: 10.1096/fj.08-105940 PubMedCrossRefGoogle Scholar
  7. 7.
    Dickson EW, Lorbar M, Porcaro WA, Fenton RA, Reinhardt CP, Gysembergh A, Przyklenk K (1999) Rabbit heart can be “preconditioned” via transfer of coronary effluent. Am J Physiol 277:H2451–H2457PubMedGoogle Scholar
  8. 8.
    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 96:11507–11512. doi: 10.1073/pnas.96.20.11507 PubMedCrossRefGoogle Scholar
  9. 9.
    Hausenloy DJ, Baxter G, Bell R, Bøtker HE, Davidson SM, Downey J, Heusch G, Kitakaze M, Lecour S, Mentzer R, Mocanu MM, Ovize M, Schulz R, Shannon R, Walker M, Walkinshaw G, Yellon DM (2010) Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations. Basic Res Cardiol 105:677–686. doi: 10.1007/s00395-010-0121-4 PubMedCrossRefGoogle Scholar
  10. 10.
    Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, Ashley E, Vichare S, Di Salvo C, Kolvekar S, Hayward M, Keogh B, MacAllister RJ, Yellon DM (2007) Effect of remote ischaemic preconditioning on myocardial injury in patients undergoing coronary artery bypass graft surgery: a randomised controlled trial. Lancet 370:575–579. doi: 10.1016/S0140-6736(07)61296-3 PubMedCrossRefGoogle Scholar
  11. 11.
    Hausenloy DJ, Yellon DM (2007) Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection. Heart Fail Rev 12:217–234. doi: 10.1007/s10741-007-9026-1 PubMedCrossRefGoogle Scholar
  12. 12.
    Hayward R, Nossuli TO, Scalia R, Lefer AM (1997) Cardioprotective effect of interleukin-10 in murine myocardial ischemia–reperfusion. Eur J Pharmacol. 334:157–163. doi: 10.1016/S0014-2999(97)01149-7 PubMedCrossRefGoogle Scholar
  13. 13.
    Heusch G, Musiolik J, Kottenberg E, Peters J, Jakob H, Thielmann M (2012) STAT5 activation and cardioprotection by remote ischemic preconditioning in humans: short communication. Circ Res 110:111–115. doi: 10.1161/CIRCRESAHA.111.259556 PubMedCrossRefGoogle Scholar
  14. 14.
    Jones SP, Trocha SD, Lefer DJ (2001) Cardioprotective actions of endogenous IL-10 are independent of iNOS. Am J Physiol Heart Circ Physiol 281:H48–H52PubMedGoogle Scholar
  15. 15.
    Karuppasamy P, Chaubey S, Dew T, Musto R, Sherwood R, Desai J, John L, Shah AM, Marber MS, Kunst G (2011) Remote intermittent ischemia before coronary artery bypass graft surgery: a strategy to reduce injury and inflammation? Basic Res Cardiol 106:511–519. doi: 10.1007/s00395-011-0185-9 PubMedCrossRefGoogle Scholar
  16. 16.
    Konstantinov IE, Arab S, Kharbanda RK, Li J, Cheung MM, Cherepanov V, Downey GP, Liu PP, Cukerman E, Coles JG, Redington AN (2004) The remote ischemic preconditioning stimulus modifies inflammatory gene expression in humans. Physiol Genomics 19:143–150. doi: 10.1152/physiolgenomics.00046.2004 PubMedCrossRefGoogle Scholar
  17. 17.
    Kottenberg E, Thielmann M, Bergmann L, Heine T, Jakob H, Heusch G, Peters J (2012) Protection by remote ischemic preconditioning during coronary artery bypass graft surgery with isoflurane but not propofol—a clinical trial. Acta Anaesthesiol Scand 56:30–38. doi: 10.1111/j.1399-6576.2011.02585.x PubMedCrossRefGoogle Scholar
  18. 18.
    Krishnamurthy P, Rajasingh J, Lambers E, Qin G, Losordo DW, Kishore R (2009) IL-10 inhibits inflammation and attenuates left ventricular remodeling after myocardial infarction via activation of STAT3 and suppression of HuR. Circ Res 104:e9–e18. doi: 10.1161/CIRCRESAHA.108.188243 PubMedCrossRefGoogle Scholar
  19. 19.
    Li J, Xuan W, Yan R, Tropak MB, Jean-St-Michel E, Liang W, Gladstone R, Backx PH, Kharbanda RK, Redington AN (2011) Remote preconditioning provides potent cardioprotection via PI3K/Akt activation and is associated with nuclear accumulation of β-catenin. Clin Sci (Lond) 120:451–462. doi: 10.1042/CS20100466 CrossRefGoogle Scholar
  20. 20.
    Lim SY, Yellon DM, Hausenloy DJ (2010) The neural and humoral pathways in remote limb ischemic preconditioning. Basic Res Cardiol 105:651–655. doi: 10.1007/s00395-010-0099-y PubMedCrossRefGoogle Scholar
  21. 21.
    Loukogeorgakis SP, Panagiotidou AT, Broadhead MW, Donald A, Deanfield JE, MacAllister RJ (2005) Remote ischemic preconditioning provides early and late protection against endothelial ischemia–reperfusion injury in humans: role of the autonomic nervous system. J Am Coll Cardiol. 46:450–456. doi: 10.1016/j.jacc.2005.04.044 PubMedCrossRefGoogle Scholar
  22. 22.
    Manukyan MC, Alvernaz CH, Poynter JA, Wang Y, Brewster BD, Weil BR, Abarbanell AM, Herrmann JL, Crowe BJ, Keck AC, Meldrum DR (2011) Interleukin-10 protects the ischemic heart from reperfusion injury via the STAT3 pathway. Surgery. 150:231–239. doi: 10.1016/j.surg.2011.05.017 PubMedCrossRefGoogle Scholar
  23. 23.
    McCord JM (1985) Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312:159–163PubMedCrossRefGoogle Scholar
  24. 24.
    Meador BM, Krzyszton CP, Johnson RW, Huey KA (2008) Effects of IL-10 and age on IL-6, IL-1β, and TNF-α responses in mouse skeletal and cardiac muscle to an acute inflammatory insult. J Appl Physiol 104:991–997. doi: 10.1152/japplphysiol.01079.2007 PubMedCrossRefGoogle Scholar
  25. 25.
    Mocanu MM, Yellon DM (2007) PTEN, the Achilles’ heel of myocardial ischaemia/reperfusion injury? Br J Pharmacol 150:833–838. doi: 10.1038/sj.bjp.0707155 PubMedCrossRefGoogle Scholar
  26. 26.
    Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683–765. doi: 10.1146/annurev.immunol.19.1.683 PubMedCrossRefGoogle Scholar
  27. 27.
    Moses MA, Addison PD, Neligan PC, Ashrafpour H, Huang N, McAllister SE, Lipa JE, Forrest CR, Pang CY (2005) Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism. Am J Physiol 289:R1609–R1617. doi: 10.1152/ajpregu.00395.2005 Google Scholar
  28. 28.
    Murry CE, Jennings RB, Reimer KA (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124–1136. doi: 10.1161/01.CIR.74.5.1124 PubMedCrossRefGoogle Scholar
  29. 29.
    Nemenoff RA, Simpson PA, Furgeson SB, Kaplan-Albuquerque N, Crossno J, Garl PJ, Cooper J, Weiser-Evans MC (2008) Targeted deletion of PTEN in smooth muscle cells results in vascular remodeling and recruitment of progenitor cells through induction of stromal cell-derived factor-1α. Circ Res 102:1036–1045. doi: 10.1161/CIRCRESAHA.107.169896 PubMedCrossRefGoogle Scholar
  30. 30.
    O’Farrell AM, Liu Y, Moore KW, Mui AL (1998) IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways. EMBO J 17:1006–1018. doi: 10.1093/emboj/17.4.1006 PubMedCrossRefGoogle Scholar
  31. 31.
    Parajuli N, Yuan Y, Zheng X, Bedja D, Cai ZP (2012) Phosphatase PTEN is critically involved in post-myocardial infarction remodeling through the Akt/interleukin-10 signaling pathway. Basic Res Cardiol 107:248. doi: 10.1007/s00395-012-0248-6 PubMedCrossRefGoogle Scholar
  32. 32.
    Peters J (2011) Remote ischaemic preconditioning of the heart: remote questions, remote importance, or remote preconditions? Basic Res Cardiol 106:507–509. doi: 10.1007/s00395-011-0187-7 PubMedCrossRefGoogle Scholar
  33. 33.
    Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P (1993) Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 87:893–899. doi: 10.1161/01.CIR.87.3.893 PubMedCrossRefGoogle Scholar
  34. 34.
    Schwartz Longacre L, Kloner RA, Arai AE, Baines CP, Bolli R, Braunwald E, Downey J, Gibbons RJ, Gottlieb RA, Heusch G, Jennings RB, Lefer DJ, Mentzer RM, Murphy E, Ovize M, Ping P, Przyklenk K, Sack MN, Vander Heide RS, Vinten-Johansen J, Yellon DM, National Heart, Lung, and Blood Institute, National Institutes of Health (2011) New horizons in cardioprotection: recommendations from the 2010 National Heart, Lung, and Blood Institute Workshop. Circulation 124:1172–1179. doi: 10.1161/CIRCULATIONAHA.111.032698 PubMedCrossRefGoogle Scholar
  35. 35.
    Sharma S, Yang B, Xi X, Grotta JC, Aronowski J, Savitz SI (2011) IL-10 directly protects cortical neurons by activating PI-3 kinase and STAT-3 pathways. Brain Res. 1373:189–194. doi: 10.1016/j.brainres.2010.11.096 PubMedCrossRefGoogle Scholar
  36. 36.
    Shimizu M, Saxena P, Konstantinov IE, Cherepanov V, Cheung MM, Wearden P, Zhangdong H, Schmidt M, Downey GP, Redington AN (2010) Remote ischemic preconditioning decreases adhesion and selectively modifies functional responses of human neutrophils. J Surg Res. 158:155–161. doi: 10.1016/j.jss.2008.08.010 PubMedCrossRefGoogle Scholar
  37. 37.
    Smith RM, Suleman N, Lacerda L, Opie LH, Akira S, Chien KR, Sack MN (2004) Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc Res 63:611–616. doi: 10.1016/j.cardiores.2004.06.019 PubMedCrossRefGoogle Scholar
  38. 38.
    Staples KJ, Smallie T, Williams LM, Foey A, Burke B, Foxwell BM, Ziegler-Heitbrock L (2007) IL-10 induces IL-10 in primary human monocyte-derived macrophages via the transcription factor Stat3. J Immunol 178:4779–4785PubMedGoogle Scholar
  39. 39.
    Steensrud T, Li J, Dai X, Manlhiot C, Kharbanda RK, Tropak M, Redington A (2010) Pretreatment with the nitric oxide donor SNAP or nerve transection blocks humoral preconditioning by remote limb ischemia or intra-arterial adenosine. Am J Physiol 299:H1598–H1603. doi: 10.1152/ajpheart.00396.2010 Google Scholar
  40. 40.
    Suleman N, Somers S, Smith R, Opie LH, Lecour SC (2008) Dual activation of STAT-3 and Akt is required during the trigger phase of ischaemic preconditioning. Cardiovasc Res 79:127–133. doi: 10.1093/cvr/cvn067 PubMedCrossRefGoogle Scholar
  41. 41.
    Tamareille S, Mateus V, Ghaboura N, Jeanneteau J, Croué A, Henrion D, Furber A, Prunier F (2011) RISK and SAFE signaling pathway interactions in remote limb ischemic preconditioning in combination with local ischemic postconditioning. Basic Res Cardiol 106:1329–1339. doi: 10.1007/s00395-011-0210-z PubMedCrossRefGoogle Scholar
  42. 42.
    Tapuria N, Junnarkar S, Abu-Amara M, Fuller B, Seifalian AM, Davidson BR (2012) Modulation of microcirculatory changes in the late phase of hepatic ischaemia-reperfusion injury by remote ischaemic preconditioning. HPB (Oxford). 14:87–97. doi: 10.1111/j.1477-2574.2011.00407.x CrossRefGoogle Scholar
  43. 43.
    Thielmann M, Kottenberg E, Boengler K, Raffelsieper C, Neuhaeuser M, Peters J, Jakob H, Heusch G (2010) Remote ischemic preconditioning reduces myocardial injury after coronary artery bypass surgery with crystalloid cardioplegic arrest. Basic Res Cardiol 105:657–664. doi: 10.1007/s00395-010-0104-5 PubMedCrossRefGoogle Scholar
  44. 44.
    Thielmann M (2012) Remote ischemic preconditioning in cardiac surgery: caught between clinical relevance and statistical significance? Basic Res Cardiol 107:259. doi: 10.1007/s00395-012-0259-3 CrossRefGoogle Scholar
  45. 45.
    Tokuno S, Hinokiyama K, Tokuno K, Löwbeer C, Hansson LO, Valen G (2002) Spontaneous ischemic events in the brain and heart adapt the hearts of severely atherosclerotic mice to ischemia. Arterioscler Thromb Vasc Biol 22:995–1001. doi: 10.1161/01.ATV.0000017703.87741.12 PubMedCrossRefGoogle Scholar
  46. 46.
    Wagner R, Piler P, Bedanova H, Adamek P, Grodecka L, Freiberger T (2010) Myocardial injury is decreased by late remote ischaemic preconditioning and aggravated by tramadol in patients undergoing cardiac surgery: a randomised controlled trial. Interact Cardiovasc Thorac Surg. 11:758–762. doi: 10.1510/icvts.2010.243600 PubMedCrossRefGoogle Scholar
  47. 47.
    Wolska BM, Solaro RJ (1996) Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry. Am J Physiol 271:H1250–H1255PubMedGoogle Scholar
  48. 48.
    Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, Bolli R (2007) Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription 1/3 pathway. Circulation 116:535–544. doi: 10.1161/CIRCULATIONAHA.107.689471 PubMedCrossRefGoogle Scholar
  49. 49.
    Yang Z, Zingarelli B, Szabó C (2000) Crucial role of endogenous interleukin-10 production in myocardial ischemia/reperfusion injury. Circulation 101:1019–1026. doi: 10.1161/01.CIR.101.9.1019 PubMedCrossRefGoogle Scholar
  50. 50.
    Young PJ, Dalley P, Garden A, Horrocks C, La Flamme A, Mahon B, Miller J, Pilcher J, Weatherall M, Williams J, Young W, Beasley R (2012) A pilot study investigating the effects of remote ischemic preconditioning in high-risk cardiac surgery using a randomised controlled double-blind protocol. Basic Res Cardiol 107:256. doi: 10.1007/s00395-012-0256-6 CrossRefGoogle Scholar
  51. 51.
    Zhang QG, Han D, Xu J, Lv Q, Wang R, Yin XH, Xu TL, Zhang GY (2006) Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt. Neuroscience 143:431–444. doi: 10.1016/j.neuroscience.2006.07.049 PubMedCrossRefGoogle Scholar
  52. 52.
    Zhou J, Wulfkuhle J, Zhang H, Gu P, Yang Y, Deng J, Margolick JB, Liotta LA, Petricoin E 3rd, Zhang Y (2007) Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proc Natl Acad Sci USA 104:16158–16163. doi: 10.1073/pnas.0702596104 PubMedCrossRefGoogle Scholar
  53. 53.
    Zu L, Bedja D, Fox-Talbot K, Gabrielson KL, Van Kaer L, Becker LC, Cai ZP (2010) Evidence for a role of immunoproteasomes in regulating cardiac muscle mass in diabetic mice. J Mol Cell Cardiol. 49:5–15. doi: 10.1016/j.yjmcc.2010.02.007 PubMedCrossRefGoogle Scholar
  54. 54.
    Zu L, Zheng X, Wang B, Parajuli N, Steenbergen C, Becker LC, Cai ZP (2011) Ischemic preconditioning attenuates mitochondrial localization of PTEN induced by ischemia–reperfusion. Am J Physiol 300:H2177–H2186. doi: 10.1152/ajpheart.01138.2010 Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Zheqing P. Cai
    • 1
    Email author
  • Nirmal Parajuli
    • 1
  • Xiaoxu Zheng
    • 1
  • Lewis Becker
    • 1
  1. 1.Division of Cardiology, Department of MedicineJohns Hopkins University School of MedicineBaltimoreUSA

Personalised recommendations