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Caspase Inhibition Via A3 Adenosine Receptors: A New Cardioprotective Mechanism Against Myocardial Infarction

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Abstract

Purpose

2-CL-IB-MECA, (A3 adenosine receptor agonist)(A3AR) mediated cardioprotection is well documented although the associated intracellular signalling pathways remain unclear. Here we demonstrate a role of the pro-survival signalling pathways MEK1/2-ERK1/2 and PI3K/AKT and their effect on modifying Caspase-3 activity in A3AR mediated cardioprotection.

Methods

Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/hypoxia and reperfusion/reoxygenation, respectively. 2-CL-IB-MECA (1 nM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of either the PI3K inhibitor Wortmannin (5 nM) or MEK1/2 inhibitor UO126 (10 μM). Heart tissues were harvested for assessment of p-ERK1/2(Thr202/Tyr204) or p-AKT (Ser-473) status or underwent infarct size assessment. Cardiac myocytes underwent flow-cytometric analysis for apoptosis, necrosis, cleaved-caspase 3/p-BAD (Ser-112 and Ser-136) activity post-reoxygenation.

Results

2-CL-IB-MECA significantly reduced infarct size compared to non-treated controls, where co-administration with either of the kinase inhibitors abolished the infarct sparing effects. Administration of 2-CL-IB-MECA at reperfusion significantly upregulated the status of p-ERK1/2 and p-AKT compared to time matched controls in a UO126 and Wortmannin sensitive manner respectively. 2-CL-IB-MECA when administered throughout reoxygenation significantly reduced apoptosis, necrosis, cleaved-caspase 3 activity and increased p-BAD (Ser-112) and p-BAD (Ser-136) activity in myocytes subjected to hypoxia/reoxygenation injury. The cytoprotective effect was abolished by co-administration with the kinase inhibitors Wortmannin and/or UO126.

Conclusions

We have described the molecular mechanisms associated with A3AR mediated cardioprotection indicating a role for the pro-survival signalling pathways that decrease caspase-3 activity. These observations provide novel insight into the pharmacological effects of A3ARs in ameliorating myocardial ischaemia/reperfusion injury.

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References

  1. Van Wylen DG, Schmit TJ, Lasley RD, Gingell RL, Mentzer Jr RM. Cardiac microdialysis in isolated rat hearts: interstitial purine metabolites during ischemia. Am J Physiol. 1992;262(6 Pt 2):H1934–8.

    PubMed  Google Scholar 

  2. Tucker AL, Linden J. Cloned receptors and cardiovascular responses to adenosine. Cardiovasc Res. 1993;27:62–7.

    Article  CAS  PubMed  Google Scholar 

  3. Ely SW, Mentzer Jr RM, Lasley RD, Lee BK, Berne RM. Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J Thorac Cardiovasc Surg. 1985;90:549–56.

    CAS  PubMed  Google Scholar 

  4. Olafsson B, Forman MB, Puett DW, et al. Reduction of reperfusion injury in the canine preparation by intracoronary adenosine: importance of the endothelium and the no-reflow phenomenon. Circulation. 1987;76:1135–45.

    Article  CAS  PubMed  Google Scholar 

  5. Toombs CF, McGee S, Johnston WE, Vinten-Johansen J. Myocardial protective effects of adenosine. Infarct size reduction with pretreatment and continued receptor stimulation during ischemia. Circulation. 1992;86:986–94.

    Article  CAS  PubMed  Google Scholar 

  6. Lasley RD, Mentzer Jr RM. Protective effects of adenosine in the reversibly injured heart. Ann Thorac Surg. 1995;60:843–6.

    Article  CAS  PubMed  Google Scholar 

  7. Ge ZD, van der Hoeven D, Maas JE, Wan TC, Auchampach JA. A(3) adenosine receptor activation during reperfusion reduces infarct size through actions on bone marrow-derived cells. J Mol Cell Cardiol. 2010;49:280–6.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Maddock HL, Mocanu MM, Yellon DM. Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury. Am J Physiol Heart Circ Physiol. 2002;283:H1307–13.

    CAS  PubMed  Google Scholar 

  9. Liu GS, Thornton J, Van Winkle DM, Stanley AW, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991;84:350–6.

    Article  CAS  PubMed  Google Scholar 

  10. Ge ZD, Peart JN, Kreckler LM, et al. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide] reduces ischemia/reperfusion injury in mice by activating the A3 adenosine receptor. J Pharmacol Exp Ther. 2006;319:1200–10.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. McIntosh VJ, Lasley RD. Adenosine receptor-mediated cardioprotection: are all 4 subtypes required or redundant? J Cardiovasc Pharmacol Ther. 2012;17:21–33.

    Article  CAS  PubMed  Google Scholar 

  12. Headrick JP, Lasley RD. Adenosine receptors and reperfusion injury of the heart. Handb Exp Pharmacol. 2009;193:189–214.

    Article  CAS  PubMed  Google Scholar 

  13. Jordan JE, Thourani VH, Auchampach JA, Robinson JA, Wang NP, Vinten-Johansen J. A(3) adenosine receptor activation attenuates neutrophil function and neutrophil-mediated reperfusion injury. Am J Physiol. 1999;277(5 Pt 2):H1895–905.

    CAS  PubMed  Google Scholar 

  14. Maddock HL, Gardner NM, Khandoudi N, Bril A, Broadley KJ. Protection from myocardial stunning by ischaemia and hypoxia with the adenosine A3 receptor agonist. IB-MECA. Eur J Pharmacol. 2003;477:235–45.

    Article  CAS  PubMed  Google Scholar 

  15. Wan TC, Ge ZD, Tampo A, Mio Y, Bienengraeber MW, Tracey WR, et al. The A3 adenosine receptor agonist CP-532,903 [N6-(2,5-dichlorobenzyl)-3′-aminoadenosine-5′-N-methylcarboxamide] protects against myocardial ischemia/reperfusion injury via the sarcolemmal ATP-sensitive potassium channel. J Pharmacol Exp Ther. 2008;324:234–43.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Auchampach JA, Ge ZD, Wan TC, Moore J, Gross GJ. A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs. Am J Physiol Heart Circ Physiol. 2003;285:H607–13.

    CAS  PubMed  Google Scholar 

  17. Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ, Freeman TC. Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol. 1996;118:1461–8.

    Article  CAS  PubMed  Google Scholar 

  18. Morrison RR, Teng B, Oldenburg PJ, Katwa LC, Schnermann JB, Mustafa SJ. Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes. Am J Physiol Heart Circ Physiol. 2006;291:H1875–82.

    Article  CAS  PubMed  Google Scholar 

  19. Matot I, Weiniger CF, Zeira E, Galun E, Joshi BV, Jacobson KA. A3 adenosine receptors and mitogen-activated protein kinases in lung injury following in vivo reperfusion. Crit Care. 2006;10:R65.

    Article  PubMed  Google Scholar 

  20. Rivo J, Zeira E, Galun E, Matot I. Activation of A3 adenosine receptor provides lung protection against ischemia-reperfusion injury associated with reduction in apoptosis. Am J Transplant. 2004;4:1941–8.

    Article  CAS  PubMed  Google Scholar 

  21. Rivo J, Zeira E, Galun E, Matot I. Activation of A3 adenosine receptors attenuates lung injury after in vivo reperfusion. Anesthesiology. 2004;101:1153–9.

    Article  CAS  PubMed  Google Scholar 

  22. Von Lubitz DK, Simpson KL, Lin RC. Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke. Ann N Y Acad Sci. 2001;939:85–96.

    Article  Google Scholar 

  23. Von Lubitz DK, Lin RC, Boyd M, Bischofberger N, Jacobson KA. Chronic administration of adenosine A3 receptor agonist and cerebral ischemia: neuronal and glial effects. Eur J Pharmacol. 1999;367:157–63.

    Article  Google Scholar 

  24. Gao Z, Li BS, Day YJ, Linden J. A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukemia 2H3 mast cells from apoptosis. Mol Pharmacol. 2001;59:76–82.

    CAS  PubMed  Google Scholar 

  25. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001;81:807–69.

    CAS  PubMed  Google Scholar 

  26. Hausenloy DJ. Signalling pathways in ischaemic postconditioning. Thromb Haemost. 2009;101:626–34.

    CAS  PubMed  Google Scholar 

  27. Hausenloy DJ, Mocanu MM, Yellon DM. Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning. Cardiovasc Res. 2004;63:305–12.

    Article  CAS  PubMed  Google Scholar 

  28. Hausenloy DJ, Yellon DM. Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection. Heart Fail Rev. 2007;12:217–34.

    Article  CAS  PubMed  Google Scholar 

  29. Shiryaev A, Moens U. Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: menage a trois or menage a quatre? Cell Signal. 2010;22:1185–92.

    Article  CAS  PubMed  Google Scholar 

  30. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res. 2004;61:448–60.

    Article  CAS  PubMed  Google Scholar 

  31. Germack R, Griffin M, Dickenson JM. Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes. J Mol Cell Cardiol. 2004;37:989–99.

    Article  CAS  PubMed  Google Scholar 

  32. Germack R, Dickenson JM. Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes. J Mol Cell Cardiol. 2005;39:429–42.

    Article  CAS  PubMed  Google Scholar 

  33. Fox R, Aubert M. Flow cytometric detection of activated caspase-3. Methods Mol Biol. 2008;414:47–56.

    CAS  PubMed  Google Scholar 

  34. Abbracchio MP, Cattabeni F. Brain adenosine receptors as targets for therapeutic intervention in neurodegenerative diseases. Ann N Y Acad Sci. 1999;890:79–92.

    Article  CAS  PubMed  Google Scholar 

  35. Abe J, Baines CP, Berk BC. Role of mitogen-activated protein kinases in ischemia and reperfusion injury : the good and the bad. Circ Res. 2000;86:607–9.

    Article  CAS  PubMed  Google Scholar 

  36. Yue TL, Wang C, Gu JL, et al. Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res. 2000;86:692–9.

    Article  CAS  PubMed  Google Scholar 

  37. Schulte G, Fredholm BB. Signaling pathway from the human adenosine A(3) receptor expressed in Chinese hamster ovary cells to the extracellular signal-regulated kinase 1/2. Mol Pharmacol. 2002;62:1137–46.

    Article  CAS  PubMed  Google Scholar 

  38. Graham S, Combes P, Crumiere M, Klotz KN, Dickenson JM. Regulation of p42/p44 mitogen-activated protein kinase by the human adenosine A3 receptor in transfected CHO cells. Eur J Pharmacol. 2001;420:19–26.

    Article  CAS  PubMed  Google Scholar 

  39. Tracey WR, Magee W, Masamune H, Oleynek JJ, Hill RJ. Selective activation of adenosine A3 receptors with N6-(3-chlorobenzyl)-5′-N-methylcarboxamidoadenosine (CB-MECA) provides cardioprotection via KATP channel activation. Cardiovasc Res. 1998;40:138–45.

    Article  CAS  PubMed  Google Scholar 

  40. Bullard AJ, Govewalla P, Yellon DM. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. Basic Res Cardiol. 2005;100:397–403.

    Article  CAS  PubMed  Google Scholar 

  41. Lim SY, Davidson SM, Paramanathan AJ, Smith CC, Yellon DM, Hausenloy DJ. The novel adipocytokine visfatin exerts direct cardioprotective effects. J Cell Mol Med. 2008;12:1395–403.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Park SS, Zhao H, Jang Y, Mueller RA, Xu Z. N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide confers cardioprotection at reperfusion by inhibiting mitochondrial permeability transition pore opening via glycogen synthase kinase 3 beta. J Pharmacol Exp Ther. 2006;318:124–31.

    Article  CAS  PubMed  Google Scholar 

  43. Jin YC, Kim KJ, Kim YM, et al. Anti-apoptotic effect of magnolol in myocardial ischemia and reperfusion injury requires extracellular signal-regulated kinase1/2 pathways in rat in vivo. Exp Biol Med. 2008;233:1280–8.

    Article  CAS  Google Scholar 

  44. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol. 2005;288:H971–6.

    Article  CAS  PubMed  Google Scholar 

  45. Danial NN. BAD: undertaker by night, candyman by day. Oncogene. 2008;27 Suppl 1:S53–70.

    Article  CAS  PubMed  Google Scholar 

  46. Scabini M, Stellari F, Cappella P, Rizzitano S, Texido G, Pesenti E. In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation. Apoptosis. 2011;16:198–207.

    Article  CAS  PubMed  Google Scholar 

  47. Schulman D, Latchman DS, Yellon DM. Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway. Am J Physiol Heart Circ Physiol. 2002;283:H1481–8.

    CAS  PubMed  Google Scholar 

  48. Brar BK, Jonassen AK, Stephanou A, Santilli G, Railson J, Knight RA, et al. Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway. J Biol Chem. 2000;275:8508–14.

    Article  CAS  PubMed  Google Scholar 

  49. Jonassen AK, Sack MN, Mjos OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res. 2001;89:1191–8.

    Article  CAS  PubMed  Google Scholar 

  50. Guo WP, Fu XG, Jiang SM, Wu JZ. Neuregulin-1 regulates the expression of Akt, Bcl-2, and Bad signaling after focal cerebral ischemia in rats. Biochem Cell Biol. 2010;88:649–54.

    Article  CAS  PubMed  Google Scholar 

  51. Mehrhof FB, Muller FU, Bergmann MW, et al. In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein. Circulation. 2001;104:2088–94.

    Article  CAS  PubMed  Google Scholar 

  52. Thourani VH, Nakamura M, Ronson RS, Jordan JE, Zhao ZQ, Levy JH, et al. Adenosine A(3)-receptor stimulation attenuates postischemic dysfunction through K(ATP) channels. Am J Physiol. 1999;277(1 Pt 2):H228–35.

    CAS  PubMed  Google Scholar 

  53. Yang XM, Krieg T, Cui L, Downey JM, Cohen MV. NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. J Mol Cell Cardiol. 2004;36:411–21.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was funded by the British Heart Foundation PhD scholarship grantFS/03/018/15145.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Correspondence to Helen L. Maddock.

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Hussain, A., Gharanei, A.M., Nagra, A.S. et al. Caspase Inhibition Via A3 Adenosine Receptors: A New Cardioprotective Mechanism Against Myocardial Infarction. Cardiovasc Drugs Ther 28, 19–32 (2014). https://doi.org/10.1007/s10557-013-6500-y

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  • DOI: https://doi.org/10.1007/s10557-013-6500-y

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