Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 May 15;101(10):2182–2198. doi: 10.1172/JCI1258

Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved.

Y Qiu 1, P Ping 1, X L Tang 1, S Manchikalapudi 1, A Rizvi 1, J Zhang 1, H Takano 1, W J Wu 1, S Teschner 1, R Bolli 1
PMCID: PMC508806  PMID: 9593774

Abstract

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.

Full Text

The Full Text of this article is available as a PDF (516.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong S. C., Hoover D. B., Delacey M. H., Ganote C. E. Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes. J Mol Cell Cardiol. 1996 Jul;28(7):1479–1492. doi: 10.1006/jmcc.1996.0138. [DOI] [PubMed] [Google Scholar]
  2. Armstrong S., Downey J. M., Ganote C. E. Preconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and calphostin C, a protein kinase C inhibitor. Cardiovasc Res. 1994 Jan;28(1):72–77. doi: 10.1093/cvr/28.1.72. [DOI] [PubMed] [Google Scholar]
  3. Armstrong S., Ganote C. E. Preconditioning of isolated rabbit cardiomyocytes: effects of glycolytic blockade, phorbol esters, and ischaemia. Cardiovasc Res. 1994 Nov;28(11):1700–1706. doi: 10.1093/cvr/28.11.1700. [DOI] [PubMed] [Google Scholar]
  4. Baxter G. F., Goma F. M., Yellon D. M. Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium. Basic Res Cardiol. 1997 Jun;92(3):159–167. doi: 10.1007/BF00788633. [DOI] [PubMed] [Google Scholar]
  5. Baxter G. F., Goma F. M., Yellon D. M. Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium. Br J Pharmacol. 1995 May;115(2):222–224. doi: 10.1111/j.1476-5381.1995.tb15866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baxter G. F., Marber M. S., Patel V. C., Yellon D. M. Adenosine receptor involvement in a delayed phase of myocardial protection 24 hours after ischemic preconditioning. Circulation. 1994 Dec;90(6):2993–3000. doi: 10.1161/01.cir.90.6.2993. [DOI] [PubMed] [Google Scholar]
  7. Bolli R., Bhatti Z. A., Tang X. L., Qiu Y., Zhang Q., Guo Y., Jadoon A. K. Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide. Circ Res. 1997 Jul;81(1):42–52. doi: 10.1161/01.res.81.1.42. [DOI] [PubMed] [Google Scholar]
  8. Bolli R., Manchikalapudi S., Tang X. L., Takano H., Qiu Y., Guo Y., Zhang Q., Jadoon A. K. 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. 1997 Dec;81(6):1094–1107. doi: 10.1161/01.res.81.6.1094. [DOI] [PubMed] [Google Scholar]
  9. Brew E. C., Mitchell M. B., Rehring T. F., Gamboni-Robertson F., McIntyre R. C., Jr, Harken A. H., Banerjee A. Role of bradykinin in cardiac functional protection after global ischemia-reperfusion in rat heart. Am J Physiol. 1995 Oct;269(4 Pt 2):H1370–H1378. doi: 10.1152/ajpheart.1995.269.4.H1370. [DOI] [PubMed] [Google Scholar]
  10. Burckhartt B., Yang X. M., Tsuchida A., Mullane K. M., Downey J. M., Cohen M. V. Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits. Cardiovasc Res. 1995 May;29(5):653–657. [PubMed] [Google Scholar]
  11. Cohen M. V., Yang X. M., Downey J. M. Conscious rabbits become tolerant to multiple episodes of ischemic preconditioning. Circ Res. 1994 May;74(5):998–1004. doi: 10.1161/01.res.74.5.998. [DOI] [PubMed] [Google Scholar]
  12. Goto M., Liu Y., Yang X. M., Ardell J. L., Cohen M. V., Downey J. M. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995 Sep;77(3):611–621. doi: 10.1161/01.res.77.3.611. [DOI] [PubMed] [Google Scholar]
  13. Haessler R., Kuzume K., Chien G. L., Wolff R. A., Davis R. F., Van Winkle D. M. Anaesthetics alter the magnitude of infarct limitation by ischaemic preconditioning. Cardiovasc Res. 1994 Oct;28(10):1574–1580. doi: 10.1093/cvr/28.10.1574. [DOI] [PubMed] [Google Scholar]
  14. Herbert J. M., Augereau J. M., Gleye J., Maffrand J. P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990 Nov 15;172(3):993–999. doi: 10.1016/0006-291x(90)91544-3. [DOI] [PubMed] [Google Scholar]
  15. Kaibuchi K., Fukumoto Y., Oku N., Takai Y., Arai K., Muramatsu M. Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C. J Biol Chem. 1989 Aug 15;264(23):13489–13496. [PubMed] [Google Scholar]
  16. Kitakaze M., Node K., Minamino T., Komamura K., Funaya H., Shinozaki Y., Chujo M., Mori H., Inoue M., Hori M. Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase. Circulation. 1996 Feb 15;93(4):781–791. doi: 10.1161/01.cir.93.4.781. [DOI] [PubMed] [Google Scholar]
  17. Kuzuya T., Hoshida S., Yamashita N., Fuji H., Oe H., Hori M., Kamada T., Tada M. Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia. Circ Res. 1993 Jun;72(6):1293–1299. doi: 10.1161/01.res.72.6.1293. [DOI] [PubMed] [Google Scholar]
  18. Li X. Y., McCay P. B., Zughaib M., Jeroudi M. O., Triana J. F., Bolli R. Demonstration of free radical generation in the "stunned" myocardium in the conscious dog and identification of major differences between conscious and open-chest dogs. J Clin Invest. 1993 Aug;92(2):1025–1041. doi: 10.1172/JCI116608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Y., Kloner R. A. Does protein kinase C play a role in ischemic preconditioning in rat hearts? Am J Physiol. 1995 Jan;268(1 Pt 2):H426–H431. doi: 10.1152/ajpheart.1995.268.1.H426. [DOI] [PubMed] [Google Scholar]
  20. Liu Y., Cohen M. V., Downey J. M. Chelerythrine, a highly selective protein kinase C inhibitor, blocks the anti-infarct effect of ischemic preconditioning in rabbit hearts. Cardiovasc Drugs Ther. 1994 Dec;8(6):881–882. doi: 10.1007/BF00877409. [DOI] [PubMed] [Google Scholar]
  21. Liu Y., Tsuchida A., Cohen M. V., Downey J. M. Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J Mol Cell Cardiol. 1995 Mar;27(3):883–892. doi: 10.1016/0022-2828(95)90038-1. [DOI] [PubMed] [Google Scholar]
  22. Liu Y., Ytrehus K., Downey J. M. Evidence that translocation of protein kinase C is a key event during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol. 1994 May;26(5):661–668. doi: 10.1006/jmcc.1994.1078. [DOI] [PubMed] [Google Scholar]
  23. Maldonado C., Qiu Y., Tang X. L., Cohen M. V., Auchampach J., Bolli R. Role of adenosine receptors in late preconditioning against myocardial stunning in conscious rabbits. Am J Physiol. 1997 Sep;273(3 Pt 2):H1324–H1332. doi: 10.1152/ajpheart.1997.273.3.H1324. [DOI] [PubMed] [Google Scholar]
  24. Marber M. S., Latchman D. S., Walker J. M., Yellon D. M. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation. 1993 Sep;88(3):1264–1272. doi: 10.1161/01.cir.88.3.1264. [DOI] [PubMed] [Google Scholar]
  25. Marber M. S., Yellon D. M. Myocardial adaptation, stress proteins, and the second window of protection. Ann N Y Acad Sci. 1996 Sep 30;793:123–141. doi: 10.1111/j.1749-6632.1996.tb33510.x. [DOI] [PubMed] [Google Scholar]
  26. Mitchell M. B., Meng X., Ao L., Brown J. M., Harken A. H., Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res. 1995 Jan;76(1):73–81. doi: 10.1161/01.res.76.1.73. [DOI] [PubMed] [Google Scholar]
  27. Newton A. C. Protein kinase C: structure, function, and regulation. J Biol Chem. 1995 Dec 1;270(48):28495–28498. doi: 10.1074/jbc.270.48.28495. [DOI] [PubMed] [Google Scholar]
  28. Ping P., Zhang J., Qiu Y., Tang X. L., Manchikalapudi S., Cao X., Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res. 1997 Sep;81(3):404–414. doi: 10.1161/01.res.81.3.404. [DOI] [PubMed] [Google Scholar]
  29. Przyklenk K., Sussman M. A., Simkhovich B. Z., Kloner R. A. Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation. 1995 Sep 15;92(6):1546–1557. doi: 10.1161/01.cir.92.6.1546. [DOI] [PubMed] [Google Scholar]
  30. Qiu Y., Rizvi A., Tang X. L., Manchikalapudi S., Takano H., Jadoon A. K., Wu W. J., Bolli R. Nitric oxide triggers late preconditioning against myocardial infarction in conscious rabbits. Am J Physiol. 1997 Dec;273(6 Pt 2):H2931–H2936. doi: 10.1152/ajpheart.1997.273.6.H2931. [DOI] [PubMed] [Google Scholar]
  31. Rehring T. F., Friese R. S., Cleveland J. C., Meng X., Robertson F. G., Harken A. H., Bannerjee A. Alpha-adrenergic preservation of myocardial pH during ischemia is PKC isoform dependent. J Surg Res. 1996 Jun;63(1):324–327. doi: 10.1006/jsre.1996.0269. [DOI] [PubMed] [Google Scholar]
  32. Sakamoto J., Miura T., Goto M., Iimura O. Limitation of myocardial infarct size by adenosine A1 receptor activation is abolished by protein kinase C inhibitors in the rabbit. Cardiovasc Res. 1995 May;29(5):682–688. [PubMed] [Google Scholar]
  33. Schwartz L. M., Jennings R. B., Reimer K. A. Premedication with the opioid analgesic butorphanol raises the threshold for ischemic preconditioning in dogs. Basic Res Cardiol. 1997 Apr;92(2):106–114. doi: 10.1007/BF00805571. [DOI] [PubMed] [Google Scholar]
  34. Simkhovich B. Z., Przyklenk K., Hale S. L., Patterson M., Kloner R. A. Direct evidence that ischemic preconditioning does not cause protein kinase C translocation in rabbit heart. Cardiovasc Res. 1996 Dec;32(6):1064–1070. doi: 10.1016/s0008-6363(96)00181-2. [DOI] [PubMed] [Google Scholar]
  35. Speechly-Dick M. E., Grover G. J., Yellon D. M. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res. 1995 Nov;77(5):1030–1035. doi: 10.1161/01.res.77.5.1030. [DOI] [PubMed] [Google Scholar]
  36. Speechly-Dick M. E., Mocanu M. M., Yellon D. M. Protein kinase C. Its role in ischemic preconditioning in the rat. Circ Res. 1994 Sep;75(3):586–590. doi: 10.1161/01.res.75.3.586. [DOI] [PubMed] [Google Scholar]
  37. Steinberg S. F., Goldberg M., Rybin V. O. Protein kinase C isoform diversity in the heart. J Mol Cell Cardiol. 1995 Jan;27(1):141–153. doi: 10.1016/s0022-2828(08)80014-4. [DOI] [PubMed] [Google Scholar]
  38. Strasser R. H., Braun-Dullaeus R., Walendzik H., Marquetant R. Alpha 1-receptor-independent activation of protein kinase C in acute myocardial ischemia. Mechanisms for sensitization of the adenylyl cyclase system. Circ Res. 1992 Jun;70(6):1304–1312. doi: 10.1161/01.res.70.6.1304. [DOI] [PubMed] [Google Scholar]
  39. Sun J. Z., Tang X. L., Knowlton A. A., Park S. W., Qiu Y., Bolli R. Late preconditioning against myocardial stunning. An endogenous protective mechanism that confers resistance to postischemic dysfunction 24 h after brief ischemia in conscious pigs. J Clin Invest. 1995 Jan;95(1):388–403. doi: 10.1172/JCI117667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sun J. Z., Tang X. L., Park S. W., Qiu Y., Turrens J. F., Bolli R. Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs. J Clin Invest. 1996 Jan 15;97(2):562–576. doi: 10.1172/JCI118449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tang X. L., Qiu Y., Park S. W., Sun J. Z., Kalya A., Bolli R. Time course of late preconditioning against myocardial stunning in conscious pigs. Circ Res. 1996 Sep;79(3):424–434. doi: 10.1161/01.res.79.3.424. [DOI] [PubMed] [Google Scholar]
  42. Triana J. F., Li X. Y., Jamaluddin U., Thornby J. I., Bolli R. Postischemic myocardial "stunning". Identification of major differences between the open-chest and the conscious dog and evaluation of the oxygen radical hypothesis in the conscious dog. Circ Res. 1991 Sep;69(3):731–747. doi: 10.1161/01.res.69.3.731. [DOI] [PubMed] [Google Scholar]
  43. Tsuchida A., Liu Y., Liu G. S., Cohen M. V., Downey J. M. alpha 1-adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res. 1994 Sep;75(3):576–585. doi: 10.1161/01.res.75.3.576. [DOI] [PubMed] [Google Scholar]
  44. Vahlhaus C., Schulz R., Post H., Onallah R., Heusch G. No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ Res. 1996 Sep;79(3):407–414. doi: 10.1161/01.res.79.3.407. [DOI] [PubMed] [Google Scholar]
  45. Yang X. M., Baxter G. F., Heads R. J., Yellon D. M., Downey J. M., Cohen M. V. Infarct limitation of the second window of protection in a conscious rabbit model. Cardiovasc Res. 1996 May;31(5):777–783. doi: 10.1016/0008-6363(96)00026-0. [DOI] [PubMed] [Google Scholar]
  46. Yang X. M., Sato H., Downey J. M., Cohen M. V. Protection of ischemic preconditioning is dependent upon a critical timing sequence of protein kinase C activation. J Mol Cell Cardiol. 1997 Mar;29(3):991–999. doi: 10.1006/jmcc.1996.0344. [DOI] [PubMed] [Google Scholar]
  47. Ytrehus K., Liu Y., Downey J. M. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol. 1994 Mar;266(3 Pt 2):H1145–H1152. doi: 10.1152/ajpheart.1994.266.3.H1145. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES