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ISSN 2522-9028 (Print)
ISSN 2522-9036 (Online)

Fiziologichnyi Zhurnal

is a scientific journal issued by the

Bogomoletz Institute of Physiology
National Academy of Sciences of Ukraine

Editor-in-chief: V.F. Sagach

The journal was founded in 1955 as
1955 – 1977 "Fiziolohichnyi zhurnal" (ISSN 0015 – 3311)
1978 – 1993 "Fiziologicheskii zhurnal" (ISSN 0201 – 8489)
1994 – 2016 "Fiziolohichnyi zhurnal" (ISSN 0201 – 8489)
2017 – "Fiziolohichnyi zhurnal" (ISSN 2522-9028)

Fiziol. Zh. 2006; 52(3): 15-24

Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatalcardiomyocytes

V.E. Dosenko, V.S.Nagibin, L.V.Tumanovskaya, V.Yu. Zagoriy, A.A. Moibenko, J. Vaage

    Department of Experimental Cardiology, BogomoletzInstitute of Physiology, Kyiv, Ukraine;Department of Surgery and Institute of Experimental MedicalResearch, Ulleval University Hospital, Oslo, Norway


A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardio- myocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mМ) was added to the culture medium just before the cycles of postconditioning. Percentages of living, necrotic, and apoptotic cells were determined by staining with bisBenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin- like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. Postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established, that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.


  1. Досенко В.Е., Загорий В.Ю., Мойбенко А.А. Влияние протеасомного протеолиза на активность NO-синтазы в изолированных тромбоцитах // Укр.біохім. журнал. – 2005. – 77. – С.39 – 42.
  2. Тумановська Л.В., Досенко В.Є., Нагібін В.С. та ін. Апоптотична, аутофагічна та онкотична загибель кардіоміоцитів при аноксії–реоксигенації // Фізіол.журн. – 2004. – 50. – С.11–19.
  3. Веремеенко К.Н., Досенко В.Е., Нагибин В.С., Кизим А.И., Мойбенко А.А. Протеолитические ферменты и апоптоз // Укр. биохим. журн. – 2003. – Т. 75, №6. –С.20–34.
  4. Azhar G., Liu L., Zhang X., Wei J.Y. Influence of age on hypoxia/reoxygenation-induced DNA fragmentation and bcl-2, bcl-xl, bax and fas in the rat heart and brain// Mech. Ageing Dev. – 1999. – 112. – P.5–25.
  5. Bao J., Sato K., Li M. et al. PR-39 and PR-11 peptides inhibit ischemia-reperfusion injury by blocking proteasome-mediated I kappa B alpha degradation //Amer. J. Physiol. Heart Circulat. Physiol. – 2001. –281. – H2612–H2618.
  6. Bulteau A.L., Lundberg K.C., Humphries K.M. et al. Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion // J. Biol. Chem. – 2001. – 276. – P.30057–30063.
  7. Campbell B., Adams J., Shin Y.K., Lefer A.M. Cardio- protective effects of a novel proteasome inhibitor following ischemia and reperfusion in the isolated perfusedrat heart // J. Mol. Cell. Cardiol. – 1999. – 31. – P.467–476.
  8. Cao C., Leng Y., Liu X. et al. Catalase is regulated by ubiquitination and proteosomal degradation. Role of the c-Abl and Arg tyrosine kinases // Biochemistry. –2003. – 42. – P.10348–10353.
  9. 9. Cepinskas G., Lush C.W., Kvietys P.R. Anoxia/ reoxygenation-induced tolerance with respect to polymorphonuclear leukocyte adhesion to cultured endothelial cells. A nuclear factor-kappa B-mediatedphenomenon // Circulat. Res. – 1999. – 84. – P.103–112.
  10. 10. Chandra J., Niemer I., Gilbreath J. et al. Proteasome inhibitors induce apoptosis in glucocorticoid-resistant chronic lymphocytic leukemic lymphocytes // Blood. – 1998. – 92. – P.4220–4229.
  11. Das S., Powell S.R., Wang P. et al. Cardioprotection with palm tocotrienol: antioxidant activity of tocotrienol is linked with its ability to stabilize proteasomes // Amer. J. Physiol. Heart Circ. Physiol. –2005. – 289. – H361–H367.
  12. Dosenko V.E., Nagibin V.S., Tumanovskaya L.V. et al. Postconditioning prevents apoptotic, necrotic and au- tophagic cardiomyocyte cell death in culture // Fiziol.Zh. – 2005. – 51. – P.12–17.
  13. Goldberg A.L. Protein degradation and protection against misfolded or damaged proteins // Nature. – 2003. – 426. – P.895–899.
  14. Hausenloy D.J., Mocanu M.M., Yellon D.M. Cross- V.E. Dosenko, V.S.Nagibin, L.V.Tumanovskaya, V.Yu. Zagoriy, A.A. Moibenko, J. Vaage talk between the survival kinases during early reperfusion: its contribution to ischemic precondition- ing // Cardiovasc. Res. – 2004. – 63. – P.305–312.
  15. Hirsch T., Dallaporta B., Zamzami N. et al. Proteasome activation occurs at an early, premitochondrial step of thymocyte apoptosis // J. Immunol. – 1998. – 161. – P.35–40.
  16. Hoffman E.K., Wilcox H.M., Scott R.W., Siman R. Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked tofamilial amyotrophic lateral sclerosis // J. Neurol. Sci. –1996. – 139. – P.15–20.
  17. Hyun D.H., Lee M., Halliwell B., Jenner P. Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitratedproteins // Neurochem. – 2003. – 86. – P.363–373.
  18. Itoh M., Takaoka M., Shibata A. et al. Preventive ef- fect of lactacystin, a selective proteasome inhibitor, on ischemic acute renal failure in rats // J. Pharmacol.Exp. Ther. – 2001. – 298. – P.501–507.
  19. 19. Kin H., Zhao Z.Q., Sun H.Y. et al. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion // Cardiovasc. Res. – 2004. – 62. – P.74–85.
  20. 20. Kukan M. Emerging roles of proteasomes in ischemia- reperfusion injury of organs // J. Physiol. Pharmacol. – 2004. – 55. – P.3–15.
  21. Lepine S., Lakatos B., Courageot M.P. et al. Sphin-gosine contributes to glucocorticoid-induced apoptosis of thymocytes independently of the mitochondrial pathway // J. Immunol. – 2004. – 173. – P.3783–3790.
  22. Lockshin R.A., Zakeri Z. Apoptosis, autophagy, and more // Int. J. Biochem. Cell. Biol. – 2004. – 36. – P.2405–2419.
  23. MacFarlane M., Merrison W., Bratton SB., Cohen GM. Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro// J. Biol. Chem. 2002. – 277. – P.36611–36616.
  24. Marshansky V., Wang X., Bertrand R. et al. Protea- somes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromisefunctioning of the electron transport chain in leukemiccells // J. Immunol. 2001. – 166. – P.3130–3142.
  25. Munafo D.B., Colombo M.I. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation // J. Cell. Science. – 2001. – –114. – P.3619–3629.
  26. Nagoshi T., Matsui T., Aoyama T., et al. PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury // J. Clin. Invest. – 2005. – 115. – P.2128–2138.
  27. Nolan B., Kim R., Duffy A. et al. Inhibited neutrophil apoptosis: proteasome dependent NF-kappaB trans- location is required for TRAF-1 synthesis // Shock. –2000. – 14. – P.290–294.
  28. Pradillo J.M., Romera C., Hurtado O. et al. TNFR1 upregulation mediates tolerance after brain ischemic preconditioning // J. Cereb. Blood. Flow. Metab. –2005. – 25. – P.193–203.
  29. 29. Ravid T., Hochstrasser M. NF-kappaB signaling: flip- ping the switch with polyubiquitin chains // Curr. Biol. – 2004. – 14. – R898–R900.
  30. 30. Reinecke H., Zhang M., Bartosek T., Charles E.M. Survival, integration, and differentiation of cardiomyo- cyte grafts // Circulation. – 1999. – 100. – P.193–202.
  31. Schulz R., Cohen M.V., Behrends M. et al. Signal transduction of ischemic preconditioning // Cardiovasc. Res. – 2001. – 52. – P.181–98.
  32. Stangl K., Gunther C., Frank T. et al. Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes andrenders early cardiac protection // Biochem. Biophys. Res. Commun. – 2002. – 291. – P.542–549.
  33. Townsend P.A., Cutress R.I., Carroll C.J. et al. BAG-1 proteins protect cardiac myocytes from simulated ischemia/reperfusion-induced apoptosis via an alternatemechanism of cell survival independent of the proteasome// J. Biol. Chem. – 2004. – 279. – P.20723–20728.
  34. Tsang A., Hausenloy D.J., Mocanu M.M., Yellon D.M. Postconditioning: a form of «modified reperfusion» protects the myocardium by activating the phospha- tidylinositol 3-kinase-Akt pathway // Circulat. Res. –2004. – 95. – P.230–232.
  35. Vinten-Johansen J., Zhao Z.Q., Zatta A.J. et al. Postconditioning – A new link in nature’s armor against myocardial ischemia–reperfusion injury // Basic. Res.Cardiol. – 2005. – 100. – P.295–310.
  36. Vugmeyster Y., Borodovsky A., Maurice M.M. et al. The ubiquitin-proteasome pathway in thymocyte apoptosis: caspase-dependent processing of the deubiquitinating enzyme USP7 (HAUSP) // Mol.Immunol. – 2002. – 39. – P.431–441.
  37. Yang X.M., Proctor J.B., Cui L. et al. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways // J.Amer. Coll. Cardiol. – 2004. – 44. – P.1103–1110.
  38. Zhang L., Zhang Z.G., Zhang R.L. et al. Postischemic (6-Hour) treatment with recombinant human tissue plasminogen activator and proteasome inhibitor PS– 519 reduces infarction in a rat model of embolic focal cerebral ischemia // Stroke. – 2001. – 32. – P.2926–2931.
  39. 39. Zhao Z.Q., Corvera J.S., Halkos M.E. et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning / / Amer. J. Physiol. – 2003. – 285. – P.H579–H588.

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