| [Download PDF]
|Year : 2010 | Volume
| Issue : 4 | Page : 219--223
Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart
Kavita Garg, Harlokesh N Yadav, Manjeet Singh, PL Sharma
Department of Pharmacology, I.S.F College of Pharmacy, Moga - 142 001, Punjab, India
P L Sharma
Department of Pharmacology, I.S.F College of Pharmacy, Moga - 142 001, Punjab
Objective : The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.
Materials and Methods : The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent.
Results : The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 μM), a protein kinase-C (PKC) inhibitor, or AG490 (5 μM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning.
Conclusion : Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.
|How to cite this article:|
Garg K, Yadav HN, Singh M, Sharma P L. Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart.Indian J Pharmacol 2010;42:219-223
|How to cite this URL:|
Garg K, Yadav HN, Singh M, Sharma P L. Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart. Indian J Pharmacol [serial online] 2010 [cited 2021 Dec 5 ];42:219-223
Available from: https://www.ijp-online.com/text.asp?2010/42/4/219/68421
Ischemic preconditioning (IPC) is an endogenous protective phenomenon in which brief episodes of sublethal ischemia followed by reperfusion increase the resistance of the myocardium to subsequent sustained ischemia of longer duration.  It has been reported that IPC produces cardioprotection by the activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT), , phosphatidylinositol-3-kinase (PI-3Kinase)  and protein kinase-C (PKC).  Erythropoietin (Epo) is a glycoproteinious hormone of molecular weight 30 kDa, produced by the kidney, which regulates the proliferation, differentiation and maturation of erythrocytes.  Its plasma levels increase during hypoxia through hypoxia-inducible factor-1 (HIF-1).  Moreover, it is also produced by the brain, liver, uterus, trophoblasts, astrocytes and macrophage of the bone marrow.  Apart from erythropoiesis, Epo enhances the activity of stem cells,  reverses vasospasm,  also used in the treatment of chronic renal failure,  protects the brain against ischemic injury  and preserves the integrity of the endothelium.  It has been reported that pretreatment with Epo produces cardioprotection against I/R-induced injury by JAK/STAT,  PI-3K/Akt  and PKC  signaling pathways.
It has been reported that both IPC and Epo produce cardioprotection through common signaling pathways: PI-3K, PKC and JAK/STAT. ,,,,,,,,, Glycogen synthase kinase-3β (GSK-3β) is a downstream common pathway of the PI-3K, PKC and JAK/STAT  pathways and its phosphorylation is noted to produce cardioprotection. , However, Nishihara et al. reported that administration of Epo with IPC exerts additional cardioprotection, mediated through enhanced phosphorylation of GSK-3β, and suggested that the Epo-mediated primary signaling pathway is different from that of the IPC-induced signaling cascade. Thus, the objective of this study was to determine the relative role of Epo-induced cell survival signaling pathways, including PI-3K, PKC and JAK/STAT, in isolated rat heart.
Materials and Methods
The experimental protocol used in the present study was approved by the Institutional Animal Ethics Committee.
Drugs and chemicals:
Human recombinant Epo (alpha) (Gennova Biopharmaceuticals Ltd., Pune, India), Wortmannin, chelerythrine and AG490 (Sigma Aldrich [P] Ltd., Bangalore, India) were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in K-H solution was 0.02%. All other reagents used in this study were of analytical grade and were always freshly prepared before use.
Isolated rat heart preparation:
Rats were administered heparin (500 IU/L, i.p.) 20 min before their sacrifice by cervical dislocation. The heart was rapidly excised and immediately mounted on Langendorff's apparatus  and was retrogradely perfused at a constant pressure of 80 mmHg with Kreb's Henseleit (KH) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl 2 2.5 mM; MgSO 4 .7H 2 O 1.2 mM; KH 2 PO 4 1.2 mM; C 6 H 12 O 6 11 mM), pH 7.4 and bubbled with 95% O 2 and 5% CO 2 . The flow rate was maintained at 7-9 ml/min using a Hoffman's screw. The heart was enclosed in a double-wall jacket, the temperature of which was maintained by circulating water heated at 37°C. IPC was produced by closing the inflow of K-H solution for 5 min followed by 5 min of reperfusion. Four such episodes were employed. Global ischemia was produced for 30 min followed by 120 min of reperfusion. The coronary effluent was collected before ischemia, immediately, 5 min and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB).
Four cycles, each cycle consisting of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution (free of Epo), and followed by 30 min global ischemia and 120 min reperfusion were used.
Assessment of myocardial injury:
The myocardial infarct size was measured using the triphenyltetrazolium chloride (TTC) staining method while the level of LDH (Siemens Medical Solution Diagnostics Ltd., Baroda, India) and CK-MB (Nicholas Piramal India Ltd., Mumbai, India) in coronary effluents was estimated using commercially available kits. Values of LDH and CK-MB were expressed in international units per liter (IU/L).
Assessment of myocardial infarct size:
The heart was removed from the Langendorff's apparatus. Both the atria and the root of the aorta were excised and the ventricles were kept overnight at a temperature of -4°C. Frozen ventricles were sliced into uniform sections of about 1-2 mm thickness. The slices were incubated in 1% w/v TTC at 37°C in 0.2M tris-chloride buffer for 30 min. The normal myocardium was stained brick red while the infarcted portion remained unstained. Infarct size was measured by the volume method. 
A diagrammatic representation of the experimental protocol is shown in [Figure 1].
In all groups, isolated rat heart was perfused with K-H solution and allowed to stabilize for 10 min. Group 1: (Sham control; n = 6) After stabilization, the heart was perfused continuously with K-H buffer for 190 min without subjecting it to global ischemia. Group 2: (Vehicle control: n = 6) After stabilization, the heart was perfused for 5 min with K-H containing DMSO (0.02%) followed by 5 min perfusion with K-H solution free of DMSO; four such cycles were repeated. Then, the preparation was subjected to 150 min of perfusion. Group 3: (Ischemia-reperfusion control; n = 6) After stabilization, the heart was subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group 4: (IPC control; n = 6) Four cycles of IPC were given just after stabilization. Each cycle comprised of 5 min ischemia and 5 min reperfusion with K-H solution and then followed by 30 min global ischemia and 120 min reperfusion. Group 5: (Epo preconditioning: n = 6) Just after stabilization, the heart was subjected to four cycles of Epo preconditioning, each cycle comprising of 5 min perfusion with K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution free of Epo and further followed by 30 min of global ischemia and 120 min of reperfusion. Group 6: (Epo preconditioning in Wortmannin-perfused rat heart; n = 6) During 10 min of stabilization, the heart was perfused with K-H solution containing 100 nM of Wortmannin followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (100 nM) Wortmannin. Then, the preparation was subjected to 30 min global ischemia followed by 120 min reperfusion. Group 7: (Epo preconditioning in Chelerythrine-perfused rat heart; n = 6) During stabilization, the heart was perfused with K-H solution containing 1 μM Chelerythrine followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (1 μM) Chelerythrine followed by 30 min global ischemia and 120 min reperfusion. Group 8: (Epo preconditioning in AG490-perfused rat heart; n = 6) The heart was perfused with K-H solution containing 5 μM AG490 during the stabilization followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (5 μM) AG490 further followed by 30 min of global ischemia and 120 min of reperfusion.
All values were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Sigmastat Software. The values were statistically analysed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Value of P <0.05 was considered as statistically significant.
Effect of IPC, Epo preconditioning and pharmacological interventions on myocardial infarct size:
Both IPC and Epo preconditioning significantly attenuated ischemia-reperfusion-induced increase in myocardial infarct size. Administration of Wortmannin or Chelerythrine or AG490 significantly (P < 0.05) reversed the Epo preconditioning-induced decrease in myocardial infarct size [Figure 2]. The difference in the inter-se efficacy of the three inhibitors were not statistically significant (P > 0.05).
Effect of IPC, Epo preconditioning and pharmacological interventions on release of LDH
Both IPC and Epo preconditioning significantly attenuated ischemia-reperfusion-induced increase in release of LDH in the coronary effluent. Administration of Wortmannin or Chelerythrine or AG490 significantly (P < 0.05) reversed the Epo preconditioning-induced decrease in the release of LDH in the coronary effluent [Figure 3]. The difference in the inter-se efficacy of the three inhibitors was not statistically significant (P > 0.05).
Effect of IPC, Epo preconditioning and pharmacological interventions on release of CK-MB
Both IPC and Epo preconditioning significantly attenuated ischemia-reperfusion-induced increase in release of CK-MB in the coronary effluent. Administration of Wortmannin or Chelerythrine or AG490 significantly (P < 0.05) reversed the Epo preconditioning-induced decrease in the release of CK-MB in the coronary effluent [Figure 4]. The difference in the inter-se efficacy of the three inhibitors was not statistically significant (P > 0.05).
Both pharmacological preconditioning and IPC remain the validated approaches to minimize the ischemia-reperfusion-induced damage of the myocardium. Also, the cardioprotective effect of Epo has been well documented in different animal models, including isolated cardiomyocytes,  isolated Langendorff heart preparation  and coronary artery ligation models.  The reported molecular mechanism of Epo-induced cardioprotection is believed to be almost identical to that of IPC-induced cardioprotection.
IPC produces cardioprotection by phosphorylation and inhibition of GSK-3β.  It has been reported that JAK/STAT, PI-3K and PKC are the common upstream kinases of GSK-3β and that IPC produces cardioprotection by activation of the JAK/STAT, , PI-3K  and PKC  pathways. In the present study, four cycles of Epo preconditioning produced cardioprotection, measured in terms of I/R-induced decrease in infarct size and decrease in the release of LDH and CK-MB. Moreover, it has been documented that early perfusion of Epo protects the ischemia-reperfusion-induced injury by JAK/STAT,  PI-3K  and PKC.  Hence, it may be suggested that the observed Epo preconditioning-induced cardioprotection may be mediated through the same pathways.
Recently, Nishihara et al. reported that administration of Epo with IPC produces additional cardioprotection in the rat heart against I/R-induced injury by enhancing the phosphorylation of GSK-3β and also that the relative importance of PKC and PI-3K pathways differs in the Epo-induced and IPC-induced cardioprotection against ischemia-reperfusion injury. It may be that cardioprotective stimuli of Epo-induced preconditioning may not be parallel to stimuli induced by IPC. GSK-3β is a unique downstream target  of various signaling pathways, viz. PI-3K, PKC and JAK/STAT, ,,,,,,,,, observed during IPC- and Epo-induced cardioprotection. In our study, pretreatment with Wortmannin (PI-3K inhibitor) or Chelerythrine (PKC inhibitor) or AG490 (JAK2 inhibitor) significantly and almost equally attenuated the Epo preconditioning-induced decrease in myocardial infarct size and release of LDH and CK-MB in the coronary effluent, indicating that Epo simultaneously activates the JAK/STAT, PI-3K and PKC pathways, which play an important role in Epo-preconditioning-induced cardioprotection. There was no statistically significant differences in the inter-se efficacy of the three inhibitors used. Thus, we found no relative difference in the role of PKC, PI-3K and JAK2 in Epo-induced cardioprotection. This difference between our results and those of Nishihara et al. may be due to the difference in the experimental models used. The activation of any one of these pathways is sufficient to inactivate the common downstream GSK-3β that protects the cardiac myocytes from ischemia-reperfusion injury in rat hearts.
Based on the results of the present study, it may be concluded that Epo-induced preconditioning was almost as effective as IPC (P > 0.05) and EPO preconditioning-induced cardioprotection in the isolated rat heart is mediated through an interplay of the JAK-2, PI-3K and PKC pathways. The order of effectiveness for attenuation of erythropoietin preconditioning was Wort>Che>AG490 in the dosages used, but these inter-se differences in the efficacy were not statistically significant.
This work is dedicated to the memory of our esteemed colleague Prof. Manjeet Singh, who expired on 30.3.2009, while this study was in progress. We are grateful to Mr. Praveen Garg, Chairman, I.S.F. College of Pharmacy, Moga, for this support and encouragement during the conduct of this study.
|1||Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.|
|2||Bolli R, Dawn B, Xun YT. Emerging role of the JAK/STAT pathway as a mechanism of protection against ischemia-reperfusion injury. J Mol Cell Cardiol 2001;33:1893-6.|
|3||Bolli R, Dawan B, Xuan YT. Role of the JAK/STAT pathway in protection against myocardial ischemia-reperfusion injury. Trends Cardiovasc Med 2003;13:72-9.|
|4||Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of glycogen synthase kinase - 3β during preconditioning through a phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. Circ Res 2002;90:377-9.|
|5||Hassouna A, Matata BM, Galinanes M. PKC-ε is upstream and PKC-α is downstream of mito KATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium. Am J Physiol Cell Physiol 2004;287:1418-25.|
|6||Moritz KM, Lim GB, Wintour EM. Developmental regulation of EPO and erythropoiesis. Am J Physiol Regul Integr Comp Physiol 1997;273:R1829-44.|
|7||Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D, Gaston B S. Nitrosothiols signal the ventilatory response to hypoxia. Nature 2001;413:171-4.|
|8||Vogt C, Pentz S, Rich IN. A role for the macrophage in normal hemopoiesis: III. In vitro and in vivo EPO gene expression in macrophages detected by in situ hybridization. Exp Hematol 1989; 17:391-7.|
|9||Shingo T, Sorokan ST, Shimazaki T, Weiss S. EPO regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci 2001;21:9733-43.|
|10||Grasso G, Buemi M, Alafaci C. Beneficial effects of systemic administration of recombinant human EPO in rabbits subjected to subarachnoid hemorrhage. Proc Natl Acad Sci USA 2002;99:5627-31.|
|11||Kausz A, Watkins SL, Hansen C, Godwin DA, Palmer RB, Brandt JR. Intraperitoneal EPO in children on peritoneal dialysis: A study of pharmacokinetics and efficacy. Am J Kidney Dis 1999;34:651-6.|
|12||Brines ML, Ghezzi P, Keenan S, Agnello D, Lanerolle NC, Cerami C, et al. EPO crosses the blood brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 2000;97:10526-31.|
|13||Chong ZZ, Kang JQ, Maiese K. Angiogenesis and plasticity: Role of EPO in vascular systems. J Hematother Stem Cell Res 2002;11:863-71.|
|14||Baker JE. EPO mimics ischemic preconditioning. Vascul Pharmacol 2005;42:233-41.|
|15||Rafiee P, Shi Y, Su J, Pritchard KA, Jr Tweddell JS, Baker JE. Erythropoietin protects the infant heart against ischemia-reperfusion injury by triggering multiple signaling pathways. Basic Res Cardiol 2005;100:187-97.|
|16||Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of EPO-mediated cardioprotection during ischemia-reperfusion injury: Role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J 2005;19:1323-5.|
|17||Fryer RM, Pratt PF, Hsu AK, Gross GJ. Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced carardioprotection. J pharmacol Exp Ther 2001;296:642-9.|
|18||Hattori R, Maulik N, Otani H, Zhu L, Cordis G. Engelman RM, et al. Role of STAT3 in ischemic preconditioning. J Mol Cell Cardiol 2001;33:1929-36.|
|19||Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, et al. Mitochondrial PKCε and MAPK form signaling modules in the murine heart: Enhanced mitochondrial PKCε-MAPK interactions and differential MAPK activation in PKC-induced cardioprotection. Circ Res 2002;90:390-7.|
|20||Armstrong SC. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res 2004;61:427-36.|
|21||Shi Y, Rafiee P, Su J, Pritchard Jr KA, Tweddell JS, Baker JE. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels. Basic Res Cardiol 2004;99:173-82.|
|22||Smith RM, Suleman N, Lacerda L, Opie LH, Akira S, Chien KR, et al. Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc Res 2004;63:611-6.|
|23||Gross ER, Hsu AK, Gross GJ. The JAK/STAT pathway is essential for opoid-induced cardioprotection: JAK2 as a mediator of STAT3, AKT, and GSK-3beta. Am J Physiol Heart Circ Physiol 2006;291:H827-34.|
|24||Downey JM, Davis AM, Cohen MV. Signalling pathways in ischemic preconditioning. Heart Fail Rev 2007;12:181-8.|
|25||Gross ER, Gross GJ. Ischemic preconditioning and myocardial infarction: An update and perspective. Drug Discovery Today: Disease Mechanisms 2007;4:159-63.|
|26||Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H, et al. EPO affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3β. Am J Physiol Heart Circ Physiol 2006;291:748-55.|
|27||Langendorff O. Untersuchungen an uberlebenden Saugethierherzen. Pflugers Arch 1895;61:291-332.|
|28||Chopra K, Singh M, Kaul N, Andrabi KI, Ganguly NK. Decrease Decrease of myocardial infarct size with desferrioxamine: Possible role of oxygen free radicals in its ameliorative effect. Mol Cell Biochem 1992;13:71-6.|
|29||Wright GL, Hanlon P, Amin K, Steenbergen C, Murphy E, Arcasoy MO. Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury. FASEB J 2004;18:1031-3.|
|30||Bullard AJ, Govewalla P, Yellon DM. Erythropoietin protect the myocardium against ischemia-reperfusion induced injury in vitro and in vivo. Basic Res Cardiol 2005;100:397-403.|
|31||Van der Meer P, Lipsic E, Henning RH, Boddeus K, van der velden J, Voors AA, et al. Erythropoietin induces neovascularization and improves cardiac function in rat with heart failure after myocardial infarction. J Am Coll Cardiol 2005;46:125-33.|
|32||Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, et al. GSK-3 beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004;113:1535-49.|
|33||Chan CY, Chen YS, Lec H H, Huang HS, Lai YL, Chen CF, et al. EPO protects post-ischemic hearts by preventing extracellular matrix degradation: Role of JAK2- ERK pathway. Life Sci 2007;81:717-23.|
|34||Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for EPO-mediated acute protection against myocardial ischemia-reperfusion injury. Circulation 2004;109:2050-3.|