Cardioprotection by Ischemic Postconditioning Is Lost in Aged and STAT3-Deficient Mice

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1 Cardioprotection by Ischemic Postconditioning Is Lost in Aged and -Deficient Mice Kerstin Boengler, Astrid Buechert, Yvonne Heinen, Christin Roeskes, Denise Hilfiker-Kleiner, Gerd Heusch, Rainer Schulz Abstract The cardioprotection by ischemic preconditioning is lost in aged wild-type and in (signal transducer and activator of transcription 3)-deficient mice. The aim of the present study was to analyze whether or not ischemic postconditioning () was effective in aged mice hearts and whether was dependent on. Young (3 months) and aged ( 13 months) C57Bl6/J mice underwent 3 minutes of ischemia and 2 hours of reperfusion without or with (3 cycles of 1 seconds of ischemia/1 seconds of reperfusion [3 1] or 5 cycles of 5 seconds of ischemia/5 seconds of reperfusion [5 5] at the beginning of reperfusion). In young mice, both 3 1 and 5 5 reduced infarct size (IS), whereas in aged mice, only 5 5 was effective in reducing IS. In young mice, 3 1 increased the phosphorylated over total (phosphorylated /) ratio at 1 minutes of reperfusion in the postconditioned anterior wall compared with the control posterior wall. In aged mice hearts, total and phosphorylated / in the anterior wall at reperfusion were reduced compared with young mice hearts. In young mice hearts subjected to 3 1 but pretreated with the JAK-2 inhibitor AG-49, phosphorylated / was reduced in the anterior wall compared with untreated young mice hearts, and IS reduction by 3 1 was abolished. Furthermore, in young mice with a cardiomyocyte-restricted deletion of, 3 1 failed to reduce IS, whereas 5 5 reduced IS. Thus, cardioprotection by is dependent on the postconditioning protocol in aged and -deficient hearts. The reduced levels of with increasing age may contribute to the age-related loss of. (Circ Res. 28;12: ) Key Words: signal transducer and activator of transcription 3 ischemic postconditioning aging Transient periods of ischemia/reperfusion reduce the irreversible tissue injury by a subsequent prolonged episode of ischemia/reperfusion, a phenomenon known as ischemic preconditioning (IP). Previous studies have demonstrated that IP is not effective in C57Bl6/J mice older than 13 months. 1 (signal transducer and activator of transcription 3) transduces stress signals from the plasma membrane to the nucleus, leading to changes in gene transcription. 2 Besides its role in postpartum cardiomyopathy, cardiomyocyte hypertrophy, and apoptosis, is involved in ischemia/reperfusion injury. 3 7 IP induces an increase in the protein content in the nucleus. 8 Furthermore, the phosphorylation of and the binding to the STAT target site are enhanced after IP. 8,9 The importance of for cardioprotection by IP has been clearly demonstrated in mice with a cardiacspecific deletion of, in which the IP-induced infarct size (IS) reduction was abolished. 1 In ischemic postconditioning (), the IS is reduced by short cycles of ischemia/reperfusion immediately following a sustained ischemic insult. The mechanisms and signal transduction cascade of, its age dependence, and especially the role of remain to be elucidated. In the present study, we therefore addressed the following questions: (1) whether or not is effective in aged mice hearts; and (2) whether the cardioprotection is dependent on the presence and phosphorylation of. Materials and Methods The present study was performed with approval from the Bioethical Committee of the district of Düsseldorf, Germany, and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No , revised 1996). C57Bl6/J mice of different ages (young, 3 months; aged, 13 months) were purchased from Charles River Laboratories (Kisslegg, Germany) or were retired breeders from our own animal facility. Mice with a cardiomyocyte-restricted deletion of ( KO) were generated by breeding -floxed mice ( flox/flox ) with -myosin heavy chain promoter/cre recombinase transgenic mice ( MHC-Cre tg/ ). 11 In Vivo Mouse Model Female C57Bl6/J mice of different ages (young, 3 months; aged, 13 months) and young female KO mice were subjected to 3 minutes of ischemia and 2 hours of reperfusion without (young, n 12; aged, n 1; KO, n 8) or with by 3 cycles of 1 seconds of ischemia and 1 seconds of reperfusion (3 1: Original received July 27, 27; resubmission received September 2, 27; accepted October 15, 27. From the Institut für Pathophysiologie (K.B., A.B., Y.H., C.R., G.H., R.S.), Universitätsklinikum Essen; and Molekulare Kardiologie (D.H.-K.), Medizinische Hochschule Hannover, Germany. Correspondence to Prof Dr Rainer Schulz, Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, Essen, Germany. Rainer.schulz@uk-essen.de 28 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 132 Circulation Research January 4/18, 28 C57Bl6/J young mice Infarct size (% AAR) C57Bl6/J aged mice KO young mice Figure 1. IS (in percentages of the area at risk [AAR]) in young and aged C57Bl6/J mice and in young - deficient mice undergoing ischemia/ reperfusion alone or (3 1 seconds or 5 5 seconds) ischemia/ reperfusion number of animals young, n 1; aged, n 7; KO, n 5) or 5 cycles of 5 seconds of ischemia and 5 seconds of reperfusion (5 5: young, n 1; aged, n 7; KO, n 9) at the beginning of reperfusion. 12 Furthermore, young C57Bl6/J were treated with the JAK-2 inhibitor AG-49 (4 g/g IP, 2 minutes before ischemia) and were subjected to 3 minutes of ischemia and 2 hours of reperfusion without (n 7) or with (n 6) 3 1. The area at risk was measured by Evans blue, and the IS was determined by TTC (2,3,5-triphenyl tetrazolium chloride) staining. For determination of phosphorylation, left ventricles of C57Bl6/J mice subjected to 3 minutes of ischemia and 1 minutes of reperfusion with 3 1 were divided into the postconditioned anterior walls (AWs) and the control posterior walls (PWs) and were snap frozen in liquid nitrogen. Young mice treated with AG-49 (1 mg/kg IV, 1 minutes before ischemia, n 7), young mice treated with vehicle (2% DMSO in.9% NaCl, n 6), or aged mice treated with vehicle (n 6) were analyzed. Western Blot Analysis Western blot analysis was performed according to standard procedures on 2- g right ventricular proteins of young ( 3 months, n 18) and aged ( 13 months, n 27 to 29) C57Bl6/J mice and on 2- g left ventricular proteins derived from the postconditioned AWs or the control PWs of young (n 6), aged (n 6), and AG-49 treated (n 7) C57/Bl6/J mice. The nitrocellulose membranes were incubated with antibodies against phospho- (Ser727, Cell Signaling, Beverly, Mass), (Cell Signaling), connexin (Cx)43 (Invitrogen, Carlsbad, Calif), or mouse monoclonal anti-rabbit glyceraldehyde-3-phophate dehydrogenase (GAPDH) (Hytest, Turku, Finland). Immunoreactive signals were detected by chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce, Rockford, Ill) and quantified with Scion Image software (Scion Corp, Frederick, Md). The protein level of Cx43 was analyzed as a reference because we have previously described a decreased amount of Cx43 in C57Bl6/J mice older than 13 months. 1 Statistics The data are reported as mean values SEM. Area at risk and IS data (young versus aged versus KO versus AG-49, ischemia/ reperfusion versus ) compared by 2-way ANOVA and Fisher s least-significant difference. The ratios of phosphorylated (P)- over total (/) (young versus aged versus AG-49, AW versus PW) were compared by 2-way ANOVA and Fisher s least-significant difference. Western blot data of and Cx43 in young and aged right ventricles were compared by unpaired Student s t test. A value of P.5 was considered to indicate a significant difference. Results The area at risk (in percentage of the left ventricle) was not different between groups (young ischemia/reperfusion, ; young 3 1, ; young 5 5, ; aged ischemia/reperfusion, ; aged 3 1, ; aged 5 5, ; KO ischemia/reperfusion, ; KO 3 1, ; -KO 5 5, , young ischemia/ reperfusion AG-49, ; young 3 1 AG- 49, ). The IS (in percentage of the area at risk) was reduced in young C57Bl6/J mice from in controls to by 3 1 and to by 5 5 (Figure 1). In aged C57Bl6/J mice, however, only 5 5 was effective in reducing the IS ( ), and there was no difference in the IS between control mice ( ) and mice undergoing 3 1 ( ). Young mice with a cardiomyocyte-restricted deletion of ( KO) underwent ischemia/reperfusion alone or (3 1 and 5 5). The IS was significantly reduced by 5 5 ( ), but not by 3 1 ( ) compared with control hearts ( , Figure 1). The impact of phosphorylation on cardioprotection by was analyzed by subjecting young AG-49 treated mice to ischemia/reperfusion alone or to 3 1. The IS was slightly, but not significantly reduced by AG-49 alone ( , n 7; Figure 2), but 3 1 did not further reduce IS (52.6 5, n 6). Western blot analysis on right ventricular proteins from C57Bl6/J mice showed a decrease of in aged to % (P.5) and of total to % (P.5) of that in young mice, which were set as 1% (Figure 3). The protein level of Cx43 in aged hearts was reduced to %.

3 Boengler et al Aging,, and Postconditioning 133 Infarct size (% AAR) ischemia/ reperfusion C57Bl6/J young mice C57Bl6/J young mice + AG number of animals Figure 2. IS (in percentages of the area at risk [AAR]) in young C57Bl6/J control mice and in mice treated with the JAK-2 inhibitor AG-49 undergoing ischemia/reperfusion alone or 3 1. To assess phosphorylation induced by, young, aged, and AG-49 treated mice underwent 3 minutes of ischemia and 1 minutes of reperfusion with induced an increase in the ratio of / in the postconditioned AWs of young vehicle-treated mice ( arbitrary units) compared with the control PWs ( arbitrary units; P.5) of the left ventricle (Figure 4). Whereas a trend toward an increase of / in the AWs compared with the PWs was found both in aged and AG-treated C57BL6/J mice, the ratio of / in the AWs (aged, arbitrary units; young AG-49 treated, arbitrary units) was significantly reduced compared with the AWs of young vehicle-treated mice. Discussion The present study demonstrates that the postconditioning algorithms 3 1 and 5 5 are equally effective in reducing IS in young C57Bl6/J mice. In aged C57Bl6/J mice A 43 kda young aged Cx43 B Protein/GAPDH (% of young mice) A B / (a.u.) 2 1,8 1,6 1,4 1,2 1,8,6,4,2 > 13 months AW PW AW PW AW PW and KO mice, only 5 5 was cardioprotective. The inhibition of JAK-2 by AG-49 abolished the IS reduction by 3 1. Furthermore, 3 1 induced a phosphorylation of, whereas in the aged heart and in + AG-49 AW PW AW PW AW PW > 13 months + AG-49 young aged Figure 4. A, Western blot analysis was performed for phosphorylated (Ser727) and total on protein extracts derived from the postconditioned AWs and the control PWs of left ventricles of young and aged vehicle-treated C57Bl6/J mice and of young C57Bl6/J mice treated with AG-49 undergoing 3 minutes of ischemia and 1 minutes of reperfusion with 3 1. B, Bar graphs representing the ratios of / in the AWs and PWs of C57Bl6/J mice undergoing 3 minutes of ischemia and 1 minutes of reperfusion with 3 1. a.u. indicates arbitrary units. 36 kda GAPDH n=19 n=27 Cx43 n=29 Figure 3. A, Western blot analysis was performed for phosphorylated (Ser727), total, Cx43, and GAPDH on protein extracts from the right ventricles of young and aged C57Bl6/J mice. B, Bar graphs representing the immunoreactivities of, total, and Cx43 normalized to GAPDH in percentages of young mice.

4 134 Circulation Research January 4/18, 28 the AG-49 treated heart, the amount of protein was reduced. has been first described in a dog model, however, it is also effective in mice, rats, rabbits, pigs, and humans (reviewed elsewhere 13 ). For cardioprotection by, both the number of postconditioning cycles and, perhaps more importantly, the duration of reocclusion appear to be important (for review see elsewhere 14 ). In isolated buffer-perfused mouse hearts, 6 postconditioning cycles (1 seconds of ischemia and 1 seconds of reperfusion) were more effective in improving postischemic systolic and diastolic function than 3 postconditioning cycles. 15 The occurrence of protection was proposed to depend on a delay in adenosine washout. However, in in situ rat hearts, IS reduction with by 3 cycles of 1 seconds of ischemia and 1 seconds of reperfusion and 6 cycles of 1 seconds of ischemia and 1 seconds of reperfusion was similar. 16 When analyzing the impact of the duration of the postconditioning stimuli in rat hearts in situ, 6 cycles of 3 seconds of ischemia and 3 seconds of reperfusion did not reduce IS, whereas shortening of the postconditioning cycles to 1 seconds of ischemia and 1 seconds of reperfusion induced a significant IS reduction compared with ischemia/reperfusion alone. 17 The benefit of more and shorter postconditioning cycles has been confirmed in isolated rabbit hearts, in which postconditioning with 6 cycles of 1 seconds of ischemia and 1 seconds of reperfusion was more effective in reducing IS than 4 cycles of 3 seconds of ischemia and 3 seconds of reperfusion. 18 In the present study, we compared the protective effects of 3 1 and 5 5 and found a similar IS reduction in young C57Bl6/J mice. However, in aged C57Bl6/J mice, only 5 5 was effective in reducing IS. This indicates that with increasing age, the intrinsic capacity of the heart to respond to a postconditioning stimulus is impaired and a different postconditioning stimulus is needed to achieve a significant IS reduction. It has already been demonstrated that does not confer cardioprotection under all pathological situations, because failed to reduce IS in hypercholesterolemic rabbits. 19 The results of the present study add to the above findings in that varying the postconditioning algorithm does not modify IS reduction in hearts of young mice but that the protection obtained in aged or genetically modified hearts depends on the postconditioning protocol. Therefore, one can speculate that a certain redundancy in signaling pathways leading to cardioprotection exists that can be recruited by different postconditioning algorithms but that such redundancy is of minor importance in young (primarily healthy) hearts but becomes more important in aged or diseased (genetically modified) hearts. The importance of for the signal transduction cascade of IP is well established. 8 1 In the present study, we addressed the question of whether or not also has an impact on cardioprotection by. Indeed, 3 1 induced an increase in phosphorylation. This is similar to IP, in which an enhancement of phosphorylation has been described. 8,9 The inhibition of JAK-2 by AG-49 induced a decrease in the phosphorylation in the postconditioned AW of the left ventricle. When studying the impact of AG-49 treatment on IS in young C57Bl6/J mice, we found a slight but not significant IS reduction by AG-49 in mouse hearts undergoing ischemia/ reperfusion alone. An even more pronounced reduction in IS by AG-49 was described in rat hearts subjected to ischemia/ reperfusion, 2 whereas in mice infarct development following 3 minutes of ischemia and 24 hours of reperfusion remained unaffected by AG-49 treatment. 8 Because AG-49 inhibits JAK-2 and thereby not only but also other downstream proteins kinases (for example, mitogen-activated protein kinases), this may help to explain its varying effect on infarct development per se. However, and most importantly, 3 1 failed to further reduce IS in AG-49 treated hearts, demonstrating the importance of phosphorylation for cardioprotection by. To directly study the impact of for cardioprotection by, -deficient mice were subjected to ischemia/ reperfusion alone or 3 1 and 5 5. In agreement with the pharmacological data, cardioprotection by 3 1 also was abolished in -deficient hearts. Therefore, the present study shows, for the first time, that is not only involved in the cardioprotection by IP but also in the IS reduction by. Because cardioprotection by 3 1 was lost in the aged mouse heart, we analyzed the protein level in myocardium of mice older than 13 months. The first evidence for a decrease of in the process of aging comes from the aged rat brain. 21 In the present study, a decrease of phosphorylated and total was detected in right ventricular proteins extracts derived from aged compared with young hearts. The Cx43 protein level, which served as a control protein, was also reduced in C57Bl6/J hearts older than 13 months, as described previously. 1 The lack of IS reduction by 3 1 in the aged mice cannot be attributed to the diminished protein level of Cx43 because in young heterozygous Cx43-deficient mice with a 5% reduction of the protein, 3 1 still reduced IS. 12 In the aged mouse heart, in which 3 1 is ineffective, the ratio of / in the postconditioned AW of the left ventricle was reduced compared with young hearts. The reduced amount of may impair the - mediated signaling cascade in the aged mouse myocardium that induces cardioprotection by. Given the complexity of the signal transduction pathway(s), we cannot explain the differences between 3 1 and 5 5 in young versus aged versus KO mice. Whereas we cannot prove causality between the reduction of in the aged mouse heart and the loss of cardioprotection by, our data demonstrating increased phosphorylation by 3 1 and loss of IS reduction in AG-49 treated mice and KO mice strongly suggest an important role of in the phenomenon of. Taken together, whereas in young C57Bl6/J mice, IS reduction is not dependent on the mode of postconditioning, in old C57Bl6/J mice, only 5 5 induces cardioprotection. is not only important for the cardioprotection by IP but also for. The reduced protein level in the aged mouse heart may contribute to the loss of cardioprotection by.

5 Boengler et al Aging,, and Postconditioning 135 Sources of Funding This work was supported by internal budgets from the Institute of Pathophysiology, University Duisburg Essen, and the School of Medicine, University of Hannover. None. Disclosures References 1. Boengler K, Konietzka I, Buechert A, Heinen Y, Garcia-Dorado D, Heusch G, Schulz R. Loss of ischemic preconditioning s cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol. 27;292:H1764 H Darnell JE Jr. STATs and gene regulation. Science. 1997;277: Hilfiker-Kleiner D, Kaminski K, Podewski E, Bonda T, Schaefer A, Sliwa K, Forster O, Quint A, Landmesser U, Doerries C, Luchtefeld M, Poli V, Schneider MD, Balligand JL, Desjardins F, Ansari A, Struman I, Nguyen NQ, Zschemisch NH, Klein G, Heusch G, Schulz R, Hilfiker A, Drexler H. A cathepsin D-cleaved 16 kda form of prolactin mediates postpartum cardiomyopathy. Cell. 27;128: Terrell AM, Crisostomo PR, Wairiuko GM, Wang M, Morrell ED, Meldrum DR. JAK/STAT/SOCS signaling circuits and associated cytokine-mediated inflammation and hypertrophy in the heart. Shock. 26; 26: Barry SP, Townsend PA, Latchman DS, Stephanpou A. Role of the JAK-STAT pathway in myocardial injury. Trends Mol Med. 27;13: Bolli R, Dawn B, Xuan Y-T. Role of the JAK-STAT pathway in protection against myocardial ischemia/reperfusion injury. Trends Cardiovasc Med. 23;13: Fischer P, Hilfiker-Kleiner D. Survival pathways in hypertrophy and heart failure: the gp13- axis. Basic Res Cardiol. 27;12: Xuan Y-T, Guo Y, Han H, Zhu Y, Bolli R. An essential role of the JAK-STAT pathway in ischemic preconditioning. Proc Nat Acad Sci USA. 21;98: Xuan Y-T, Guo Y, Zhu Y, Wang O-L, Rokosh G, Messing RO, Bolli R. Role of the protein kinase C-e-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation. 25;112: Smith RM, Suleman N, Lacerda L, Opie LH, Akira S, Chien KR, Sack MN. Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc Res. 24;63: Hilfiker-Kleiner D, Hilfiker A, Fuchs M, Kaminski K, Schaefer A, Schieffer B, Hillmer A, Schmiedl A, Ding Z, Podewski E, Poli V, Schneider MD, Schulz R, Park J-K, Wollert KC, Drexler H. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury. Circ Res. 24;95: Heusch G, Büchert A, Feldhaus S, Schulz R. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol. 26;11: Vinten-Johansen J. Postconditioning: a mechanical maneuver that triggers biological and molecular cardioprotective responses to reperfusion. Heart Fail Rev. 27;12: Vinten-Johansen J, Zhao Z-Q, Zatta AJ, Kin H, Halkos ME, Kerendi F. Postconditioning. A new link in nature s armor against myocardial ischemia-reperfusion injury. Basic Res Cardiol. 25;1: Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F, Zhao Z-Q, Guyton RA, Headrick JP, Vinten-Johansen J. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res. 25;67: Kin H, Zhao Z-Q, Sun H-Y, Wang N-P, Corvera JS, Halkos ME, Kerendi F, Guyton RA, Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res. 24;62: Tang XL, Sato H, Tiwari S, Dawn B, Bi Q, Li Q, Shirk G, Bolli R. Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions 45 min. Am J Physiol Heart Circ Physiol. 26; 291:H238 H Yang X-M, Philipp S, Downey JM, Cohen MV. Postconditioning s protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res Cardiol. 25;1: Iliodromitis EK, Zoga A, Vrettou A, Andreadou I, Paraskevaidis IA, Kaklamanis L, Kremastinos DT. The effectiveness of postconditioning and preconditioning on infarct size in hypercholesterolemic and normal anesthetized rabbits. Atherosclerosis. 26;188: Mascareno E, El-Shafei M, Maulik N, Sato M, Guo Y, Das DK, Siddiqui MAQ. JAK/STAT signaling is associated with cardiac dysfunction during ischemia and reperfusion. Circulation. 21;14: De-Fraja C, Conti L, Govoni S, Battaini F, Cattaneo E. STAT signalling in the mature and aging brain. Int J Dev Neurosci. 2;18:

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