postconditioning and intermittent bradykinin induced cardioprotection require cyclooxygenase...

Introduction

It has been reported that Postconditioning (PostC),i.e., repetitive cycles of reperfusion and coronary

occlusion following an ischemic insult, cause mas-sive salvage of the myocardium similar to that ofischemic preconditioning (IP) [35, 36, 38]. More-over, PostC involves signal transduction pathways

Claudia PennaDaniele MancardiFrancesca TullioPasquale Pagliaro

Postconditioning and intermittentbradykinin induced cardioprotectionrequire cyclooxygenase activation andprostacyclin release during reperfusion

Received: 27 July 2007Returned for 1. Revision: 6 August 20071. Revision received: 16 November 2007Returned for 2. Revision: 22 November 20072. Revision received: 23 November 2007Accepted: 27 November 2007Published online: 9 January 2008

j Abstract Postconditioning (PostC), obtained with brief intermittentcycles of ischemia alternating with reperfusion applied after the ischemicevent, has been shown to reduce infarct size. Recently, we have shownthat PostC triggering includes B2 receptor activation and its downstreampathway. Moreover, we showed that BK intermittent infusion induces acardioprotection similar to PostC. The aim of this study was toinvestigate the involvement of cyclooxygenase-(COX)-derivated prosta-glandins, such as prostacyclin (PGI2) pathway in the cardioprotectiveaction mediated by intermittent BK infusion.

Isolated rat hearts underwent 30 min ischemia and 120 min reperfu-sion. Myocardial damage was evaluated using nitro-blue-tetrazoliumstaining. The production of metabolite of PGI2, 6-keto-PGF1a, wasevaluated with EIA assay on the samples collected during reperfusion.The perfusion pressure and the left ventricular pressure were monitored.In Control hearts, the infarct size was 64% ± 4% of risk area. PostCreduced significantly the infarct size (28% ± 4% P < 0.001 Vs. Control).BK intermittent protocol to mimic PostC, attenuated infarct size(40% ± 2% P < 0.01 Vs. Control). The BK-intermittent and PostCprotections were abolished with COX-inhibition. Intermittent BK andPostC enhanced the release of prostacyclin metabolite, 6-keto-PGF1a, inthe late phase of reperfusion (i.e., 6-keto-PGF1a peaked 30 min afterprotective maneuvers). Also the stable PGI2 analogue, Iloprost, given inthe early reperfusion reduced infarct size and improved post-ischemicheart function. In conclusion, protection by PostC and intermittent BKrequires COX activation and PGI2 release during late reperfusion. Thesedata suggest that COX must not be inhibited to have PostC protection.This finding should be kept present by future clinical studies on PostC.

j Key words bradykinin B2 receptors – ischemia/reperfusion –postconditioning – prostacyclin – rat

ORIGINAL CONTRIBUTIONBasic Res Cardiol 103:368–377 (2008)DOI 10.1007/s00395-007-0695-7

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C. Penna Æ D. Mancardi Æ F. TullioDr. P. Pagliaro (&)Dipartimento di Scienze Cliniche eBiologicheUniversita di TorinoRegione Gonzole 1010043 Orbassano (TO), ItalyTel.: +39-11/6705430 ext -5450Fax: +39-11/9038639E-Mail: [email protected]

which are similar to those seen in IP [12, 16, 23–26,31–40].

Virtually in all of the species in which different PostCprotocols have been tested they have been protective,including humans [e.g., 4, 31, 32, 38], with the excep-tion of recent works conducted on a rodent model [5].In this respect, it is intriguing that PostC protection ispresent in wildtype mice [2, 13] and it is not lost inconnexin 43-deficient mice [13]; yet an age-related lossof PostC protection in mice has been described, whichseems to be related to a reduced level of signal trans-ducer and activator of transcription 3 (STAT3) withincreasing age [2]. Remote ischemic PostC has beenalso induced by cycles of few minutes of ischemia/reperfusion applied to a distal artery territory (femoralor renal artery) [15, 40]. Importantly, the protection bypostconditioning represent a long-term protective ef-fect and not a mere attenuation of event involved inearly reperfusion injury [21]. Therefore, postcondi-tioning has impressive clinical potential and to pre-cisely clarify the involved pathway signaling is ofparamount importance.

Recently, we have demonstrated that the intermit-tent infusion of bradykinin (BK) at the beginning ofreperfusion can trigger PostC-like cardioprotectionagainst infarct size via B2 BK receptors activation [24].

Cyclooxygenase enzymes (COX) are constitutivelyexpressed in most tissues, including myocardium [10,11]. It is reported that B2 BK receptors activationresults in de novo synthesis of prostacyclin (PGI2)[34], which has been demonstrated to attenuateischemia–reperfusion injury [4]. In particular, Bertiet al. [1] and Rossoni et al. [28] have reported thatboth indomethacin and aspirin aggravated ischemia-induced ventricular dysfunction in perfused rabbithearts and this was associated with inhibition of PGI2

production in the cardiac tissue. Yet, either admin-istration of the stable PGI2 analogue Iloprost [7] orprostaglandin E1 [8] during ischemia have been seento attenuate myocardial stunning in open chest dogs.

It seems that PGI2 is produced by jeopardizedmyocardium [33] and that the rate of its formationincreases particularly during the first 5–10 min ofreperfusion declining thereafter [1, 6]. Endogenousprostaglandins are also involved in ischemic precon-ditioning [10].

Therefore, we hypothesized that intermittent BKmay enhance PGI2 release during reperfusion and thatCOX activation is involved in PostC.

To verify the role of COX pathway in the cardio-protection by PostC and intermittent BK, we studiedthe effect of COX inhibition during PostC maneuversand during the intermittent infusion of BK. Moreover,we studied the effects of the administration of thestable PGI2 analogue Iloprost during early reperfu-sion. Finally, we measured the release of 6-ketoPGF1a,

a PGI2 metabolite, which is used to evaluate theactivation of B2 BK receptor, during reperfusion.

Materials and methods

Male Wistar rats (n = 105; body weight 450–550 g)received humane care in compliance with Italian law(DL-116, 27 Jan. 1992) and in accordance with theGuide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health(NIH Publication No. 85-23, revised 1996).

j Isolated heart perfusion

The methods were similar to those previously de-scribed [22–25]. In brief, each animal was anesthe-tized. The chest was opened 10 min after heparintreatment and the heart was rapidly excised. Isolatedrat hearts were weighed, attached to the perfusionapparatus and retrogradely perfused with oxygenatedKrebs–Henseleit buffer (127 mM NaCl, 17.7 mMNaHCO3, 5.1 mM KCl, 1.5 mM CaCl2, 1.26 mMMgCl2, 11 mM D-glucose and gassed with 95% O2 and5% CO2). A constant flow was adjusted with a properpump to obtain a typical coronary perfusion pressure(PP) of 80–85 mmHg during the initial part of sta-bilization. Thereafter the same flow level (9 ± 1 ml/min/g) was maintained throughout the experiment.

A small hole in the left ventricular wall alloweddrainage of the thebesian flow, and a polyvinyl-chlo-ride balloon was placed into the left ventricle andconnected to an electromanometer for the recordingof left ventricular pressure (LVP). The balloon wasfilled with saline to achieve an end-diastolic LVP of5 mmHg. Coronary perfusion pressure, coronary flowand LVP were monitored to asses the preparationconditions. LVP was used to calculate the positive(dP/dtmax) and negative (dP/dtmin) first derivative ofpressure during systole and diastole, respectively.

The hearts were electrically paced at 280 bpm andkept in a temperature-controlled chamber (37�C).

All chemicals were purchased from Sigma (USA).

j Experimental protocols (Fig. 1)

Each heart was allowed to stabilize for 20 min. Afterthe stabilization period, hearts were subjected to aspecific protocol, which included in all groups a30 min of global no-flow ischemia. A period of 120-min reperfusion followed the 30 min ischemia in allgroups (see below). Pacing was discontinued on ini-tiation of ischemia and restarted after the third min-ute of reperfusion in all groups [16, 22–25].

C. Penna et al. 369Cyclooxygenase is involved in postconditioning

Experimental protocols are described in Fig. 1.After stabilization the hearts of the Control group

(Group 1, n = 20) were exposed to 30 min ischemiaand then to 120 min reperfusion only.

In Group 2 (PostC group; n = 20) after the 30 minischemia, the hearts immediately underwent a pro-tocol of PostC. This consisted of five cycles of 10 sreperfusion and 10 s global ischemia [23–25].

In Group 3 the intermittent infusion of BK(Intermittent BK + intermittent buffer; n = 15) wasperformed after 30 min ischemia, this intermittentprotocol consisted of five cycles of 10 s of oxygenated/non-oxygenated buffer with BK (100 nM) [24, 27].

To evaluate the role of COX pathway hearts wereco-infused with an inhibitor of COX, Indomethacin(INDO, 10 lM) [27] during the initial 3 min ofreperfusion, either during the PostC maneuvers(Group 4, n = 10) or during intermittent BK protocol(Group 5, n = 10). To determine the effects of theantagonist, in Group 6 (INDO group, n = 10) indo-methacin was only infused during the initial 3 min ofreperfusion. Moreover, in Group 7 Iloprost (12 nM),an analogous of PGI2 [8, 10], was infused intermit-tently in lieu of BK (Intermittent ILOPROST + inter-mittent buffer; n = 10) after the 30 min ischemia, as

in Group 3; in Group 8 Iloprost (12 nM) was infusedin continuous (Continuous ILOPROST; n = 10) for3 min immediately after the 30 min ischemia.

Assessment of myocardial injury

Infarct areas were assessed at the end of the experi-ment as previously described [22–25]. In brief,immediately after reperfusion each heart was rapidlyremoved from the perfusion apparatus and the leftventricle (LV) was dissected into 2–3 mm circumfer-ential slices. Following 20 min of incubation at 37�Cin 0.1% solution of nitro-blue tetrazolium in phos-phate buffer, unstained necrotic tissue was carefullyseparated from stained viable tissue by an indepen-dent observer. The necrotic mass was expressed as apercentage of total left ventricular mass. In fact,though in this model the whole heart underwentnormothermic ischemia, only the LV had a fixedvolume and pre-load; therefore only the LV mass wasconsidered as a risk area [22–25].

j 6-keto-PGF1a assay

In the hearts of Group 1, Group2 and Group 3, thePGI2-releasing capacity of the isolated heart wasmeasured by evaluating the concentration of its stablemetabolite 6-keto-PGF1a in perfusate samples, col-lected before ischemia and after 3, 5, 10, 30, 60, 90 and120 min of reperfusion. The amount of 6-keto-PGF1areleased was determined with an enzyme-immuno-assay kit (Amersham International plc, UK).

j Statistical analysis

All data are expressed as means ± SEM One-wayANOVA and Newman–Keuls multiple comparisontest (for post-ANOVA comparisons) have been usedto compare infarct size. One-way ANOVA has beenalso used to compare area under the curve of 6-keto-PGF1a release. Two-way ANOVA (variables treatmentgroups and time point) and one-way ANOVA formultiple measure (post test Newman–Keuls multiplecomparison test) have been used for the analysis of PPand LVP data. A P value <0.05 was considered sta-tistically significant.

Results

j Infarct size (Fig. 2)

Total infarct size, expressed as a percentage of leftventricular mass, was 64% ± 4% in Control Group 1.PostC (Group 2) significantly (P < 0.001) reduced

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Fig. 1 Experimental design. The isolated, Langendorff-perfused hearts werestabilized for 20 min, and then subjected to 30 min of normothermic globalischemia followed by 120 min of reperfusion. PostC postconditioning; BKBradykinin; INDO Indomethacin; ILOPROST analogous of prostacyclin. For furtherexplanation see text

370 Basic Research in Cardiology, Vol. 103, No. 4 (2008)� Steinkopff Verlag 2008

infarct size to 28% ± 4% of risk area. This high in-farct size in Control group and the strong protectiveeffect of PostC are features of the constant flow modelof isolated rat heart, as suggested in our previouswork [23].

In intermittent BK, Group 3, the infarct size was40% ± 2%; a value similar to that obtained with PostCand significantly (P < 0.01) lower than that of ControlGroup 1. The cardioprotective effect of PostC and thatof intermittent-BK was abolished during the co-infu-sion with the COX-inhibitor, INDO. In fact, the infarctareas of Group 4 (PostC + INDO, 65% ± 2%) andGroup 5 (intermittent BK + INDO, 57% ± 6%) weresimilar to that of the Control Group 1.

The treatment with INDO alone did not signifi-cantly change infarct size, which was 57 ± 6, i.e.,similar to that of Control Group 1, and significantlyhigher than that of intermittent BK (P < 0.05) andPostC (P < 0.001) groups (Fig. 2).

Iloprost reduced infarct size both when infusedintermittently (Group 7, Infarct size 39 ± 6, P < 0.01

Vs. Control) and in continuous for three min (Group8, infarct size 42 ± 7, P < 0.05 Vs. Control).

j Perfusion pressure and left ventricular pressure(Table 1; Figs. 3, 4, 5)

Perfusion pressure is represented as percent variationwith respect to baseline level (Fig. 3). Perfusionpressure increased in all groups during reperfusion.Two-way ANOVA revealed that there is not interac-tion between the considered variables (treatmentgroups and time point), whereas both treatmentgroups and time point affect the results (P < 0.01 forboth). The post ANOVA comparison revealed that theonly group that showed a significantly (P < 0.01)blunted increase in pressure (i.e., a reduced post-ischemic vasoconstriction) was the Intermittent Ilo-prost group (Group 7).

Systolic function is represented by develop LVPand dP/dtmax, which are reported as percent varia-tion with respect to baseline level in Fig. 4. For bothparameters the two-way ANOVA revealed that thereis not interaction between the considered variables(treatment groups and time point), whereas bothtreatment groups and time point affect the results(P < 0.01 for both). In reperfusion, the left ven-tricular function was not statistically differentamong groups. The post ANOVA comparison re-vealed that the only group that performed signifi-cantly better than Control group (P < 0.01) in termsof dP/dtmax (Fig. 4B) was the Intermittent Iloprostgroup.

Diastolic function is represented by diastolic LVPand dP/dtmin, which are reported in Fig. 5 as mmHgand as percent variation with respect to baseline level,respectively. For both parameters the two-wayANOVA revealed that there is no interaction betweenthe considered variables (treatment groups and timepoint), whereas both treatment groups and time pointaffect the results (P < 0.01 for both). The post ANO-VA comparison revealed that in reperfusion therewere groups that reached higher diastolic LVP (higher

Table 1 Baseline hemodynamic parameters

PP (mmHg) LVEDP (mmHg) Dev LVP (mmHg) dP/dtmax (mmHg/s) dP/dtmin (mmHg/s)

Control (Group 1) 87 ± 4 4.8 ± 0.9 80 ± 3 1950 ± 60 )1,578 ± 130PostC (Group 2) 82 ± 2 4.2 ± 0.7 78 ± 5 2021 ± 111 )1,788 ± 122Intermittent BK (Group 3) 86 ± 3 4.0 ± 1.0 75 ± 4 2187 ± 121 )1,679 ± 120PostC + INDO (Group 4) 89 ± 2 4.7 ± 0.7 70 ± 6 2078 ± 174 )1,580 ± 164Intermittent BK + INDO (Group 5) 82 ± 2 4.2 ± 0.7 78 ± 5 2019 ± 120 )1,754 ± 144INDO (Group 6) 86 ± 3 4.3 ± 0.4 75 ± 4 2188 ± 114 )1,689 ± 120Intermittent ILOPROST (Group 7) 80 ± 2 5.0 ± 0.7 81 ± 3 1965 ± 89 )1,779 ± 94ILOPROST 3 min (Group 8) 82 ± 2 4.5 ± 0.9 82 ± 3 1987 ± 93 )1,680 ± 85

PP perfusion pressure; LVEDP left ventricular end diastolic pressure; Dev LVP developed left ventricular pressure; dP/dtmax maximum rate of increase of LVP duringsystole; dP/dtmin maximum rate of decrease of LVP during diastole. Other acronyms as text

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Fig. 2 Infarct size. The amount of necrotic tissue is expressed as percent of theleft ventricle (LV), which is considered the risk area. PostC postconditioning; BKBradykinin; INDO Indomethacin; ILOPROST analogous of prostacyclin. NS notsignificant; ***P < 0.001, **P < 0.01 and *P < 0.05 Vs. Control; #P < 0.001Vs. PostC and NS Vs. Control. For further explanation see text


contracture) and others that reached lower levels ofdiastolic LVP (less contracture) than Control group.Again the group that performed significantly betterthan Control group (P < 0.01) in terms of both dia-stolic LVP (Fig. 4A) and dP/dtmin (Fig. 4B) was theIntermittent Iloprost group, which showed less con-tracture and an increased lusitropism. Yet, the groupthat performed worst was the INDO group, both interms of contracture (diastolic LVP) and relaxation(dP/dtmin).

j 6-keto PGF1a release during reperfusion (Fig. 6)

To easily compare the three groups in which 6-ketoPGF1a release was measured, in Fig. 6 results are pre-sented as normalized data. In the Control group, therelease of 6-keto PGF1a slightly increased during theinitial part of reperfusion, then started to decline after30 min of reperfusion, reaching a value not differentfrom baseline level at the end of reperfusion. On thecontrary, in the Intermittent BK group the release of 6-keto PGF1a started to increase after BK-treatment,showing a sharp increase at 30 min reperfusion.Thereafter it slightly declined, and at the end ofreperfusion was still higher than baseline. A similartrend was observed in PostC group. The analysis of the

area under the curve revealed that the Control curvewas significantly lower than that of Intermittent BKcurve (P < 0.05), but not different from that of PostCcurve. The areas under Intermittent BK curve andPostC curve were not statistically different.

Discussion

We have previously shown that cardioprotectiontriggered by postconditioning involves endogenousactivation of B2 receptors. Using intermittent infusionof BK in the early phase of reperfusion, we triggeredcardioprotection akin to postconditioning. We dem-onstrated that B2 receptors, nitric oxide, protein ki-nase G, mitochondrial KATP channels and reactiveoxygen species are also integral to the protectiontriggered by intermittent BK [24].

In the present study our goals were (1) to show thatcardioprotection triggered by postconditioning orintermittent BK involves activation of COX; (2) toshow that intermittent BK trigger the release of PGI2

and (3) to show that PGI2 analogous can mimicpostconditioning.

Here, we show that the cardioprotection inducedby PostC or by intermittent BK infusion is dependent

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Fig. 3 Perfusion pressure. Percent variation ofperfusion pressure with respect to baseline level foreach group, during the 30 min ischemia and 120 minreperfusion. Time 0 correspond to the beginning ofreperfusion. PostC postconditioning; BK Bradykinin;INDO Indomethacin; ILOPROST analogous ofprostacyclin. *P < 0.01 between Control andIloprost intermittent groups. For further explanationsee text


on COX activation and augmentation of PGI2 releasein the late phase of reperfusion. Moreover, Iloprost, astable PGI2 analogous, given in early reperfusion in-duced a cardioprotective effect either when infusedintermittently or when infused in continuous.

Regardless of the fact that protective stimuli(Intermittent BK or PostC) are applied only for fewseconds at the beginning of reperfusion the endoge-nous release of PGI2 metabolite is enhanced duringthe late period of reperfusion. It has been, previously,reported that during reperfusion in non-protectedhearts the rate of PGI2 formation increases particu-larly during the first 5–10 min of reperfusion declin-ing rapidly thereafter [1, 6]. Here we confirm thisfinding. We suggest that the potentially protectiveeffect of PGI2 release cannot operate in naive ische-

mia/reperfusion because the enhanced productionduring the first phase of reperfusion rapidly decline inthe second phase of reperfusion, when reperfusioninjury occurs. On the contrary the PostC maneuversand intermittent BK triggers the above reportedprotective cascade [24], and may allow COX activityand PGI2 release in the late phase of reperfusion, thuspreventing reperfusion injury. In other words, wesuggest that PostC or Intermittent BK triggers pro-tection, which allows COX activity and subsequentPGI2 formation.

It is worth of note that the protective effect ofIntermittent BK on infarct size seems to be less thanthat produced by PostC; yet the Intermittent BKseems to increase prostacyclin to a greater level thanPostC. Although these differences are not statistically


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Fig. 4 Systolic function. A Percent variation ofDeveloped left ventricular (LV) pressure with respectto baseline level for each group, during the 30 minischemia and 120 min reperfusion. B Percentvariation of first derivative of LV pressure duringsystole (dP/dtmax) with respect to baseline level foreach group, during the 30 min ischemia and 120 minreperfusion Time 0 correspond to the beginning ofreperfusion. PostC postconditioning; BK Bradykinin;INDO Indomethacin; ILOPROST analogous ofprostacyclin. *P < 0.01 between Control andIloprost intermittent groups. For further explanationsee text


significant, these data are in line with the fact thatother factors (e.g., adenosine and opioids), other thanBK and COX, are involved in the PostC-inducedprotection [e.g., 12, 26, 35, 39].

Moreover, Intermittent BK and Iloprost (inter-mittently and in continuous) achieved similar degreeof protection in terms of infarct size reduction. Onceagain suggesting that other factors, other than BK andprostaglandin, are involved in the PostC-inducedprotection.

The fact that Iloprost may induce protection inreperfusion is in agreement with previous studieswhich have shown a protective role of prostaglandins inreperfusion [7, 8, 10]. With respect to this, it isintriguing that the involvement of COX has been veryrecently demonstrated in the additive effect of latepreconditioning and PostC protection [29]. However,the fact that Iloprost is able to induce protection wheninfused intermittently and in continuous in the earlyreperfusion, whereas the endogenous prostaglandin

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Fig. 5 Diastolic function. A LV diastolic (mmHg)during the 30 min ischemia and 120 min reperfusion.B Percent variation of first derivative of LV pressureduring diastole (dP/dtmin) with respect to baselinelevel for each group, during the 30 min ischemia and120 min reperfusion Time 0 correspond to thebeginning of reperfusion. PostC postconditioning; BKBradykinin; INDO Indomethacin; ILOPROST analogousof prostacyclin. *P < 0.01 between Control andIloprost intermittent groups; #P < 0.01 betweenControl and INDO groups. For further explanationsee text


release is enhanced in the late phase of reperfusion byPostC, suggests different mechanisms of protection byendogenous prostaglandin(s) release triggered byPostC and infusion of exogenous prostaglandin ana-logue. Differences might be due to molecule stabilitydifferences and/or pharmacodynamic features; furtherstudies may clarify the reasons of these differences.

The more robust end-point in analyzing I/R injuryis infarct size [9, 20]. Therefore, we focused ourattention on myocardial damages evaluated by infarctsize. As for preconditioning [9, 20], systolic function(developed LV pressure and dP/dtmax) may not be anappropriate end-point to study PostC protection. Theimprovement of function during reperfusion in pre-conditioned rat hearts has been attributed to thereduction of adenosine release during this phase [9].On the contrary, in post-conditioned hearts an accu-mulation of adenosine has been reported [16]. Thesedifferences may explain our findings of an uncleareffect on systolic function by PostC treatment. Yet, itis hard to distinguish the impairment of function dueto necrosis and/or to stunning of viable tissue. Inorder to understand the role of PostC on myocardialstunning appropriate studies are required. Moreover,it has been suggested that in rodent models contrac-ture development, rather than systolic function, maybe a more appropriate end-point to understand theprotective effects of cardioprotective maneuvers [9].In the present study the only conditions that clearlyimproves post-ischemic heart performance is thetreatment with Intermittent Iloprost, which also im-

proved contracture. On the other hand, the onlytreatment that clearly worsens diastolic heart perfor-mance is Indomethacin treatment. On the base ofthese effects on contracture, but keeping in mind themodel limits above reported, we can argue that COXis involved not only in reducing infarct size, but alsoin improving heart function recovery of viable tissue.This is also in line with the reduced post-ischemicvasoconstriction (less increase in PP) observed in thepresent study. Moreover, the beneficial effect by Ilo-prost is in agreement with the involvement ofendogenous prostaglandins in ischemic precondi-tioning [10], with the anti-stunning effect by Iloprostobserved in a canine model of ischemia/reperfusion[8] and with the involvement of COX in late precon-ditioning, which clearly improves post-ischemicstunning [29].

Differently from previous studies [8, 10, 17, 19,26, 30, 37], which used BK or other agents in con-tinuous for a long period during reperfusion, wewere able to reproduce PostC by means of few sec-onds (50 s in total) treatment with BK only whengiven in an intermittent manner in the very earlyperiod of reperfusion [24] and we confirm that latePGI2 release is mandatory to limit reperfusion injuryby PostC.

Another novelty of the present study is that theinhibitor, indomethacin, has been given for 3 minonly to prevent the triggering of protection by PostCor intermittent BK, confirming that the initial phaseof reperfusion is of paramount importance for trig-

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Ischemia ReperfusionFig. 6 6-keto PGF1a release. The PGI2-releasingcapacity of the isolated heart is estimated by thepercent variation of the concentration of its stablemetabolite 6-keto-PGF1a in perfusate samples duringbaseline and reperfusion. Each value is represented aspercent with respect to the mean baseline data ofeach group. Time 0 correspond to the beginning ofreperfusion. PostC postconditioning; BK Bradykinin.For further explanation see text


gering cardioprotective pathways. Also the stable PGI2

analogue, iloprost, is able to induce protection whenapplied for few minutes at the beginning of reperfu-sion.

Kinins are produced during ischemia reperfusionby the action of a kininogenase activated by the aci-dosis. Recently, PostC has been seen to induce car-dioprotection by maintaining acidosis during the firstminutes of reperfusion as reoxygenated myocardiumproduces reactive oxygen species that activate pro-tective signaling [3]. Bradykinin was believed to bethe only kinin acting on the B2 receptors in rats;however, it has been demonstrated that isolated rathearts also releases Arg-kallidin, a kallidin-like pep-tide, which acts on B2 receptors too [14, 18, 19].Importantly, Arg-kallidin release increases duringischemia and mediates IP and preconditioning-likeprotection, via B2 receptor activation, in isolated rathearts [18, 19]. Whether the endogenous mediator ofPostC protection is BK or Arg-kallidin was beyond theaims of the present study.

In conclusion, the major new findings in this studyare: (1) postconditioning triggered cardioprotectioninvolves endogenous activation of COX and release ofPGI2; (2) intermittent BK infusion also triggers theCOX activation and induces PGI2 formation in the latephase of reperfusion; (3) exogenous infusion of thestable PGI2 analogue, Iloprost, is able to induce car-dioprotection when infused in the early reperfusioneither continuously or intermittently.

We suggest that protection exerted by postcon-ditioning and intermittent BK is not solely depen-

dent on the activation of the so called RISK(reperfusion injury salvage kinase [12, 24, 25, 38],but it also depends on the late release of PGI2. Thatis, during PostC maneuvers the heart may releasekinins that accumulate in an intermittent mannerand triggers a protective pathway that lead to aprotected state, which include COX activation andPGI2 release.

j Clinical implication

One goal in applying reperfusion therapeutic strate-gies is to pharmacologically mimic postconditioning.It is likely that one drug or maneuver cannot target allof the mechanisms of reperfusion injury, but a com-bined therapy integrating pharmacological agents(e.g., BK and Iloprost) and maneuvers, such asintermittence of infusion, in the very early phase ofreperfusion may provide a broader spectrum ap-proach to attenuate reperfusion injury. From a clini-cal point of view, it is important that ischemia is notnecessary, whereas intermittence of drug infusion isable to trigger PostC. Equally important, COX mustnot be inhibited to obtain cardioprotection by ische-mic postconditioning during reperfusion.

j Acknowledgments We thank Regione Piemonte; Compagnia diSan Paolo’’; the Consorzio Interuniversitario per le Ricerche Car-diovascolari (CIRC) and the Italian Ministry of University andResearch (MIUR, PRIN). We Thank Dr. Giovanni Berta forinsightful suggestions.

References

1. Berti F, Rossoni G, Magni F, Caruso D,Omini C, Puglisi L, Galli G (1988)Nonsteroidal antiinflammatory drugsaggravate acute myocardial ischemia inthe perfused rabbit heart: a role forprostacyclin. J Cardiovas Pharmacol12:438–444

2. Boengler K, Buechert A, Heinen Y,Roeskes C, Hilfiker-Kleiner D, HeuschG, Schulz R (2008) Cardioprotection byischemic postconditioning is lost inaged and STAT3-deficient mice. CircRes 102:131–135

3. Cohen MV, Yang XM, Downey JM(2007) The pH hypothesis of postcon-ditioning: staccato reperfusion rein-troduces oxygen and perpetuatesmyocardial acidosis. Circulation115:1895–1903

4. Darling CE, Solari PB, Smith CS, Fur-man MI, Przyklenk K (2007) �Postcon-ditioning’ the human heart: multipleballoon inflations during primaryangioplasty may confer cardioprotec-tion. Basic Res Cardiol 102:274–278

5. Dow J, Kloner RA (2007) Postcondi-tioning does not reduce myocardialinfarct size in an in vivo regionalischemia rodent model. J CardiovascPharmacol Ther 12:153–163

6. Engels W, Van Bilsen M, De Groot MJ,Lemmens PJ, Willemsen PH, RenemanRS, Van der Vusse GJ (1990) Ischemiaand reperfusion induced formation ofeicosanoids in isolated rat hearts. Am JPhysiol 258:H1865–H1871

7. Farber NE, Gross GJ (1989) Prosta-glandin E1 attenuates postischemiccontractile dysfunction after brief cor-onary occlusion and reperfusion. AmHeart J 118:17–24

8. Farber NE, Pieper GM, Thomas JP,Gross GJ (1988) Beneficial effects ofiloprost in the stunned canine myo-cardium. Circ Res 62:204–215

9. Gelpi RJ, Morales C, Cohen MV, Dow-ney JM (2002) Xanthine oxidase con-tributes to preconditioning’spreservation of left ventricular devel-oped pressure in isolated rat heart:developed pressure may not be anappropriate end-point for studies ofpreconditioning. Basic Res Cardiol97:40–46

10. Gres P, Schulz R, Jansen J, Umschlag C,Heusch G (2002) Involvement ofendogenous prostaglandins in ischemicpreconditioning in pigs. CardiovascRes 55:626–632

11. Grosser T, Fries S, FitzGerald GA(2006) Biological basis for the cardio-vascular consequences of COX-2 inhi-bition: therapeutic challenges andopportunities. J Clin Invest 116:4–15


12. Hausenloy DJ, Tsang A, Yellon DM(2005) The reperfusion injury salvagekinase pathway: a common target forboth ischemic preconditioning andpostconditioning. Trends CardiovascMed 15:69–75

13. Heusch G, Buchert A, Feldhaus S,Schulz R (2006) No loss of cardiopro-tection by postconditioning in conn-exin 43-deficient mice. Basic ResCardiol 101:354–356

14. Hilgenfeldt U, Stannek C, Lukasova M,Schnolzer M, Lewicka S (2005) Rattissue kallikrein releases a kallidin-likepeptide from rat low-molecular-weightkininogen. Br J Pharmacol 146:958–963

15. Kerendi F, Kin H, Halkos ME, Jiang R,Zatta AJ, Zhao ZQ, Guyton RA, Vinten-Johansen J (2005) Remote postcondi-tioning. Brief renal ischemia andreperfusion applied before coronaryartery reperfusion reduces myocardialinfarct size via endogenous activationof adenosine receptors. Basic Res Car-diol 100:404–412

16. Kin H, Zatta AJ, Lofye MT, AmersonBS, Halkos ME, Kerendi F, Zhao ZQ,Guyton RA, Headrick JP, Vinten-Jo-hansen J (2005) Postconditioning re-duces infarct size via adenosinereceptor activation by endogenousadenosine. Cardiovasc Res 67:124–133

17. Leesar MA, Stoddard MF, Manchikala-pudi S, Bolli R (1999) Bradykinin-in-duced preconditioning in patientsundergoing coronary angioplasty. J AmColl Cardiol 34:639–660

18. Liu X, Lukasova M, Zubakova R, Lew-icka S, Hilgenfeldt U (2005) A kallidin-like peptide is a protective cardiac ki-nin, released by ischaemic precondi-tioning of rat heart. Br J Pharmacol146:952–957

19. Liu X, Lukasova M, Zubakova R, Lew-icka S, Hilgenfeldt U (2006) Kallidin-like peptide mediates the cardiopro-tective effect of the ACE inhibitorcaptopril against ischaemic reperfusioninjury of rat heart. Br J Pharmacol148:825–832

20. Lochner A, Genade S, Moolman JA(2003) Ischemic preconditioning: in-farct size is a more reliable endpointthan functional recovery. Basic ResCardiol 98:337–346

21. Mykytenko J, Kerendi F, Reeves JG, KinH, Zatta AJ, Jiang R, Guyton RA,Vin-ten-Johansen J, Zhao ZQ (2007) Long-term inhibition of myocardial infarc-tion by postconditioning duringreperfusion. Basic Res Cardiol 102:90–100

22. Pagliaro P, Mancardi D, Rastaldo R,Penna C, Gattullo D, Miranda KM,Feelisch M, Wink DA, Kass DA, Paol-occi N (2003) Nitroxyl affords thiol-sensitive myocardial protective effectsakin to early preconditioning. FreeRadic Biol Med 34:33–43

23. Penna C, Cappello S, Mancardi D,Raimondo S, Rastaldo R, Gattullo D,Losano G, Pagliaro P (2006) Post-con-ditioning reduces infarct size in theisolated rat heart: role of coronary flowand pressure and the nitric oxide/cGMP pathway. Basic Res Cardiol101:168–179

24. Penna C, Mancardi D, Rastaldo R, Lo-sano G, Pagliaro P (2007) Intermittentactivation of bradykinin B2 receptorsand mitochondrial KATP channelstrigger cardiac postconditioningthrough redox signaling. CardiovascRes 75:168–177

25. Penna C, Rastaldo R, Mancardi D,Raimondo S, Cappello S, Gattullo D,Losano G, Pagliaro P (2006) Post-con-ditioning induced cardioprotection re-quires signaling through a redox-sensitive mechanism, mitochondrialATP-sensitive K+ channel and proteinkinase C activation (see comment).Basic Res Cardiol 101:180–189

26. Philipp S, Yang XM, Cui L, Davis AM,Downey JM, Cohen MV (2006) Post-conditioning protects rabbit heartsthrough a protein kinase C-adenosineA2b receptor cascade. Cardiovasc Res70:308–314

27. Rastaldo R, Paolocci N, Chiribiri A,Penna C, Gattullo D, Pagliaro P (2001)Cytochrome P-450 metabolite of ara-chidonic acid mediates bradykinin-in-duced negative inotropic effect. Am JPhysiol Heart Circ Physiol 280:H2823–H2832

28. Rossoni G, Berti M, Colonna VD, Ber-nareggi M, Del Soldato P, Berti F (2000)Myocardial protection by the nitrode-rivative of aspirin, NCX 4016: in vitroand in vivo experiments in the rabbit.Ital Heart J 1:146–155

29. Sato H, Bolli R, Rokosh GD, Bi Q, Dai S,Shirk G, Tang XL (2007) The cardio-protection of the late phase of ischemicpreconditioning is enhanced by post-conditioning via a COX-2-mediatedmechanism in conscious rats. Am JPhysiol Heart Circ Physiol 293:H2557–2564

30. Schulz R, Post H, Vahlhaus C, HeuschG (1998) Ischemic preconditioning inpigs: a graded phenomenon: its relationto adenosine and bradykinin. Circula-tion 98:1022–1029

31. Sivaraman V, Mudalgiri NR, Di SalvoC, Kolvekar S, Hayward M, Yap J, Ke-ogh B, Hausenloy DJ, Yellon DM (2007)Postconditioning protects human atrialmuscle through the activation of theRISK pathway. Basic Res Cardiol102:453–459

32. Staat P, Rioufol G, Piot C, Piot C, CottinY, Cung TT, L’Huillier I, Aupetit JF,Bonnefoy E, Finet G, Andre-Fouet X,Ovize M (2005) Postconditioning thehuman heart. Circulation 112:2143–2148

33. van Bilsen M, Engels W, van der VusseGJ, Reneman RS (1989) Significance ofmyocardial eicosanoid production. MolCell Biochem 88:113–121

34. Veeravalli KK, Akula A, Kota MK(2003) Nitric oxide- and prostaglandin-mediated cardioprotection by bradyki-nin in myocardial ischemia and reper-fusion injury. Pol J Pharmacol 55:1021–1029

35. Vinten-Johansen J, Zhao ZQ, Zatta AJ,Kin H, Halkos ME, Kerendi F (2005)Postconditioning–a new link in nat-ure’s armor against myocardial ische-mia–reperfusion injury. Basic ResCardiol 100:295–310

36. Yang XM, Downey JM, Cohen MV(2005) Postconditioning’s protection isnot dependent on circulating bloodfactors or cells but requires PI3-kinaseand guanylyl cyclase activation. BasicRes Cardiol 100:57–63

37. Yang XP, Liu YH, Scicli GM, Webb CR,Carretero OA (1997) Role of kinins inthe cardioprotective effect of precon-ditioning: study of myocardial ische-mia/reperfusion injury in B2 kininreceptor knockout mice and kinino-gen-deficient rats. Hypertension30:735–740

38. Yellon DM, Opie LH (2006) Postcon-ditioning for protection of the infarc-ting heart. Lancet 367:456–458

39. Zhao ZQ, Corvera JS, Halkos ME, Ke-rendi F, Wang NP, Guyton RA, Vinten-Johansen J (2003) Inhibition of myo-cardial injury by ischemic postcondi-tioning during reperfusion:comparison with ischemic precondi-tioning. (erratum appears in Am JPhysiol Heart Circ Physiol. 2004Jan;286(1):H477). Am J Physiol HeartCirc Physiol 285:H579–H588

40. Zhao ZQ, Vinten-Johansen J (2006)Postconditioning: reduction of reper-fusion-induced injury. Cardiovasc Res70:200–211


postconditioning and intermittent bradykinin induced cardioprotection require cyclooxygenase...

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