RAPID COMMUNICATION
Bradykinin elicits "second window" myocardial protection in rat heart through an NO-dependent mechanism

The Hatter Institute, University College London, London WC1E 6DB, United Kingdom

	ABSTRACT

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Bradykinin is an important endogenous mediator exerting acute protective effects in the ischemic myocardium. The aims of this study were to investigate whether exogenously administered bradykinin could evoke delayed myocardial protection and to determine whether any protection observed might be dependent on nitric oxide (NO) generation. Conscious rats received bradykinin (40 µg/kg iv) or saline, preceded 15-20 min earlier by the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg ip) or saline. Twenty-four hours later, hearts were Langendorff perfused and subjected to 35 min of regional ischemia and 120 min of reperfusion. Infarct size was assessed using tetrazolium staining and expressed as a percentage of the risk zone. Bradykinin pretreatment reduced the infarct-to-risk ratio from 53.5 ± 3.2% to 29.1 ± 4.7% (P < 0.01). The administration of L-NAME before bradykinin abrogated the delayed protection (infarct size 52.3 ± 5.0%) but alone did not influence infarct size (53.5 ± 4.8%). These results are the first to demonstrate that bradykinin can evoke a delayed ("second window") enhancement of myocardial tolerance to ischemia, an action that is dependent on the early generation of NO.

infarct size; nitric oxide synthase; preconditioning

	INTRODUCTION

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THE TEMPORAL CHARACTERIZATION of cardiac ischemic preconditioning has disclosed two periods of enhanced ischemic tolerance. A period of protection, called classical or early preconditioning, is established immediately after the preconditioning ischemia and wanes within 2-3 h. A subsequent period of protection, termed "delayed," "second window," or "late" preconditioning, is manifested 12-24 h following the initial preconditioning stimulus and can last up to 72 h (1, 6, 22, 24). Early preconditioning is dependent on the generation of several diffusible factors that act as initiators or triggers of the adaptive response (9). Wall et al. (34) showed that classical preconditioning in a rabbit infarct model was abrogated in the presence of Hoe-140 (icatibant), a specific bradykinin B₂ receptor antagonist, and was mimicked by infusion of exogenous bradykinin. Subsequently, protection conferred by endogenous and administered bradykinin has been reported in numerous other models, including rats and dogs, and also in human myocardium (8, 13, 15, 21, 28, 29, 23, 32, 33, 36).

Second window preconditioning, like classical preconditoning, is dependent on the generation of various mediators during the period of antecedent ischemia. Adenosine A₁ receptor activation and nitric oxide (NO) both contribute independently as cotriggers for the acquisition of delayed protection (3, 4, 6, 7, 26). Despite extensive evidence for the role of bradykinin in early preconditioning, it is not known whether bradykinin triggers delayed protection. Accordingly, the principal aim of this study was to investigate whether pretreatment with bradykinin elicits a delayed phase of myocardial protection. NO has been demonstrated convincingly to act as a cotrigger of delayed preconditioning. Furthermore, NO has been previously implicated in some studies as a mediator of the early cardioprotective actions of bradykinin (for examples, see Refs. 13, 28, 33, 36). Therefore, we hypothesized that bradykinin triggers delayed protection in the heart through a mechanism involving the generation of NO. In this study, rats were treated with exogenously administered bradykinin, and responses to ischemia-reperfusion were examined 24 h later.

	METHODS

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Animals and Materials

Male Sprague-Dawley rats were used in this study. All animals had free access to water and pellet chow and were treated in accordance with the Guidelines on the Operation of the Animals (Scientific Procedures) Act, 1986, published by the Stationery Office (London, UK). Bradykinin acetate, N-nitro-L-arginine methyl ester (L-NAME), Evans blue, and triphenyltetrazolium chloride were obtained from Sigma (Poole, Dorset, UK). All other reagents were of analytic quality.

Treatment Protocols

The experimental treatment protocols are illustrated in Fig. 1. Twenty-four hours before infarct induction, hearts were randomly assigned to one of the following four treatment groups:

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Experimental treatment protocols. On day 1 conscious rats were pretreated with 0.9% saline (SAL) or N-nitro-L-arginine methyl ester (L-NAME) by intraperitoneal injection. After 15-20 min, they were given bradykinin (BK) or 0.9% SAL by intravenous injection. After 24 h, hearts were excised, Langendorff perfused, and subjected to 35 min left coronary occlusion (hatched bar) followed by reperfusion and infarct size assessment. See text for description of the groups.

Group 1: saline + saline (control). Rats received 0.5 ml iv saline by dorsal tail vein injection, preceded 15-20 min earlier by 0.5 ml ip saline. Animals were returned to their cages for 24 h before the infarct protocol described below.

Group 2: saline + bradykinin. Rats received 40 µg/kg iv bradykinin, preceded 15-20 min earlier by 0.5 ml ip saline. Animals were left for 24 h as described above.

Group 3: L-NAME + bradykinin. Rats received 40 µg/kg iv bradykinin, preceded 15-20 min earlier by 10 mg/kg ip L-NAME. Animals were left for 24 h as described above.

Group 4: L-NAME + saline. Animals received 0.5 ml iv saline, preceded 15-20 min earlier by 10 mg/kg ip L-NAME. Animals were left for 24 h as described above.

The dose of bradykinin was selected from previously published work that characterized the hemodynamic effects of intravenous bradykinin in dose ranges of 5-80 µg/kg (17). In preliminary experiments, we compared doses of 20 and 40 µg/kg. The hemodynamic response to the 40 µg/kg dose was judged to be at the limit of what was tolerable. L-NAME at 2.5-30 mg/kg ip has previously been shown to produce rapid inhibition of NO synthase (NOS) isoforms lasting ~6 h (10, 17, 18, 31, 35).

Isolated Heart Perfusion Protocol

Twenty four hours after the pretreatments, animals were deeply anesthetized with pentobarbital sodium (50 mg/kg ip). Heparin (300 IU) was concurrently administered intraperitoneally. Hearts were excised and placed in ice-cold buffer solution to arrest contraction. They were rapidly mounted on a Langendorff apparatus and perfused retrogradely via the ascending aorta with a modified Krebs-Henseleit buffer composed of (in mM) 118 NaCl, 25 NaHCO₃, 11 D-glucose, 4.7 KCl, 1.22 MgSO₄, 1.21 KH₂PO₄, and 1.84 CaCl₂ (pH 7.3-7.5 when equilibrated with with 95% O₂-5% CO₂). Perfusion pressure was maintained at 80 mmHg throughout the experiments. Heart temperature was maintained at 37°C (±1°C). A latex isovolumic balloon was inserted into the left ventricle via a small incision of the left atrial appendage and was inflated to give a preload of 5-10 mmHg. The balloon catheter was linked to a pen recorder for measurement of developed pressure and heart rate. All hearts were stabilized for 15-20 min before the institution of ischemia. Myocardial infarction was induced by occlusion of the left main coronary artery for 35 min, followed by 2 h of reperfusion.

Infarct Size Evaluation

On completion of the reperfusion period, the left coronary artery was reoccluded, and Evans blue dye was infused via the aorta to differentiate the ischemic zone from the nonischemic zone. After being frozen at 20°C for 1-4 h, hearts were sliced into 2-mm transverse sections from apex to base. Slices were then incubated with 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) at 37°C for a period of 10-15 min. Triphenyltetrazolium chloride reacts with viable tissue, producing a red formazan derivative, which is distinct from the white necrotic tissue once fixed in 10% formalin for 24 h. Myocardial slices were traced onto transparent acetate sheets in a blinded fashion. Areas of the infarcted and risk tissue were determined using computer-assisted planimetry. Tissue volumes were then calculated (area × 2 mm thickness) and expressed as the ratio of infarcted-risk tissue (I/R%).

Hemodynamic Effects of Bradykinin Pretreatment

Hemodynamic effects of the agents administered to rats on day 1 (bradykinin, L-NAME) were examined in a separate cohort of anesthetized animals. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (Harvard apparatus; Edenbridge, UK). Rats were ventilated with room air supplemented with oxygen at 70-75 cycles/min. The right carotid artery was cannulated to monitor blood pressure and heart rate via a Lectromed pressure transducer (Lectromed; Letchworth, UK) connected to a pen recorder. The right jugular vein was also cannulated for bradykinin infusion. Body temperature was maintained around 37°C (±1°C) with the use of a heating pad. Arterial pH, PCO₂, and PO₂ were monitored using a blood gas system (AVL 995 pH/blood gas analyzer, AVL Medical Instruments; Stonebridge, UK). All rats were stabilized for a period of 10-15 min before the administration of substances. L-NAME (10 mg/kg) or saline was given intraperitoneally 15-20 min before bradykinin (40 µg/kg) or saline intravenously.

Statistical Analysis

All values are expressed as means ± SE. Differences in infarct size data were evaluated using one-way analysis of variance (ANOVA) followed by Fisher's protected least significant difference test. Hemodynamic parameters were examined with repeated-measures ANOVA. Differences in group mean values were considered significant when P < 0.05.

	RESULTS

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A total of 49 animals were used in this study. Thirty- one animals were used for the infarct study. All animals survived the pretreatment phase; during Langendorff perfusion, one heart was excluded due to a persistent bradyarrhythmia during the reperfusion period. Therefore, we reported the data for 30 successfully completed infarct size experiments. An additional 18 animals were used for acute hemodynamic measurements.

Infarct Size Data

Body weights and heart weights were comparable among the experimental groups (Table 1). There were no significant differences in myocardial risk zones among the groups. Myocardial infarct volume was significantly smaller in animals pretreated with an intravenous bolus of bradykinin, an effect abolished with prior administration of L-NAME. Infarct size, normalized as a percentage of risk zone volume (I/R%), is shown in Fig. 2. Hearts from saline-treated control rats exhibited an infarct size (expressed as I/R%) of 53.5 ± 3.2%. This value is consistent with our previous experience of this model. Treatment with 40 µg/kg bradykinin 24 h before infarction caused a prominent reduction in infarct size to 29.1 ± 4.7%, P < 0.01. This protective effect was completely abrogated with the prior administration of L-NAME (52.3 ± 5.0%, P < 0.05). Finally, treatment with L-NAME and intravenous saline did not influence infarct size (53.5 ± 4.8%, P < 0.05)

View this table: [in this window] [in a new window]   Table 1.   Body weight, heart wet weight, myocardial infarct, and risk zone volume, variable among experimental groups

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Infarct-to-risk zone ratio. *P < 0.01 vs. saline + saline group.

Hemodynamic Data During Infarct Protocol

Coronary flow rate (CFR) and rate-pressure product (RPP) (developed pressure × heart rate) data during the infarct procedure are depicted in Fig. 3. In the group treated with bradykinin alone (no L-NAME), CFR at baseline was significantly greater compared with the other experimental groups. This effect on CFR, implying a reduction of coronary vascular tone, was not seen in hearts pretreated with L-NAME and bradykinin (group 3). During ischemia and the remainder of the experimental protocol, however, there were no significant differences among the groups, although during reperfusion there was a tendency toward higher coronary flow rates in the bradykinin-treated group. RPP during preischemic stabilization and throughout the experiments did not differ significantly among the groups (Fig. 3B). This finding is consistent with our own and other previous observations using the model.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: changes in coronary flow rate during the ischemia-reperfusion protocol. *P < 0.05 and P < 0.1 vs. saline control (group 1) at corresponding time point (repeated-measures ANOVA). B: cardiac contractile performance during ischemia-reperfusion protocols summarized as rate-pressure product.

Hemodynamic Effects of Bradykinin In Vivo

Changes in mean arterial pressure (MAP) induced by bradykinin, L-NAME, or saline pretreatment are illustrated in Fig. 4. MAP at baseline did not differ significantly among the groups. After the administration of bradykinin (40 µg/kg), there was an immediate reduction in MAP by ~40% within 10 s. This hypotensive action was rapidly reversed, and the MAP returned to near-baseline levels within 2 min. The application of bradykinin after L-NAME administration produced a similar effect. The reduction in MAP induced by bradykinin was slightly attenuated by prior treatment with L-NAME, suggesting that the systemic hypotensive effect of bradykinin was only partially NO dependent. L-NAME alone did not significantly alter MAP. Heart rate (data not shown) was also monitored throughout the drug administration, but no significant alterations were observed.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effects of bradykinin (BK) treatment on mean arterial pressure in pentobarbital-anesthetized rats. Baseline values were obtained immediately before intravenous injection of BK (40 µg/kg) or saline. Standard error of the mean was 2-6 mmHg (bars removed for clarity). *P < 0.01 vs. saline + saline group. n = 3-5 animals per group.

	DISCUSSION

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The major findings of the present study can be summarized as follows: 1) systemic treatment with bradykinin evoked a delayed cardioprotective response 24 h later; 2) nonselective NOS inhibition at the time of bradykinin administration abolished the delayed protection afforded by bradykinin; and 3) the protective response was independent of the acute hemodynamic actions of bradykinin because L-NAME blocked the delayed cardioprotective effect of bradykinin but did not abrogate the hypotensive response to the peptide apparent many hours after the peptide has been catalytically eliminated. To our knowledge, the present study is the first to report that bradykinin induces a delayed ("second window") cardioprotective response.

Bradykinin and Delayed Preconditioning

A current paradigm for delayed ischemic preconditioning invokes a primary role of several diffusible mediators generated in the myocardium as a result of transient ischemia-reperfusion. Despite its clear role as a trigger of classic preconditioning, bradykinin has not been examined in relation to delayed preconditioning. We (19) have reported recently that pretreatment with the angiotensin-converting enzyme (ACE) inhibitor perindoprilat potentiated a subthreshold preconditioning stimulus sufficiently to induce delayed preconditioning in pig hearts. Although our study with perindoprilat did not provide direct evidence for the involvement of bradykinin, the result was compatible with the hypothesis that bradykinin (or other peptides catalytically inactivated by ACE) might be implicated in triggering the delayed phase of preconditioning. It remains to be determined whether endogenous bradykinin is a primary trigger of delayed preconditioning, for example, by using pharmacological inhibitors of bradykinin B₂ receptors or bradykinin B₂ receptor-knockout mice. However, the present study provides confirmation that bradykinin is capable of eliciting a delayed cardioprotective response.

The "NO hypothesis" of delayed preconditioning advanced by Bolli (6) postulates that NO acts as both a trigger and a distal mediator of delayed myocardial protection. Delayed preconditioning against myocardial stunning and infarction was abrogated by treatment with a NOS inhibitor during the preconditioning phase in rabbit hearts (7, 26). Furthermore, pretreatment with the NO donors diethylenetriamine-NO and S-nitroso-N-pencillamine induced protection against both myocardial infarction and stunning 24 h later (26, 27). In addition, delayed ischemic preconditioning was not demonstrable in inducible NOS knockout mice, providing strong evidence for the further involvement of NO as a distal mediator in delayed preconditioning (16). Our findings that bradykinin induces delayed protection through an NOS-dependent mechanism are compliant with the prevailing mechanistic view of delayed preconditioning. Figure 5 presents a schematic proposal for the delayed protection stimulated by bradykinin. We propose that bradykinin B₂ receptor activation stimulates NOS to produce NO from the endogeous substrate L-arginine (25). L-NAME inhibits the enzyme and blocks the production of NO. We used a nonselective NOS inhibitor L-NAME at a dose (10 mg/kg ip) that has previously been reported to reliably inhibit both constitutive NOS isoforms (endothelial NOS and neuronal NOS) and the inducible isoform. Although at present we cannot state conclusively which constitutive NOS isoform is the source of NO in the delayed cardioprotection, it is likely to be endothelial NOS rather than neuronal NOS. We hypothesize that the NO generated as a result of bradykinin B₂ receptor activation could stimulate cardiac myocyte adaptation through a cascade of intracellular events similar to those invoked for "second window" ischemic preconditioning (6). It remains to be seen whether NO generation via inducible NOS, a key mechanism in ischemia-induced delayed preconditioning, is an additional distal mechanism in bradykinin-induced delayed protection.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Schematic proposal for the induction of a delayed cardioprotective response following application of bradykinin. Immediate activation of nitric oxide (NO) synthase (NOS) as a result of bradykinin B2 receptor activation leads to the generation of NO. The most likely NOS isoform is endothelial NOS (eNOS). According to prevailing the "second window" preconditioning hypothesis, NO could subsequently trigger an adaptive response in cardiac myocytes, involving the activation of protein kinase C (PKC) isoforms and other kinases. The subsequent induction of unknown mediators of protection would result in enhanced tolerance to ischemia 24 h following application of bradykinin.

Role of NO in Acute Versus Delayed Cardioprotective Effects of Bradykinin

Although our data suggest a key role of NO generation in the mediation of bradykinin-induced delayed cardioprotection, there is no consensus that NO is a mediator of bradykinin-induced acute cardioprotection. For example, Vegh et al. (33) showed that the antiarrhythmic actions of bradykinin treatment in the canine heart was abolished by L-NAME. Shoelkens and Linz (28) showed that the functional and metabolic effects of bradykinin in the isolated rat heart were abolished by L-NAME. Similarly, Feng et al. (13) reported that bradykinin pretreatment improved recovery of ventricular and coronary vascular function by a mechanism that was blocked by L-NAME. However, in contrast, Goto et al. (15) found that bradykinin-induced acute infarct-limiting effect in rabbit heart was not abrogated by L-NAME. Similarly, Bugge and Ytrehus (8) found that bradykinin-induced acute cardioprotection in rat heart was not modified by N^G-nitro-L-arginine, an NOS inhibitor with a similar pharmacological profile to L-NAME. Thus there may well be important species and end-point variations in the role of NO in mediating the acute cardioprotective actions of bradykinin, with some models showing NO dependency. These observations may point to critical divergences in the mechanisms mediating the acute and delayed actions of bradykinin.

Vascular Effects of Bradykinin Treatment

The immediate systemic depressor action of bradykinin lasted around 1-2 min and was not significantly attenuated by L-NAME. Although bradykinin-induced vasodilatation may be endothelium dependent, the contribution of NO-mediated responses may vary among vascular beds. Our observation that the systemic hypotensive effect of bradykinin was not abrogated by L-NAME is consistent with previous observations in conscious rats (14, 17). We also observed that basal coronary flow rate was increased in hearts from bradykinin-pretreated animals. Hearts from animals that received cotreatment with L-NAME did not exhibit this basal reduction in coronary vascular tone. Previous studies of delayed cardioprotection elicited by various trigger stimuli do not reveal robust patterns of altered coronary flow. For example, Cornelussen et al. (11) reported that heat stress pretreatment enhanced baseline CFR 24 h later in the isolated working heart model. Baxter et al. reported (2) that the gram-negative bacterial endotoxin derivative monophosphoryl lipid A augmented coronary flow in rabbit heart 24 h later. Vatner et al. (20) reported that transient ischemia augmented coronary endothelium-dependent responses 24 h later in the canine heart. On the other hand, we did not find that A₁ agonist treatment caused an increase in CFR in either the rat (12) or rabbit heart (5). Tosaki et al. (30) did not observe an enhancement of coronary flow as a consequence of pretreatment with monophosphorl lipid A. The relationship between this bradykinin-induced decrease in coronary tone and tissue injury during ischemia-reperfusion are not known. During coronary occlusion, coronary flow rate decreased similarly in all groups and, whereas during reperfusion there was a tendency toward higher flow rates in the bradykinin-pretreated group, this was not statistically significant. Thus the biological significance of alterations in coronary vessel tone and reactivity brought about by various triggers of delayed cardioprotection, and the molecular mechanisms underlying these changes, is unclear at present and warrants further investigation in more appropriate and sensitive models.

In conclusion, this study is the first to show that bradykinin can elicit a delayed preconditioning-like effect in myocardium, an action that appears to be dependent on the activation of NOS. The delayed cardioprotective effect instigated by a single bolus of bradykinin points to a novel physiological action of this peptide. This observation encourages us to believe that, during brief periods of ischemia, endogenous bradykinin could play a role in triggering a "second window" preconditioning response.

	ACKNOWLEDGEMENTS

The authors acknowledge the technical assistance of Nick Davies during animal preparation. We thank the Hatter Foundation for continued support.

	FOOTNOTES

Z. Ebrahim was supported by a British Heart Foundation PhD studentship (FS/98075). The work was also supported by British Heart Foundation Programme Grant RG/98002.

Address for reprint requests and other correspondence: G. F. Baxter, The Hatter Institute & Centre for Cardiology, Univ. College London Hospital and Medical School, Grafton Way, London WC1E 6DB, UK (E-mail: g.baxter{at}ucl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 February 2001; accepted in final form 11 April 2001.

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