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Cardiovascular Research 2007 75(1):168-177; doi:10.1016/j.cardiores.2007.03.001
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Copyright © 2007, European Society of Cardiology

Intermittent activation of bradykinin B2 receptors and mitochondrial KATP channels trigger cardiac postconditioning through redox signaling

Claudia Pennaa, Daniele Mancardia, Raffaella Rastaldob, Gianni Losanob and Pasquale Pagliaroa,*

aDipartimento di Scienze Cliniche e Biologiche, dell'Università di Torino, Italy
bDipartimento di Neuroscienze (sez. Fisiologia) dell'Università di Torino, Italy

* Corresponding author. Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Regione Gonzole 10, 10043 ORBASSANO (TO), Italy. Tel.: +39 11 6705430/7710; fax: +39 11 9038639. pasquale.pagliaro{at}unito.it

Received 11 September 2006; revised 2 March 2007; accepted 5 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Objective Postconditioning (PostC) maneuvers allow post-ischemic accumulation of autacoids, which trigger protection. We tested if PostC-triggering includes bradykinin (BK) B2 receptor activation and its downstream pathway.

Methods and results Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. Infarct size was evaluated using nitro-blue tetrazolium staining. In Control hearts infarct size was 61±5% of risk area. PostC (5 cycles of 10 s reperfusion/ischemia) reduced infarct size to 22±4% (p<0.01). PostC protection was abolished by B2 BK receptor-antagonists (HOE140 or WIN64338), nitric oxide synthase-inhibitor (L-nitro-arginine-methylester), protein kinase G (PKG)-blocker (8-bromoguanosine-3',5'-cyclic-monophosphorothioate), and mitochondrial KATP (mKATP)-blocker (5-hydroxydecanoate) each given for 3 min only. Since 3 min of BK-infusion (100 nM) did not reproduce PostC protection, protocols with Intermittent-BK infusion were used to mimic PostC: a) 5 cycles of 10 s oxygenated-no-BK/oxygenated+BK buffer; b) 5 cycles of 10 s oxygenated-no-BK/hypoxic+BK buffer. Both protocols with Intermittent-BK attenuated infarct size (36±5% and 38±4%, respectively; p<0.05 vs Control and NS vs PostC for both; NS vs each other). Intermittent-BK protection was abolished by the same antagonists used to prevent PostC protection. Intermittence of re-oxygenation only (5 cycles of 10 s oxygenated/hypoxic buffer) did not reproduce PostC. Yet, cardioprotection was triggered by intermittent mKATP activation with diazoxide, but not by intermittent reactive oxygen species (ROS) generation with purine/xanthine oxidase. ROS scavengers (N-acetyl-L-cysteine or 2-mercaptopropionylglycine), given for 3 min only, abolished PostC-, Intermittent BK-and diazoxide-induced protection.

Conclusions Intermittent targeting of specific cellular sites (i.e. BK B2 receptors and mKATP channels) during early reperfusion triggers PostC protection via ROS signaling. Since neither intermittent oxygenation nor exogenous ROS generators can trigger protection, it is likely that intermittent autacoid accumulation and ROS compartmentalization may play a pivotal role in PostC-triggering.

KEYWORDS Ischemia-reperfusion injury; Nitric oxide; KATP channel; Postconditioning; Redox signaling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
It has been reported that Postconditioning (PostC), i.e. repetitive cycles of reperfusion and coronary occlusion following an ischemic insult, cause massive salvage of the myocardium, similar to the degree of protection obtained with ischemic preconditioning (IP) [1]. PostC has also been shown to be protective in humans [2,3]. PostC, like IP, involves signal transduction pathways which are very similar [4–11]. Kin et al. [10] implicated a delayed ‘washout’ of intravascular adenosine and subsequent adenosine receptor activation as a possible initial step in PostC signaling. The involvement of reperfusion injury salvage kinases and the prevention of mitochondrial transition pore opening have also been reported in PostC [4,12,13]. We, and the other authors, have found that nitric oxide synthase (NOS), cGMP and mitochondrial K+ATP (mKATP) channels are in the pathway to protection [5,7,8]. We also showed that a reactive oxygen species (ROS) scavenger, N-acetylcysteine, eliminated PostC only when it was given during the PostC maneuvers [8]. This finding, which was confirmed by Downey and Cohen [13], involves early redox-sensitive mechanism [8] in PostC protection. Data from these studies imply that during PostC maneuvers the heart may release factors which accumulate and trigger a pathway leading to a protected state [14]. The possibility to trigger protective pathways suggests that PostC may be different from treatment during reperfusion, such as staged/controlled reperfusion [15,16] or modification of the reperfusate [17–21], which has been proposed for many years. However, we wondered how metabolite(s) normally produced and released during ischemia/reperfusion are able to trigger protection only in the presence of PostC maneuvers.

Kinins, such as bradykinin (BK) and Arg-kallidin, can act on B2 receptors [20–31] and can trigger IP [22–25,29] via NO, cGMP, protein kinase G (PKG), mKATP opening and ROS production [27]. Moreover, BK administered before or after the start of ischemia and continued throughout the reperfusion, to modify the reperfusate, has been shown to be beneficial in swine, mice and rabbits [17,20,21,31]. The above studies suggest that 1) kinins are released during ischemia/reperfusion, 2) BK and other kinins can mimic IP via B2 receptor activation and 3) the protective effect of BK can occur at reperfusion. However, it is yet to be determined whether B2 receptors are involved in PostC and whether BK is protective when administered, only for a few minutes, after reperfusion in lieu of PostC. Yet, we wondered whether implementing on metabolite, such as BK, for only a few minutes at the beginning of reperfusion, would be enough to trigger PostC protection. We hypothesize that the intermittence of oxygen bursts and/or intermittence of metabolite accumulation will change the myocardial environment to allow protective pathways to be activated and to put the heart into a protected state.

To verify the role of B2 receptors and their downstream pathway elements (NOS, PKG, mKATP and ROS) in triggering PostC we performed three series of experiments. In the first series (Experiments I), we studied whether 1) B2 receptor inhibitors can abrogate the effects of PostC; 2) antagonists of downstream pathway elements given during the initial 3 min of reperfusion during PostC maneuvers can prevent PostC protection; and 3) BK given during the initial 3 min of reperfusion in lieu of PostC can reproduce PostC protection. In the second series of experiments (Experiments II) we tested whether 1) BK and/or re-oxygenation given in intermittent manner at the beginning of reperfusion can mimic PostC; 2) inhibitors of B2 receptors and downstream pathways elements can prevent the protection induced by Intermittent BK and/or re-oxygenation. Finally, in the third series (Experiments III) we studied whether continuous or intermittent direct activation of elements of the pathway downstream to B2 receptors are able to trigger protection.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
2.1 Animals
Male Wistar rats (n=206; body weight 450–550 g) received humane care in compliance with Italian law (DL-116, Jan. 27, 1992) and in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Isolated heart perfusion
The methods were similar to those previously described [7,8,32]. In brief, each animal was anesthetized. The chest was opened 10 min after heparin treatment and the heart was rapidly excised. Isolated rat hearts were retrogradely perfused with oxygenated Krebs–Henseleit buffer (127 mM NaCl, 17.7 mM NaHCO3, 5.1 mM KCl, 1.5 mM CaCl2, 1.26 mM MgCl2, 11 mM D-glucose and gassed with 95% O2 and 5% CO2). A constant coronary flow (CF) was adjusted with a proper pump to obtain a typical coronary perfusion pressure (CPP) of 80–85 mmHg during the initial part of the stabilization. Thereafter the same flow level (9±1 ml/min/g) was maintained throughout the experiment.

A small hole in the left ventricular wall allowed drainage of the thebesian flow, and a polyvinyl–chloride balloon was placed into the left ventricle and connected to an electromanometer for recording of left ventricular pressure (LVP). The balloon was filled with saline to achieve an end-diastolic LVP of 5 mmHg. CPP, CF and LVP were monitored to assess the preparation conditions. The hearts were electrically paced at 280 bpm and kept in a temperature-controlled chamber (37 °C). Reagents necessary to assess myocardial infarction were purchased from Merck (USA). Other chemicals were purchased from Sigma (USA) or TOCRIS (UK).

2.3 Experimental protocols
Each heart was allowed to stabilize for 20 min. After the stabilization period, hearts were subjected to a specific protocol, which included in all groups 30 min of global no-flow ischemia. A period of 120 min of reperfusion followed the 30 min ischemia in all groups (see below). Pacing was discontinued on initiation of ischemia and restarted after the third minute of reperfusion in all groups [7,8,10] (Fig. 1A, B and C).


Figure 1
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  		Fig. 1 Schematic representation of protocols: A represents protocols of Experiments I (Groups 1–8); B represents protocols of Experiments II (Groups 9–16), C represents protocols of Experiments III (Groups 17–20) and protocols of Additional Control Experiments (Groups 21–33). I=10 s global ischemia; R=10 s reperfusion; Nox=10 s normoxic buffer; Ipox=10 s hypoxic buffer; Interm = Intermittent; Stab = Stabilization; DRUG = BK or DZO; other acronyms as in the text.

 
2.4 Experiments I (Groups 1–8)
Experimental protocols for Experiments I are described in Fig. 1A. After stabilization, the hearts of the Control group (Group 1, n=18) were exposed to 30 min ischemia and then to 120 min reperfusion only. In Group 2 (PostC group; n=18) after the 30 min ischemia, the hearts immediately underwent a protocol of PostC. This consisted of five cycles of 10 s reperfusion and 10 s global ischemia [7,8]. To test the role of B2 BK receptors in PostC, in Group 3 (PostC+HOE140 group, n=6), during the PostC protocol the hearts were perfused with their specific antagonist HOE140 (100 nM) [27,30]. To mimic PostC with a B2 activator, in Group 4 (3 min BK group; n=6) after 30 min ischemia the hearts were perfused with BK (100 nM) [31,33] during the initial 3 min of reperfusion.

Since B2 receptor activation can trigger IP via NO, PKG, mKATP opening and ROS production [27], we checked whether these mechanisms are also involved in PostC-triggering using either the ROS scavenger, N-acetyl-L-cysteine (NAC; 4 mM) [8,32], or the PKG blocker, 8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS; 1 µM) [34]; or the mKATP channel blocker, 5-hydroxydecanoate (5-HD; 100 µM) [8,35,36] or the NOS inhibitor, L-nitro-arginine-methylester (LNAME; 100 µM) [7]. Therefore, in Group 5 (PostC+NAC; n=5), Group 6 (PostC+Rp-8-Br-PET-cGMPS; n=6), Group 7 (PostC+5-HD; n=5) and Group 8 (PostC+LNAME; n=5) the hearts during the protocol maneuvers of PostC (i.e. during the initial 3 min of reperfusion) were perfused with a solution containing one of the above reported antagonists.

The above studies suggested that 3 min of infusion with an antagonist of B2 receptors as well as those of downstream pathway elements can prevent PostC-triggering. However, BK given for 3 min only, in lieu of PostC, is not able to trigger PostC protection (see Results section). Therefore, a second series of experiments (Experiments II) was conducted to test whether the intermittent oxygen bursts and/or intermittence of BK would change the myocardial environment to allow kinins to activate protective pathways (i.e. B2 receptors and their downstream pathway elements).

2.5 Experiments II (Groups 9–16)
Experimental protocols of Experiments II are described in Fig. 1B. In Group 9 (intermittent re-oxygenation; n=5), Group 10 (Intermittent BK; n=6) and Group 11 (Intermittent BK+intermittent re-oxygenation; n=7) after 30 min ischemia the hearts were perfused with intermittent perfusion of oxygenated and/or non-oxygenated buffers containing or not containing BK. In particular, these intermittent protocols consisted of five cycles of 10 s of oxygenated/non-oxygenated buffers (Group 9), or five cycles of 10 s infusion of oxygenated buffer without and with BK (100 nM) (Group 10), or five cycles of 10 s of oxygenated buffer alternated with 10 s infusion of BK in a non-oxygenated buffer (Group 11). The oxygen content was 20–22% in the oxygenated buffer and 4–4.5% in non-oxygenated buffer.

Since a high protective effect (see Results section) was achieved with the Intermittent BK+intermittent re-oxygenation protocol (Group 11), we studied whether the antagonist of B2 receptors as well as those of their downstream pathway elements can abrogate this protective effect. Therefore, the hearts after 30 min ischemia underwent a protocol of Intermittent BK+intermittent re-oxygenation, as in Group 11, while perfused with a solution containing one of the specific antagonists at the dose above reported. In particular we used the B2 receptor antagonist in Group 12 (Intermittent BK+HOE140; n=6), the ROS scavenger in Group 13 (Intermittent BK+NAC; n=6), the PKG blocker in Group 14 (Intermittent BK+Rp-8-Br-PET-cGMPS; n=5), the mKATP channel inhibitor in Group 15 (Intermittent BK+5-HD; n=5), and the NOS inhibitor in Group 16 (Intermittent BK+LNAME; n=5).

2.6 Experiments III (Groups 17–20)
Experimental protocols of Experiments III are described in Fig. 1C. Since the results of the above experiments (see Results section) suggest that downstream steps to B2 receptor activation (e.g. mKATP channel activation and ROS production) should also be accomplished in an intermittent fashion, we tested whether a mKATP activator or an exogenous ROS source is as effective as a PostC stimulus if administrated either continuously or intermittently. To activate mKATP channels we used diazoxide (DZO, 30 µM) [36,37]. To generate ROS we infused purine/xanthine oxidase (P/XO, 1 mM and 20 U/l, respectively) [38]. Therefore, in Group 17 (3 min DZO group; n=5) and in Group 18 (3 min ROS group; n=5), diazoxide and P/XO, respectively, were infused for 3 min in continuous. In Group 19 (Intermittent DZO group; n=5) and in Group 20 (intermittent ROS group; n=5), diazoxide and P/XO were infused in intermittent fashion as in Group 11.

2.7 Additional control experiments (Groups 21–33)
Experimental protocols of additional control experiments are also described in Fig. 1C. To verify the effects of a single bout of BK with a dose equal to the total bradykinin administered in the Intermittent BK studies, we performed experiments in which 100 mM BK were infused for 50 s continuously (Group 21; 50 s BK group; n=5). To confirm B2 receptor involvement, we used another B2 antagonist, WIN64338, which is structurally unrelated to HOE140. Thus in Group 22 (PostC+WIN64338 group, n=5), during the PostC protocol the hearts were perfused with 100 µM of WIN64338 [39]. In Group 23 (Intermittent BK+WIN64338; n=5) the same concentration of WIN64338 was given during intermittent-BK infusion. To confirm ROS involvement, we used another ROS scavenger, 2-mercaptopropionylglycine (MPG). Thus in Group 24 (PostC+MPG group, n=5), during the PostC protocol the hearts were perfused with 300 µM of MPG [36,40]. In Group 25 (Intermittent BK+MPG; n=5) and in Group 26 (Intermittent DZO+MPG; n=5) the same concentration of MPG was given during intermittent-drug infusion.

To determine the effects of the antagonists themselves, in Group 27 (HOE140 group, n=6), Group 28 (WIN64338 group, n=6), Group 29 (NAC group, n=5), Group 30 (MPG group, n=5), Group 31 (Rp-8-Br-PET-cGMPS group; n=5), Group 32 (5-HD group; n=5), and Group 33 (LNAME group; n=6) after 30 min ischemia the hearts were perfused, during the initial 3 min of the reperfusion, with one of the antagonist reported above.

Since diazoxide and WIN64338 had been dissolved in DMSO 0.04% [36,37], in 4 additional hearts we tested intermittent DMSO, which was ineffective (protocol and data not shown). The doses of the drugs used were selected because they have commonly and successfully been used previously, and because these drugs have shown to be potent antagonists of B2 receptors, NOS, mKATP channel and PKG activation or ROS scavengers [7,8,30–40].

2.8 Assessment of myocardial injury
At the end of the experiments all hearts were removed from the apparatus for the analysis of infarct size. Infarct areas were assessed as previously described [7,8,32]. Briefly, immediately after reperfusion each heart was rapidly removed from the perfusion apparatus and the left ventricle (LV) was dissected into 2–3 mm circumferential slices. Following 20 min of incubation at 37 °C in 0.1% (w/v) solution of nitro-blue tetrazolium in phosphate buffer, unstained necrotic tissue was carefully separated from stained viable tissue by an independent observer. The necrotic mass was expressed as a percentage of the total left ventricular mass. In fact, though in this model the whole heart underwent normothermic ischemia, only the LV had a fixed volume and pre-load; therefore only the LV mass was considered as risk area. Lactate dehydrogenase (LDH) release was assessed as previously described [7,8].

2.9 Statistical analysis
All data are expressed as means±S.E.M. One-Way ANOVA and Tukey's HSD (honestly significant difference) for post-ANOVA comparisons were used to evaluate the statistical significance of the differences of the parameters between groups. A p value <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Cardiac weight (1455±12; range 1280–1780 mg, n=206) and the cardiac to body weight ratio (2.60±0.009; range 2.04–2.98 mg/g) were similar in all groups. The risk area, i.e., LV mass, was also similar in all groups (LV weight was 915±10; range 579–1125 mg).

3.1 Experiments I (Groups 1–8)
Fig. 2 shows results from Control group (Group 1), PostC group (Group2), and from groups (Groups 3–8) in which we checked the role of BK B2 receptors and their downstream pathway elements in triggering PostC protection. Total infarct size, expressed as a percentage of left ventricular mass, was 61±5% in Control Group 1. In PostC Group 2 the infarct size was 22±4%, a value significantly (p<0.01) lower than that of Control Group 1. Infarct size of Group 3 (PostC+HOE140) and that of Group 4 (3 min BK) was similar to that of the Control Group 1 (p=non significant (NS) vs control; p<0.05 vs PostC, for both) (Fig. 2).


Figure 2
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  		Fig. 2 Infarct size of Experiments I (Groups 1–8): Figure represents infarct size at the end of the experiments, expressed as percentage of left ventricle (LV) mass. For each group individual datapoints and means ±SEM are reported. **p<0.01 vs Control; # p<0.05 vs PostC.

 
To verify the involvement of downstream elements usually recruited by B2 receptor activation and PostC, the cardiac preparations were treated with specific inhibitors which block signal transduction leading to protection. The inhibitors were given during the initial 3 min reperfusion, while performing PostC (Groups 5–8). Each of the inhibitors abolished the infarct sparing effect induced by PostC. These results suggest that both B2 receptors and their downstream pathway elements are involved in PostC. However, during early reperfusion a simple implementation of BK is not enough to mimic PostC.

3.2 Experiments II (Groups 9–16)
Fig. 3 shows data from groups (Groups 9–11) in which we checked the role of intermittent oxygen burst and/or Intermittent BK in inducing protection. Fig. 3 also shows data of the groups (Groups 12–16) in which during Intermittent BK+intermittent re-oxygenation we tested the involvement of B2 receptors and the involvement of the downstream pathway elements. For comparative purpose, data from Control (Group 1) and PostC (Group 2) are re-reported in Fig. 3.


Figure 3
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  		Fig. 3 Infarct of Experiments II (Groups 9–16). Figure represents infarct size at the end of the experiments, expressed as percentage of left ventricle (LV) mass. Data of Control (Group 1) and PostC (Group 2) are also reported. For each group individual datapoints and means±SEM are reported. *p<0.05 and **p<0.01 vs Control; # p<0.05 vs PostC; § p<0.05 vs Intermittent BK (either Group 10 or 11).

 
Intermittent re-oxygenation alone (Group 9) did not reproduce the infarct sparing effect of PostC. Surprisingly, both the Intermittent BK alone (Group 10) and the Intermittent BK+intermittent re-oxygenation (Group 11) reproduced the protective effect of PostC (Infarct size: 36±5% and 38±4%, respectively; p<0.05 vs Control and NS vs PostC for both; NS vs each other). When each inhibitor was given during the initial 3 min reperfusion, while performing Intermittent BK infusion (Groups 12–16), the infarct sparing effect triggered by Intermittent BK was abolished. The infarct size of Intermittent BK and PostC groups was also significantly (p<0.05) smaller that that of all antagonist groups (Groups 12–16) (Fig. 3).

3.3 Experiments III (Groups 17–20)
Fig. 4 shows data from groups (Groups 17–20) in which we checked whether the mKATP activator, DZO, or the exogenous ROS source, P/XO, were effective as a PostC stimulus when given continuously for 3 min or intermittently. For comparative purpose, data from Control (Group 1), PostC (Group 2) and Intermittent BK (Group 11) are re-reported in Fig. 4.


Figure 4
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  		Fig. 4 Infarct of Experiments III (Groups 17–20). Figure represents infarct size at the end of the experiments, expressed as percentage of left ventricle (LV) mass. Data of Control (Group 1), PostC (Group 2) and Intermittent BK (Group 11) are also reported. For each group individual datapoints and means±SEM are reported. *p<0.05 and **p<0.01 vs Control; # p<0.05 vs PostC; § p<0.05 vs Intermittent BK; {infty} p<0.05 vs Intermittent DZO.

 
Neither 3 min diazoxide (3 min DZO, Group 17) nor 3 min P/XO (3 min ROS, Group 18) given in continuous were able to reproduce the infarct sparing effect of PostC. Yet, the protective effect of PostC was reproduced by intermittent diazoxide (Group 19; Infarct size=35±3%, p<0.05 vs Control, NS vs PostC and NS vs Intermittent BK), but not by intermittent P/XO (Group 20, Intermittent ROS) (Fig. 4).

3.4 Additional control experiments (Groups 21–33)
Fig. 5A and B show data from additional control experiments (Groups 21–33). For comparative purpose data of Control (Group 1), PostC (Group 2) and Intermittent BK (Group 11) are re-reported in both Fig. 5A and B.


Figure 5
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  		Fig. 5 Infarct of Additional Control Experiments (Groups 21–26, Panel A and Groups 27–33 Panel B). A and B represent infarct size at the end of the experiments, expressed as percentage of left ventricle (LV) mass. Data of Control (Group 1), PostC (Group 2) and Intermittent BK (Group 11) are also reported in both panels. For each group individual datapoints and means±SEM are reported. * p<0.05 and ** p<0.01 vs Control; # p<0.05 vs PostC; § p<0.05 vs Intermittent BK.

 
Fig. 5A shows that in Group 21 a single bout of BK (50 s BK continuously) was unable to trigger PostC protection. In the presence of a non-peptidic BK B2 receptor antagonist, WIN64338, neither PostC maneuvers (PostC+WIN64338, Group 22), nor Intermittent BK (Intermittent BK+WIN64338, Group 23) were able to trigger protection. Also, the ROS scavenger MPG, a sulfhydryl donor more specific for mitochondrial activity than NAC [36,40], prevented the protection by PostC (PostC+MPG, Group 24), by Intermittent BK (Intermittent BK+MPG, Group 25) and by Intermittent DZO (Intermittent DZO+MPG, Group 26). Fig. 5B shows that the treatment with each of the blockers alone (Groups 27–33) did not significantly change infarct size, which were similar to that of Control Group 1, and significantly (p<0.05 for all) higher than that of PostC and Intermittent BK groups. Infarct sparing effects of PostC, Intermittent BK and Intermittent DZO were corroborated by the reduction of LDH release during reperfusion with respect to Control Group 1 (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Acknowledgments
 References
 
Our main goals were 1) to show that cardioprotection triggered by postconditioning involves endogenous activation of B2 receptors; 2) to study which downstream signaling events must also be accomplished to trigger protection and 3) to trigger PostC protection by exogenous activation of these receptors or downstream element(s) during the early reperfusion.

We found that 1) cardioprotection triggered by PostC maneuvers involves endogenous activation of BK B2 receptors. In fact only 3 min of B2 receptor antagonists were able to prevent PostC protection; 2) blockers of downstream pathway elements (i.e. PKG, mKATP, NOS), including ROS scavengers, given for 3 min only were also able to avoid PostC protection; 3) surprisingly, it was necessary to give BK or diazoxide in an intermittent manner during early reperfusion to trigger PostC protection.

Our findings are different from classical treatment during reperfusion [17–21]. We showed that, using either agonists or antagonists during early reperfusion only, it is possible to, respectively, reproduce or avoid PostC triggering. In fact, 5 cycles of 10 s Intermittent BK or diazoxide triggered PostC like protection. Yet, antagonists of NOS, PKG, mKATP, and ROS suppressed the protective effect if given for 3 min, just to bracket PostC maneuvers or intermittent-BK infusion.

Results suggest that during PostC maneuvers the heart releases kinins that accumulate in an intermittent manner, which trigger pathways leading to a protected state. Intermittent oxygenation only does not trigger protection (Group 9). One reason could be that hypoxic reperfusion (without additional BK) is washing out the metabolites which are likely being produced during hypoxia.

Attenuation of ROS production had been proposed to contribute to PostC [1]. However, it has been reported that PostC not only attenuates oxidants and oxidant-mediated injury, but may also preserve the signaling function of ROS and NO [8,13,14]. In fact, ROS play an essential, though double-edged, role in cardioprotection. They may participate in reperfusion injury [41,42 and references therein] or may play a role as signaling elements of protection [8,13,27,36,41–46]. It has been reported that ROS are involved in IP and that exposure to ROS generating system could precondition the heart [27,36,38,43–46]. Several metabolites, including acetylcholine, bradykinin, opioids and phenylephrine, trigger preconditioning via a mKATP-ROS-dependent mechanism [27,45,46].

Despite the suppression of PostC protection by cell permeant ROS scavengers [8,13 and present study], exogenous ROS application was unable to trigger protection either when given continuously for 3 min or when given intermittently. Since only intermittent targeting of specific cellular sites, such as BK B2 receptor and mKATP channel activation, triggers protection, it is likely that autacoid intermittent accumulation and/or ROS compartmentalization may play a pivotal role in PostC-triggering. In fact ROS scavengers abolish Intermittent BK-and Intermittent DZO-protection.

We propose that PostC is triggered by an Intermittent BK B2 receptor mechanism that involves PKG, and early production of ROS via mKATP channel activation [27,36,45,46]. It seems necessary that ROS production occurs at the right moment in the right compartment (i.e. mitochondria) to participate in PostC triggering of protection against reperfusion injury. The exact mechanisms responsible for this compartment-specific response is not specifically addressed in this study but likely vary with timing of reperfusion, protocol and experimental model. It is well known that PostC, to be effective, requires a precox beginning and a species-specific algorithm [14]. It is likely that before ischemia compartmentalization it is not mandatory to trigger protection with exogenous sources of ROS [27,36,45,46]. However, during reperfusion the border between signaling effect and deleterious effect of ROS is very narrow; thus timing and compartmentalization may be very important.

Also, NO synthases have a specific subcellular compartmentalization with co-localized effectors, yet, ROS and NO generation interact and can modulate each other. In fact, ROS, NO and peroxynitrite can have beneficial or deleterious effects in function of amount, compartmentalization and timing of generation [41,42,47,48].

In summary, the data of the present study, obtained in an ex vivo preparation, imply that during the brief ischemia/reperfusion periods of postconditioning the heart releases and intermittently accumulates kinins which activate downstream enzymes (e.g. NOS/PKG). These enzymes may allow mitochondria to produce enough ROS (or the right type of ROS) in the appropriate compartment to activate downstream mediators to put the heart into a protected state. Therefore, the PostC protection is achieved only if ROS production is timely and not reduced by a scavenger of free radicals. In other words, our data suggest that among the triggers of PostC- and Intermittent BK-protection there is intermittent mKATP activation, which may produce the right type and/or amount of ROS which, in turn, contribute to the activation of intrinsic mechanisms against the deleterious effects of the subsequent reperfusion. Despite the infarct sparing effect, neither Intermittent BK, nor intermittent diazoxide nor PostC improve contractility recovery during reperfusion (data not shown), as we reported for PostC only [8]. This is also in agreement with the ineffectiveness of postconditioning against myocardial stunning [49].

4.1 Further considerations on the used antagonists and agonists
It is known that NAC may affect intra and extra-cellular free radicals. In fact, NAC is readily hydrolyzed to cysteine and is able to expand natural antioxidant defenses by increasing intra-cellular reduced glutathione concentration. Importantly, NAC has sulfhydryl groups that can directly scavenge free radicals under acidotic conditions which exist in the early post-ischemic phase of the experiments [50]. MPG also acts as a sulfhydryl donor and several studies indicate that MPG may be more specific for mitochondrial activity than NAC [36,40,50].

NOS inhibition prevented PostC protection in the in vivo rabbit [5]. Yet, in the isolated rat heart [7] NOS inhibition by a continuous infusion of similar or higher concentrations of LNAME during the entire period (i.e. for 120 min) of reperfusion attenuated (not suppressed) the protective effect induced by PostC. In the present study, 3 min of LNAME infusion completely prevented the protection induced by PostC or by Intermittent BK (Groups 8 and 16). The reasons for these differences are not clear at the moment. In the literature are reported both cardioprotective and detrimental effects of NO during reperfusion. Contradictory results about the effects of NO during reperfusion have been reviewed by Bolli in 2001 [47] and Schulz et al. in 2004 [48]. Again a reasonable explanation for the differences in results between the present and our previous study [7] is that NO effects are concentration-and time-dependent [48]. We can argue that the NOS-inhibitor given during the initial 3 min does not allow the triggering of protective mechanisms by NO/cGMP/PKG pathway, whereas the prolonged infusion of the NO-inhibitor avoids the deleterious effects of an augmented production of NO in the presence of high levels of ROS, which can occur in the late reperfusion phase and can lead to excessive peroxynitrite formation and tissue injury [41,48,50].

Bradykinin was believed to be the only kinin acting on the B2 receptors in rats. Recently, it has been demonstrated in isolated rat hearts a kallidin-like peptide, Arg-kallidin, which also acts on B2 receptors [28–30]. Importantly, Arg-kallidin release increases during ischemia and mediates IP and preconditioning-like protection, via B2 receptor activation, in isolated rat hearts [29,30]. Whether the endogenous mediator of PostC protection is BK or Arg-kallidin was beyond the aims of the present study. Whichever the case, in the present study, using two structurally unrelated B2 receptor antagonists (HOE140 or WIN64338), we show that these receptors are involved in endogenous PostC and in exogenous activation by Intermittent BK.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Acknowledgments
 References
 
We show for the first time, in isolated rat hearts, the involvement of BK B2 receptors in PostC and stress the importance of the intermittence of maneuvers in PostC triggering. Intermittent ligand accumulation and B2 receptor activation appear to be an important determinant of protection, in which the redox conditions of the myocardium play a pivotal role. In fact, BK infusion for 3 min (i.e. without intermittence) did not trigger protection. However, either PostC or the Intermittent BK triggered cardioprotection and both protections were prevented by the B2 receptors antagonists (HOE140 or WIN64338), the NOS inhibitor (LNAME) and the ROS scavengers (NAC or MPG). The downstream events of intermittent B2 receptor activation include also PKG activation and the intermittent opening of mKATP channels, which may be responsible for ROS signaling.

Time for primary review 28 days


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
References
 
We thank Miss Jennifer M. Lee for language revision, Miss Francesca Moro and Miss Francesca Tullio for the technical assistance. Funding Sources: Regione Piemonte, Compagnia di San Paolo, INRC and MIUR.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 

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