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Cardiovascular Research 2004 61(3):591-599; doi:10.1016/j.cardiores.2003.10.008
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Copyright © 2003, European Society of Cardiology

Remote preconditioning by infrarenal aortic occlusion is operative via {delta}1-opioid receptors and free radicals in vivo in the rat heart {star}

Christof Weinbrenner*,a, Falk Schulzea, László Sárvárya,b and Ruth H Strassera

aDepartment of Cardiology, Medical Clinic II, University of Technology Dresden, P.O. Box 95, Fetscherstr. 76 D-01307 Dresden, Germany
bDepartment of Cardiology, Medical Clinic III, University of Heidelberg, Heidelberg, Germany

* Corresponding author. Tel.: +49-351-4501700; fax: +49-351-4501702. cweinbre{at}web.de

Received 2 April 2003; revised 26 September 2003; accepted 5 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Summary
 References
 
Background: Ischemic preconditioning (PC) is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart (remote PC). We have previously shown that remote PC by infrarenal occlusion of the aorta [IOA] in the rat is as powerful as classical ischemic PC. This protection may be transmitted by humoral factors, and protein kinase C is a mediator in the signal transduction mechanism. Focus of the present study was to address the question whether remote preconditioning is dependent on the activation of the {delta}1-opioid receptor and/or free radicals, the infarct size was determined after either inhibition of the {delta}1-opioid receptor or scavenging free radicals. Methods and results: IOA was performed in rats by occlusion of the infrarenal aorta for 15 min followed by a 10-min reperfusion period. Infarction of the heart was induced by 30 min regional ischemia followed by 30 min of reperfusion. The area of infarct was determined by propidium iodide and the risk zone was demarcated by zinc cadmium sulfide fluorescent particles. Control hearts (30 min regional ischemia of the heart followed by 30 min of reperfusion; no IOA) had an infarct size of 54±3%, whereas classical preconditioning by three ischemia/reperfusion [I/R] cycles, 5 min each, reduced it to 12±1% of the risk zone (p<0.05). Fifteen minutes IOA with 10 min of reperfusion was highly protective and reduced the infarct size to 20±5% (p<0.05 vs. control). Inhibition of the {delta}1-opiod receptors by 7-benzylidenenaltrexone [BNTX] blocked the protection obtained by PC and IOA (41±4% and 44±2%, respectively; p<0.05 vs. the group without BNTX). BNTX in control hearts had no influence on infarct size (52±2%). Inhibition of endogenously released radicals by N-2-mercaptopropionyl glycine [MPG] blocked the infarct size reduction of IOA (46±3%; p<0.05 vs. IOA), but had no influence on the protection in classically preconditioned hearts protected by three cycles I/R (13±4%). Only if the number of the preconditioning stimuli was reduced to one was MPG able to overcome the protection (43±4%, p<0.05 vs. PC with one I/R cycle (21±4%)). Conclusion: Remote preconditioning using IOA protects the rat heart from infarction. Classical and remote PC share both the {delta}1-opioid-receptor and free radicals as common elements in their signal transduction pathways. MPG can block protection from IOA and from one, but not from three, classical preconditioning cycles. This indicates that the protection by remote preconditioning is comparable to classical PC with one I/R cycle.

KEYWORDS Preconditioning; Infarct size; Ischemia; Free radicals; Opioid receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Summary
 References
 
Murry et al. [1] first described the phenomenon of ischemic preconditioning (brief periods of ischemia interspaced with reperfusion reduce the infarct size of the heart) in 1986. In 1993, Przyklenk et al. [2] described a mechanism of protecting the anterior wall of the heart by a brief period of ischemia in a territory of the heart which is distant to the final area of infarction. This mechanism was termed "remote preconditioning" or "preconditioning on a distance". Recently, intra-cardiac remote preconditioning was questioned, however, by Nakano [3] from Downey's group who could not find a protection in the remote myocardium after regional ischemia in situ followed by global ischemia of the rabbit heart in the Langendorff model. The mechanism of remote preconditioning is not limited to one organ system either; for example McClanahan et al. [4] have shown that brief ischemia of the kidney can protect the heart from infarction. Meanwhile, it was also observed that a preconditioning stimulus of an area perfused by the mesenterial artery [5–7] or the femoral artery [8] confers protection to the heart.

Remote preconditioning was shown in several species, including rat, rabbit and canine. Recently an indication for the presence of remote preconditioning in humans was made by Günaydin et al. [9]. They showed that during coronary artery bypass surgery a tourniquet wrapped around an upper extremity in order to induce brief periods of ischemia resulted in an attenuated increase of lactate dehydrogenase, suggesting a reduction in myocyte injury. This observation also indicates a potential link to the clinical and therapeutical relevance of remote preconditioning.

The signal transduction pathways of both kinds of preconditioning share some similarities. Classical preconditioning can be mimicked by pharmacological activation of numerous G-protein coupled receptors. In remote preconditioning, antagonists of adenosine receptors [10], {alpha}1-adrenergic receptors [11], bradykinin receptors [5,12], and opioid receptors [13,14] could prevent the protection. Among the opioid receptors, it is not determined, however, which receptor-subtype is involved in remote preconditioning.

The signals from classical ischemic, pharmacological, and remote preconditioning converge on the level of protein kinase C [15]. Protein kinase C as a key enzyme in preconditioning is disputed, however, since in pigs [16] and dogs [17] inhibition of protein kinase C did not abolish protection. Only after inhibition of both protein kinase C and tyrosine kinase was protection of ischemic preconditioning completely blocked [18]. Recently, we found that protein kinase C is involved in the signaling mechanism of remote preconditioning [19]. At the same time, Wolfrum et al. [12] published that protein kinase C-{varepsilon} is the protective isoform of remote preconditioning.

In classical preconditioning free radicals are important transducers of the signaling cascade. Whether or not they are triggers or mediators of preconditioning is not clarified yet [20]. It is known that free radicals can directly stimulate kinases embedded in the signal transduction cascade of preconditioning [21,22]. Conversely, protection of preconditioning can be blocked by MPG or ascorbic acid, both scavengers of free radicals [23,24]. MPG, however, could inhibit the protection of preconditioning only if the preconditioning stimulus consists of one instead of three ischemia/reperfusion stimuli [24]. This suggests that reducing the number of preconditioning cycles weakens the power of protection. Thus, MPG can abrogate the protection. In 1997, Sandhu et al. [25] already made the observation that the strength of preconditioning depends on the repetition of preconditioning cycles. Investigations with low flow or ramp ischemia also suggest that preconditioning is a graded phenomenon [26].

In remote preconditioning, little is known about the signal transduction cascade downstream of the receptor level. Specifically, the role of free radicals has not been investigated so far.

For protecting the heart "at a distance", the previously characterized model of infrarenal occlusion of the aorta was used [19]. It is hypothesized that remote preconditioning acts similarly to classical preconditioning through activation of the {delta}1-opioid receptor. Furthermore, we compared the signal transduction pathways of classical and remote preconditioning regarding free radicals, which are important triggers and/or mediators of classical preconditioning [20].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Summary
 References
 
All procedures were in conformance 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) and were approved by the local Animal Care and Use Committee.

2.1 General surgical preparation
Male Wistar rats, 250–320 g, were anesthetized via intraperitoneal administration of thiopental (40 mg/kg). The effectiveness of the anesthesia was maintained with additional thiopental (5–10 mg/kg i.p) every 15 min if necessary. The rats were tracheotomized and ventilated with room air supplemented with oxygen at 65 breaths/min (rodent ventilator, TSE, Bad Homburg, Germany). Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5–10 cmH2O. Arterial pH, Pco2, and Po2 were monitored at baseline, 15 min of coronary occlusion or infrarenal occlusion of the aorta, and 15 min of reperfusion by a blood gas analyzer (AVL 990) and maintained within a normal physiological range. Body temperature was maintained at 37 °C by using a heating pad.

The left axillary artery was cannulated to measure the blood pressure and heart rate via a pressure transducer (MLT844, ADInstruments, Spechbach, Germany) connected to a computer (using ADInstruments PCLab). The left axillary vein was cannulated in order to infuse drugs or saline.

The abdomen was opened in the median line and the abdominal aorta was exposed after moving the small intestine to the right side of the abdomen. For occlusion of the infrarenal aorta a Biemer vessel clip (Aesculap) was placed around the aorta for the designated times.

A left thoracotomy was performed at the fourth intercostals space followed by a pericardiotomy. A 5-0 suture (Ethibond Excel, Ethicon, Germany) was passed below the left descending vein and coronary artery for later induction of coronary artery occlusion. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure and reperfusion was confirmed by epicardial hyperemia. Heart rate and blood pressure were allowed to stabilize for 10 min before the following protocols were initiated.

2.2 Study groups and experimental protocols
In the first protocol, seven groups with four to seven rats in each group were studied. Group 1 rats served as controls and received a prolonged equilibration time of 40 min prior to the 30-min period of regional ischemia followed by 30 min of reperfusion (Fig. 1a). Group 2 was ischemically preconditioned (PCx3) with three cycles of 5 min of regional ischemia by ligation of the left descending artery followed by 5 min of reperfusion before the onset of 30 min of regional ischemia. In the third group, the infrarenal aorta was occluded for 15 min followed by a 10-min reperfusion period prior to the regional ischemia of the heart (IOA). The {delta}1-selective opioid receptor antagonist BNTX was used to test for the involvement of the {delta}1-opioid receptor. It was given as a bolus with a dose of 3 mg/kg body weight intravenously 10 min prior to the intervention in control hearts (Co+BNTX), classically preconditioned hearts (PCx3+BNTX), and remotely preconditioned hearts (IOA+BNTX). To test if activation of the {delta}1-opioid receptor can confer protection we used DPDPE, a {delta}1-selective opioid receptor agonist, and gave it as a bolus (3 mg/kg body weight) 10 min prior to the infarct inducing ischemia of the heart (DPDPE).


Figure 1
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  		Fig. 1 a and b: Experimental protocol. See text for details.

 
In the second protocol, MPG, a free radical scavenger, was used in 4 additional groups to investigate the role of free radicals in remote preconditioning (Fig. 1b). The dose of MPG was selected to 20 mg/kg body weight since other groups have demonstrated that this dose does inhibit the protection by one classical preconditioning stimulus [24,27,28]. A total of 20 mg/kg MPG were given as a bolus intravenously starting 20 min prior to the interventions in control (Control+MPG), classically preconditioned hearts which were preconditioned by either one (PCx1+MPG) or three (PCx3+MPG) ischemia/reperfusion cycles, and remotely preconditioned hearts (IOA+MPG). We also protected one group by classical preconditioning where we used only one, instead of three, preconditioning cycles (PCx1).

2.3 Determination of infarct size
After 15 min of reperfusion, 10 mg/kg body weight propidium iodide at a concentration of 1 mg/ml was given as a bolus. After an additional 15 min of reperfusion, the heart was excised and quickly mounted on a Langendorff apparatus to wash out the blood. The coronary artery was reoccluded, and 1–10 µm zinc cadmium sulfide fluorescent particles (Duke Scientific Corp., Palo Alto, CA) were infused to demarcate the risk zone as the tissue without fluorescence. The heart was weighed, frozen, and cut into 2-mm-thick slices. The areas of infarct (bright red fluorescent under ultraviolet light) and risk zone (non-fluorescent under ultraviolet light) were determined by planimetry. Infarct size was expressed as a percentage of the risk zone.

2.4 Chemicals
7-Benzylidenenaltrexone (BNTX) was purchased from Tocris (Biotrend, Köln, Germany) and was dissolved in 0.9% saline to a concentration of 1 mg/ml. [D-Pen2,5]enkephaline (DPDPE) was dissolved in 0.9% saline to a concentration of 1 mg/ml and N-2-mercaptopropionyl glycine (MPG) was dissolved in 0.9% saline to a concentration of 7 mg/ml. DPDPE, MPG, and propidium iodide were purchased from Sigma (Deisenhofen, Germany). The solutions were freshly prepared prior to every experiment.

2.5 Statistics
Values are means±S.E. Two-way analysis of variance and Bonfferoni post hoc test were used to test for differences between hemodynamic values. One-way analysis of variance without repeated measures and Tukey–Kramer post hoc test were used to test for differences in infarct size. A p value <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Summary
 References
 
3.1 Hemodynamics
Baseline hemodynamic parameters were comparable in the 12 groups of animals (Table 1). Only the PCx3, PCx1, control+MPG, and PCx1+MPG groups had higher heart rates at baseline compared to control hearts. Thus the rate pressure product was also significantly different in these groups. In the PCx1 group, the mean arterial pressure was higher than in the control group at baseline. Within the groups, the mean arterial pressure dropped significantly in all groups except for the control group after 30 min of reperfusion. In some groups, this was seen already after 30 min of coronary occlusion. Administration of BNTX, DPDPE, and MPG caused a depression of the heart rate and the mean arterial pressure, albeit this was only significant in the PCx1+MPG group for the mean arterial pressure. Comparisons of the hemodynamics between the groups at coronary occlusion or reperfusion revealed no statistical difference. This we also observed in our previously published paper [19], where an improved contractile force could only be detected after 60 and 120 min of reperfusion but not after 10 min of reperfusion.


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  		Table 1 Hemodynamic data

 
3.2 Role of opioids in remote preconditioning
Infarct sizes normalized as a percentage of the ischemic (risk) zone are shown in Fig. 2. Control hearts (30 min of regional ischemia) had an infarct size of 54±3%. Rats which were treated with 15 min IOA followed by 10 min of reperfusion (IOA) had a significant infarct size reduction down to 20±5% (p<0.05 vs. control). Statistically this was not significantly different from classically preconditioned hearts (PCx3, 12±1%; p<0.05 vs. control). BNTX blocked the protection obtained from classical PC (41±4%; p<0.05, PCx3+BNTX vs. PCx3) as well as from remote preconditioning (44±2%; p<0.05, IOA+BNTX vs. IOA). Thus, the infarct size reduction effect of both classical and remote preconditioning was significantly blocked by BNTX. BNTX itself had no influence on the infarct size in control hearts (52±2%; Control+BNTX). Conversely, the {delta}1-opioid receptor agonist DPDPE could induce an infarct size reduction which was comparable to the one observed in classically and remotely preconditioned hearts (21±4%; p<0.05 vs. control).


Figure 2
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  		Fig. 2 Infarct size was normalized as a percentage of the ischemic (risk) zone in in situ rat hearts. Panel A: protection of the heart by classical and remote preconditioning: Control hearts experienced 30 min of infarct-inducing regional ischemia. Hearts were classically preconditioned (PCx3) with three cycles of 5 min of regional ischemia followed by 5 min of reperfusion prior to the 30 min of infarct-inducing regional ischemia. One group of hearts was protected with one ischemia/reperfusion cycle (PCx1). Infrarenal occlusion of the aorta (IOA) for 15 min followed by 10 min of reperfusion prior to the regional ischemia was conducted in remotely preconditioned hearts. Panel B: the {delta}1-opioid receptor is involved in remote preconditioning: 3 mg/kg DPDPE, a {delta}1-opioid receptor agonist, was given as a bolus 10 min prior to the infarct-inducing ischemia. A 3 mg/kg BNTX, a {delta}1-opioid receptor antagonist, was administered as a bolus 10 min prior to the intervention consisting of infarction or preconditioning in control, classically preconditioned, or remotely preconditioned hearts. A 20 mg/kg MPG was given intravenously as a bolus 20 min prior to the intervention in control, classically preconditioned, or remotely preconditioned hearts. Classical PC resulted in a significant reduction of infarct size compared to control hearts (*p<0.05 vs. control). IOA was comparable in reducing the infarct size (*p<0.05). DPDPE was also protective to the hearts (*p<0.05). BNTX abrogated the protection caused by classical or remote preconditioning (§p<0.05 vs. the comparable group without BNTX). Thus, the signaling of remote preconditioning depends on activation of the {delta}1-opioid receptor. Panel C: remote preconditioning depends on the liberation of free radicals: MPG, a free radical scavenger, had no influence on infarct size in control hearts. Classical preconditioning by three preconditioning stimuli could not be blocked by MPG, whereas only one single preconditioning cycle could be blocked (§p<0.05 vs. PCx1). The reduction of infarct size after one preconditioning stimulus was statistically not significantly different from the infarct size in hearts in which preconditioning was performed with three cycles (*p<0.05 vs. control). Protection by remote preconditioning, which was nearly as protective as classical preconditioning, could be inhibited by MPG (§p<0.05 vs. IOA). This suggests a signal transduction pathway of remote preconditioning which is dependent on free radicals.

 
3.3 Free radicals and remote preconditioning
Fig. 2 presents the infarct size data for the hearts of rats in which free radicals were blocked systemically by MPG. We did not test whether the selected dose of 20 mg/kg MPG blocks the released radicals, although we anticipate that it would do so since this dose of MPG has been demonstrated to block the protection of delayed preconditioning induced by the activation of the {delta}-opioid receptor [27]. Two additional studies with comparable doses of MPG (1 mg/kg for 45 min intravenously [24], or 1.5 mg/kg for 30 min [28]) have shown, that the protection by one classical preconditioning stimulus could be inhibited. None of these studies tested, however, if the released free radicals were blocked.

A bolus of 20 mg/kg MPG administered intravenously was unable to block protection obtained from classic preconditioning by three ischemia/reperfusion stimuli (13±4%; PCx3+MPG). If the number of preconditioning stimuli was reduced to one, MPG was able to block the protection from classic preconditioning (43±4%; p<0.05, PCx1+MPG vs. PCx1). Classical preconditioning of the heart with one ischemia/reperfusion stimulus was nearly as protective as preconditioning with three stimuli (21±4% and 12±1%, respectively; p=n.s., PC±1 vs. PC±3). MPG itself had no influence on the infarct size in control hearts (53±5%; Control+MPG). Remote preconditioning by 15 min infrarenal occlusion and 10 min of reperfusion could be blocked by MPG (46±3%; p<0.05, IOA+MPG vs. IOA). Thus, MPG effectively blocked the infarct size reduction of classic preconditioning by one preconditioning stimulus as well as from remote preconditioning.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Summary
 References
 
The present study demonstrates that remote preconditioning by the previously established rat model of infrarenal occlusion of the aorta was highly protective against infarction. The signal transductions of classical and remote preconditioning share {delta}1-opioid receptors as well as free radicals as common elements in their protecting pathway. The differential effects of radical scavengers in inhibiting the protection of preconditioning support the notion of a gradual effectiveness of the preconditioning process, with multiple ischemic cycles in classical preconditioning being more robust than a single classical preconditioning cycle or remote preconditioning.

It may be noteworthy that for the determination of the infarct size propidium iodide [29] was used for the staining of the infarcted tissue. The ischemic zones in our previous publication [19] were stained with triphenyltetrazolium chloride (TTC) and did not differ from the infarct sizes shown here. This method has the advantage over TTC staining that the reperfusion period can be shortened from 120 to 30 min. Also, the infarcted zone demarcates much more distinctly with propidium iodide than with TTC. On the other hand, the shorter reperfusion time does not allow making extensively hemodynamic measurements during the reperfusion. Thus, the improvement in the mean arterial pressure in protected hearts—a second parameter of protection—cannot be observed.

Others and we have previously shown that remote preconditioning can effectively reduce infarct size [19]. This protection "at a distance" seems to be transmitted by a humoral and not a neuronal factor. This assumption, however, is still a matter of debate [7,30]. We have also found that the degree of infarct size reduction of remote preconditioning by infrarenal occlusion of the aorta is dependent on the duration of the ischemia of the lower limb. Furthermore, a reperfusion period has to be necessarily interspaced between the ischemia of the lower limb and the infarct-inducing ischemia of the heart [19]. A protocol using 5 min of ischemia and reperfusion each repeated for three times failed to protect the myocardium (unpublished data). This is in concordance with data from Liem et al. [30] who could not protect the heart with repetitive cycles of mesenteric ischemia/reperfusion, either. They were successful, however, with 15 min of mesenteric ischemia followed by 10 min of reperfusion and they found an infarct size reduction similar to that of classical preconditioning. It could be excluded that the increase in mean arterial pressure upon closure of the infrarenal aorta as well as the drop in the mean arterial pressure upon reopening of the aorta is responsible for the protection of remote preconditioning. Others and we have not determined yet if the protection of remote preconditioning equals the one obtained by classical preconditioning. That was not the scope of this or the previously published work. The infarct size data, however, show a trend for smaller infarcts in classically, rather than in remotely, preconditioned hearts; albeit this was statistically not significant.

4.1 Opioid receptors are involved in remote preconditioning
Recently, it was observed that naloxone, an unspecific opioid receptor antagonist, blocks the protection obtained from remote preconditioning by mesenteric ischemia [14]. In classical ischemic preconditioning, the involvement of the opioid receptors was first described in 1995 by Schultz et al. [31]. Meanwhile, it was shown that the {delta}1-opioid receptor subtype is mainly the trigger for this protection [32]. This could be demonstrated in every species investigated so far. The involvement of the {delta}-opioid receptor in the protection of human cardiomyocytes was also shown [33]. Some authors stated, however, that the {kappa}- and not {delta}-opioid receptors are involved in the infarct size limiting and arrhythmia suppressant effect of preconditioning [34,35]. In contrast, Aitchison et al. [36] found not a reduction, but an increase in infarct size following activation of the {kappa}-opioid receptor in the rat. Coles et al. [37] observed a proarrhythmic and not an antiarrhythmic effect of the {kappa}-opioid receptor in pigs.

Therefore, the {delta}1-opioid receptor in remote preconditioning was investigated. Inhibition of the {delta}1-opioid receptor by BNTX blocked the protection from remote preconditioning. Furthermore, BNTX also inhibited the protection from classical preconditioning, which Schultz et al. [32] already showed. On the other hand, activation of the {delta}1-opioid receptor by DPDPE could mimic the protection obtained from preconditioning, as others already demonstrated [35]. Dickson et al. [13] could protect virgin acceptor hearts from infarction by the coronary effluent of previously ischemic preconditioned hearts. This effect could be blocked by the unspecific opioid receptor antagonist naloxone. The analysis of the effluent by radioimmunoassay revealed Met- and Leu-enkephalins. Administration of exogenous Met- and Leu-enkephalins to untreated isolated perfused hearts, however, resulted in no protection. Therefore they concluded that the liberated opioids may not have been exclusively or directly involved in the mechanism of "transfused" protection. Recently Patel et al. [14] could inhibit the protection from mesenteric ischemia by naloxone, supporting our findings. Miki et al. [38] investigated the role of opioids in classical ischemia. In isolated perfused rabbit hearts they were unable to block the protection. In in situ hearts naloxone only blocked the protection of classical preconditioning if preconditioning was performed with one instead of three ischemia/reperfusion cycles.

The results demonstrated here implicate that remote preconditioning is mediated by the activation of the {delta}1-opioid receptor. BNTX was able to block the protection from remote as well as from classical preconditioning. Based on the naloxone data from Miki et al. [38], it cannot be excluded, however, that preconditioning has a gradual effectiveness, with multiple cycles in classical preconditioning being more robust than a single classical preconditioning cycle or remote preconditioning. It is also unknown if the agonists causing the activation the {delta}1-opioid receptor in the heart, finally resulting in cardioprotection, are either liberated in the ischemic lower limb or generated in the heart by an as yet unknown signaling mechanism.

4.2 Free radicals are part of the signaling mechanism in remote preconditioning
In classical preconditioning the protecting signal from opioids is transduced via G-proteins and/or by free radicals to the downstream enzymes like protein kinase C or mitogen-activated protein kinase kinases, probably involving the ATP-dependent potassium channel [20]. Therefore, the involvement of free radicals in the protection mechanism of remote preconditioning was investigated.

The free radical scavenger MPG could abrogate the protection from remote preconditioning. For our knowledge, this is the first demonstration of the involvement of free radicals in the protection mechanism of remote preconditioning. For comparison, MPG was also used in classically preconditioned hearts. The finding that the protection by one, but not by three, preconditioning stimuli can be blocked by MPG is supported by the results from Baines et al. [24] in the rabbit heart.

In 1993, Richard et al. [39] showed that MPG was unable to block the protection from three preconditioning cycles. From classical preconditioning it is known that a single ischemic stimulus has to be of at least 3 min duration to induce preconditioning [40]. Increasing the number of ischemia/reperfusion stimuli provides a more effective protection of preconditioning [25]. As stated above, naloxone, the opioid receptor antagonist, blocks the protection from a single preconditioning stimulus, but not from three ischemia/reperfusion stimuli [38]. Thus, classical preconditioning with three ischemia/reperfusion cycles and remote preconditioning could be somewhat different regarding the power of protection.

Our results showed for the first time that free radicals are an element in the signal transduction pathway of remote preconditioning. The fact that MPG can block the protection from remote preconditioning and classical preconditioning with one, but not with three, ischemia/reperfusion cycles implicates that free radicals are only partially involved in the mechanism of preconditioning. Thus, stronger preconditioning stimuli could include further signaling elements of preconditioning and the protection may not be solely dependent on the liberation of free radicals. Since protection from remote preconditioning can be blocked by MPG, either remote preconditioning has no redundancy in its signal transduction pathway or the robustness of the protecting signal of remote preconditioning is weaker than the one from classical preconditioning with three ischemia/reperfusion cycles.

The source of free radicals in remote preconditioning is yet unknown. Either the radicals can be liberated from the ischemic lower limb or other mechanisms, e.g. the activation of opioid and/or other g-protein-coupled receptors by remote preconditioning, generate free radicals in the heart resulting in cardioprotection. Recently it was shown that ischemia and reperfusion of skeletal muscle lead to the appearance of hydroxyl radicals in the circulation [41] indicating that in remote preconditioning free radicals are generated in the ischemic lower limb.


   5. Summary
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Summary
References
 
In summary, remote preconditioning by infrarenal occlusion of the aorta triggers the protection of the infarcted heart. A {delta}1-selective opioid receptor antagonist and the blockade of free radicals can abolish the cardioprotection induced by remote preconditioning. This implies the involvement of the {delta}1-opioid receptor and free radicals in the signaling mechanism of remote preconditioning. Thus, opioids and free radicals are common elements in the signal transduction pathways of remote and classical preconditioning. The results suggest that both protection mechanisms behave somewhat differently regarding the strength of protection, classical preconditioning (three cycles ischemia/reperfusion) being more powerful than remote preconditioning. It cannot be excluded, however, that classical preconditioning has more redundancy in its signaling pathway than remote preconditioning. Further factors involved in the signaling mechanism of remote preconditioning have to be determined. Since it has been shown recently that remote preconditioning also occurs in humans, this phenomenon could have clinical implications.


   Acknowledgements
 
We thank Manuela Rothe for her excellent technical assistance.


   Notes
 
Time for primary review 12 days

{star} Part of this study was presented at the Annual Meeting of the American Heart Association 2002 as well as at the meeting of the German Society of Cardiology 2003. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (We 1955/2-2). Part of this work was sponsored by a grant of the University of Technology Dresden (MedDrive) and by the Doktor-Robert-Pfleger Stiftung. Back


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

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