Cardiovascular Research

Cardiovascular Research 2002 55(3):590-601; doi:10.1016/S0008-6363(02)00446-7
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway

Christof Weinbrenner^a,b,*, Manfred Nelles^b, Nicole Herzog^b, László Sárváry^a,b and Ruth H Strasser^a,b

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

^* Corresponding author. Tel.: +49-351-450-1700; fax: +49-351-450-1702 cweinbre{at}web.de

Received 30 October 2001; accepted 15 April 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Background: Ischemic preconditioning 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. To address the question whether this remote preconditioning is transduced by neuronal or humoral factors an in situ model of infrarenal occlusion of the aorta (IOA) in the rat was developed. Furthermore, the signal transduction pathways of classical and remote preconditioning regarding protein kinase C, which is one of the key enzymes in classical preconditioning, were compared. Methods and Results: Controls (30 min regional ischemia followed by 2 h of reperfusion) had an infarct size of 62±5% whereas classical preconditioning reduced it to 10±3% of the risk zone (P0.001). Fifteen minutes IOA without reperfusion of the aorta had no influence on infarct size (52±4%). When, however, IOA was performed for 15, 10, or 5 min, respectively, followed by a 10-min reperfusion period the size of myocardial infarction decreased significantly. This decrease was dependent on the duration of IOA (18±3%, 37±8%, 42±2%, respectively; P0.001 for the time-dependent linear trend in decrease of infarct size). Fifteen minutes IOA showed the strongest protection which was comparable to classical preconditioning (18±3%, P0.001 vs. control). Blockade of the nervous pathway by 20 mg/kg hexamethonium could not inhibit the protection afforded by IOA (14±4%). Using chelerythrine, a selective protein kinase C-inhibitor, at a dose of 5 mg/kg body weight, protection from remote (68±4%, P0.001 vs. 15 min IOA followed by 10 min of reperfusion without chelerythrine) as well as from classical preconditioning (56±5%, P0.001) was completely blocked. Conclusion: Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified.

KEYWORDS Receptors; Signal transduction; Preconditioning; Protein kinases; Infarction; Ischemia

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Murry et al. [1] first described the phenomenon of ischemic preconditioning (brief periods of ischemia interspaced with reperfusion) in 1986. The mechanism of reducing infarct size of the heart is well established and this protection has also been observed in a number of other organs including brain [2], liver [3], and skeletal muscles [4]. In 1993, Przyklenk et al. [5] 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 preconditioning was questioned however by Nakano [6] 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 phenomenon of remote preconditioning is not unique to the heart. Liauw et al. [7] have shown that ischemia of one skeletal muscle can protect the contralateral muscle from ischemia–reperfusion injury. The mechanism of remote preconditioning is not limited to one organ system either; for example McClanahan et al. [8] could show 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 [9–11] or the femoral artery [12] confers protection to the heart. It was proposed that remote preconditioning acts through sensory nerve stimulation in the ischemic organ, since the ganglion blockade by hexamethonium may abolish this protection [9,11]. Contrary to this observation, it was shown in vitro that the effluent during preconditioning can protect virgin isolated rabbit hearts from infarction, which supports a non-neuronal action of protection [13].

Remote preconditioning was shown in several species including rat, rabbit and canine, which is comparable to classical preconditioning. Recently an indication for the presence of remote preconditioning in humans was made by Günaydin et al. [14] who showed that during coronary artery bypass surgery a tourniquet wrapped around an upper extremity to induce brief periods of ischemia resulted in an attenuated increase of lactate dehydrogenase suggesting a reduction in myocyte injury [14]. This observation also gives a potential link to the clinical and therapeutical relevance of remote preconditioning.

The signal transduction pathway of both kinds of preconditioning share also similarities. Classical preconditioning can be mimicked by numerous G-protein coupled receptors (i.e. ₁-adrenergic, adenosine, bradykinin, opioid). The signals from classical ischemic and pharmacological preconditioning converge on the level of protein kinase C, which seems to be a key enzyme in preconditioning [15]. After the activation of protein kinase C the signal is transmitted via other protein kinases (i.e. tyrosine kinases [16], p38 mitogen activated protein kinase [17,18]) to the yet unidentified final end-effector. In the phenomenon of remote preconditioning adenosine receptors [25], ₁-adrenergic receptors [19], bradykinin receptors [9], and opioid receptors [20] have been proposed to be involved in the infarct size limiting effect since their receptor antagonists could prevent the protection obtained from preconditioning on a distance. To our knowledge nothing is known about the signal transduction cascade downstream of the receptor level, especially the role of protein kinase C in remote preconditioning has not been investigated so far.

Classical preconditioning can be induced by a single brief period of ischemia followed by reperfusion prior to the infarct inducing ischemia. In order to induce protection the preconditioning ischemia must be of at least 3 min duration [21]. Longer ischemic periods below the threshold of infarction or repetitions of ischemia–reperfusion to precondition the heart do not reduce the infarct size any further. Pharmacological inhibition of ischemic preconditioning, however, is dependent on the strength of the ischemic preconditioning stimulus since for example the bradykinin receptor antagonist HOE 140 can block protection from a single preconditioning stimulus but not from three ischemia–reperfusion stimuli [22]. This was also observed for opioid receptors using naloxone as an antagonist [23]. Whether remote preconditioning is as powerful as classical preconditioning in protecting the heart and if there exist similarities in the time course of protection is not clarified yet.

For protecting the heart on a distance, a model of infrarenal occlusion of the aorta was developed. It is hypothesized that remote preconditioning acts through an as yet unidentified humoral trigger signal to the heart. If so, it is proposed that the degree of protection of the heart is dependent on the amount of protecting substance which is produced by the trigger organ. If a humoral factor is responsible for the protection, a reperfusion period of the ischemic organ would have to be interspaced between the ischemia of the trigger organ and the ischemia of the heart since the protecting mediator would have to be transmitted to the heart by the circulation. To test if a neuronal pathway may be involved in the protection of remote preconditioning we used the ganglion blocker hexamethonium. Finally we compared the signal transduction pathways of classical and remote preconditioning regarding protein kinase C which is a key enzyme in classical preconditioning.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 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 was approved by the local Animal Care and Use Committee.

2.1 General surgical preparation
Male Wistar rats, 250–350 g, were used for the study. The rats were anesthetized via intraperitoneal administration of sodium pentobarbital (30 mg/kg). A tracheotomy was performed and the trachea was intubated with a cannula connected to a rodent ventilator (Foehr Medical Instruments, Seeheim, Germany). The rats were ventilated with room air supplemented with oxygen at 65 breaths/min. Atelectase was prevented by maintaining a positive end-expiratory pressure of 2.5 cmH₂O. Arterial pH, p_CO2, and p_O2 were monitored at baseline, 5 and 25 min of coronary occlusion, 30 and 60 min of reperfusion by a blood gas analyzer (Ciba Corning) and maintained within a normal physiological range (pH 7.35–7.45, p_CO2 30–40 mmHg, p_O2 80–120 mmHg) by adjusting the respiratory rate and/or the tidal volume. Body temperature was maintained at 37 °C by using a heating pad. In some cases the administration of sodium-bicarbonate was necessary to maintain the pH in a physiological range.

The left axillary artery was cannulated to measure the blood pressure and heart rate via a pressure transducer (DPT-6000, pvb Medizintechnik, Kirchseeon, Germany) connected to a MacLab/2e and a MacIntosh computer (using ADInstruments Chart V 3.6.8 data recording software). The left axillary vein was cannulated 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 intercostal space followed by a pericardiotomy. A 5-0 suture on a curved taper needle (5/0 Ethibond Excel, Ethicon, Germany) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle, and the ends were pulled through a small vinyl tube to form a snare. The coronary branch was occluded by tightening the snare, which was then fixed by clamping the tube with a small hemostat. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure. Reperfusion was achieved by releasing the snare and 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 6 to 8 hearts 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 2 h of reperfusion (Fig. 1a). Group 2 was ischemically preconditioned (classical PC) 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 permanently for 165 min (IOA all the time). Regional ischemia of the heart was initiated after 15 min of IOA. In group 4 the infrarenal aorta was occluded for 15 min. At the time of the reopening of the occlusion on the aorta, regional ischemia on the heart was initiated for 30 min (15 IOA no Rep). In groups 5 to 7 the infrarenal aorta was occluded for 5, 10, or 15 min, respectively, followed by a 10-min reperfusion period of the lower limb prior to the 30 min of regional ischemia of the heart (5, 10, or 15 IOA 10 Rep).

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (a, b) Experimental protocol. See text for details.

 
In the second protocol chelerythrine was used in three additional groups to investigate the role of PKC in remote preconditioning (Fig. 1b). Groups 8 to 10 were the same as the groups 1, 2, and 7 with the difference that chelerythrine at a concentration of 5 mg/kg body weight was infused intravenously over a period of 2 min. Five minutes later the specified treatment started (Chel+control, Chel+classical PC, Chel+15 IOA 10 Rep). In group 11 the involvement of the neurogenic pathway in remote preconditioning was tested. Hexamethonium (20 mg/kg body weight) was infused intravenously over a 3-min period 15 min prior to the IOA protocol (Hexa+15 IOA 10 Rep) according to the protocol used by Gho et al. [11]. In groups 12 and 13, 5% glucose, which serves as vehicle, was infused instead of chelerythrine (Table 2) in classically and remotely preconditioned hearts.

View this table: [in this window] [in a new window]   Table 2 Infarct size data

 
2.3 Exclusion criteria
A total of 74 out of 92 rats successfully completed the above protocols. Animals were excluded from data analysis if they exhibited severe hypotension or if it was not possible to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis. The exclusion of 20% of the animals from the present study was evenly distributed among the protocol groups. Five additional rats were used for the determination of the lactate levels during IOA and reperfusion.

2.4 Determination of infarct size
At the end of the experiments after 2 h of reperfusion the heart was excised and quickly mounted on a Langendorff apparatus by the aortic root. The heart was perfused at constant pressure (100 cmH₂O) with 0.9% saline to wash out the blood for 1 min. The coronary artery was reoccluded, and 1–10 µm zinc cadmium sulfide fluorescent particles (Duke Scientific, Palo Alto, CA) were infused into the perfusate to demarcate the risk zone as the tissue without fluorescence. The heart was then removed from the apparatus, weighed, frozen, and cut into 2-mm-thick slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in pH 7.4 phosphate buffer for 10 min at 37 °C followed by fixation in 10% formalin for at least 15 min. The areas of infarct (TTC negative tissue) and risk zone (nonfluorescent under ultraviolet light) were determined by planimetry. Infarct size was expressed as a percentage of the risk zone.

2.5 Lactate measurement
In five additional animals ischemia of the lower limb induced by infrarenal occlusion of the aorta was verified by measuring the lactate concentration in the venous blood after releasing the clip from the aorta. After 10 min of equilibration 500 µl of venous blood were aspirated out of the inferior vena cava in a syringe filled with 50 µl heparin (baseline). The blood loss was substituted with 500 µl of 0.9% sodium chloride solution. Then the infrarenal aorta was occluded for 15 min. Ten seconds, 2 min, 5 min, and 8 min after the release of the clip from the aorta, again samples of venous blood were drawn as described above (Fig. 3).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Augmentation of lactate in the lower limb during infrarenal occlusion of the aorta. Lactate concentration in the venous blood of the inferior vena cava. A sample was drawn after the equilibration period (baseline) and at the time points 10 s, 2 min, 5 min, and 8 min after starting the reperfusion of the lower limb after 15 min of IOA.

 
The lactate concentration was determined in the blood plasma according to the manufacturer's description using a colorimetric assay (Sigma Diagnostics, Deisenhofen, Germany). Determinations were done in triplicates.

2.6 Chemicals
Chelerythrine chloride was purchased from Alexis Biochemicals (San Diego, CA) and was dissolved in dH₂O to a concentration of 10 mg/ml. For infusion, chelerythrine was 10-fold diluted with 5% glucose solution. Hexamethonium which was dissolved in dH₂O and 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical (Deisenhofen, Germany).

2.7 Statistics
Values are presented as means±S.E.M. One-way analysis of variance with repeated measures and Tukey–Kramer post hoc tests were used to test for differences within groups for hemodynamics, and without repeated measures to test for differences in hemodynamic values as well as in infarct size (Instat, Graphpad Software, San Diego, CA). A P value <0.05 was considered significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Hemodynamics
Baseline hemodynamic parameters were comparable in the 11 groups of animals (Table 1). No significant differences concerning the heart rate were found between the groups with the exception of the 15 IOA 10 Rep group which had significantly lower baseline heart rates. The mean arterial pressure (MAP) as well as the rate pressure product (RPP) were not different at baseline for all groups. At 60 and 120 min of reperfusion the MAP was significantly enhanced in the classical PC and the 15 IOA 10 Rep group compared to the control. Also the RPP as a measurement of the recovery of the heart function was significantly enhanced in both groups compared to control at 60 and 120 min of reperfusion. Additionally the RPP was significantly augmented in the 15 IOA 10 Rep group (the protected group) compared to the 15 IOA no Rep group (the non-protected group) at 60 min of reperfusion. Thus the enhanced MAP and RPP suggest an improved recovery of contractility after regional ischemia in the classically and the remotely preconditioned hearts, which also correlates with the protection status.

View this table: [in this window] [in a new window]   Table 1 Hemodynamic data

 
Interestingly, in all groups with IOA with or without reperfusion the mean arterial blood pressure (MAP) rose about 13% immediately after the occlusion of the infrarenal aorta (Fig. 2a and b). This rise was significant only in the 15 IOA 10 Rep group (P<0.01). Within the first 2 min, however, the MAP returned to baseline values. When reopening the aorta for reperfusion of the lower limb the MAP dropped for about 34% in all IOA groups and returned to baseline values within 4 min (P<0.01 for all groups). Prior to the coronary artery occlusion all MAP values between the groups were comparable. Within the IOA groups the heart rate was slightly but significantly depressed during IOA of the aorta. Surprisingly, this heart rate depression could be prevented by inhibition of PKC by chelerythrine. Hexamethonium resulted in a significant depression of the heart rate, MAP, and RPP due to the ganglion blockade which sustained until 60 min of reperfusion.

View larger version (165K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (a) Mean arterial pressure increase with IOA and decrease with reperfusion of the aorta. Shown is a compressed exemplary original registration of the arterial pressure during IOA (upper panel) and reperfusion (lower panel). (b) Increased mean arterial pressure after the IOA initiation, pressure depression after reperfusion of the aorta. Mean arterial pressure (mmHg) was measured in the 15 IOA no Rep, 15 IOA 10 Rep, and Chel+15 IOA 10 Rep groups before IOA, immediately after the initiation of IOA, after 2, 5, and 14 min of IOA, furthermore immediately, 4, and 9 min after reopening the aorta. For comparison the MAP of the control and the PC group are shown at baseline and immediately prior to the coronary occlusion. The MAP-increase after IOA was significant in the 15 IOA 10 Rep group compared to baseline. The MAP-decrease after reperfusion of the aorta was significant compared to the MAP after 14 min of IOA in all three IOA groups.

 
3.2 Lactate
Ischemia of the lower limb was verified by measuring the lactate, which is released during 15 min of infrarenal occlusion of the aorta. Lactate as an indicator of anaerobic glycolysis was determined in the venous blood obtained from the inferior vena cava upon reperfusion of the infrarenal aorta. The concentration of lactate increased to 248% over baseline upon reperfusion (Fig. 3) and returned to baseline values within 5 to 8 min of reperfusion.

3.3 Infarct size studies
No differences in body weight or heart weight among the groups was observed (Table 2). Infarct sizes normalized as a percentage of the ischemic (risk) zone are shown in Fig. 4. Rats with 15 min IOA without reperfusion (IOA all the time, Table 2) or with reperfusion of the aorta at the time of initiation of the infarct inducing ischemia on the heart (15 min IOA no Rep, Table 2 and Fig. 4) showed an infarct size of 43±5% and 52±4%, respectively, which is comparable to that seen in control hearts (62±5%). Interspacing of a 10-min reperfusion period between 15 min of IOA and the infarct inducing ischemia of the heart (15 IOA 10 Rep), however, reduced the infarct size significantly to 18±3% (P<0.001 vs. control, P<0.01 vs. 15 IOA no Rep) which is comparable to the reduction seen in classically preconditioned hearts (classical PC, 10±3%, P<0.001 vs. control). Diminution of the duration of IOA to 10 min (10 IOA 10 Rep) still significantly reduced the infarct size. Part of the powerful protection of 15 min IOA, however, was lost (37±8%, P<0.05 vs. control). Further shortening the IOA period to 5 min (5 IOA 10 Rep) showed a trend for a reduced infarct size (42±2% P = n.s. vs. control). The reduction in infarct size with 5, 10, and 15 min of IOA showed a linear trend; this linear trend being significant (P<0.001).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Infrarenal occlusion of the aorta remotely preconditions the heart. Infarct size normalized as a percentage of the ischemic (risk) zone in in situ rat hearts. Control hearts experienced only 30 min of regional ischemia. Hearts were ischemically preconditioned (PC) with three cycles of 5 min of regional ischemia followed by 5 min of reperfusion before the 30 min of infarct inducing regional ischemia (classical PC). 15 IOA no Rep, infrarenal occlusion of the aorta (IOA) for 15 min prior to the regional ischemia. IOA was finished at the same time when the coronary occlusion started; 5, 10, 15 IOA 10 Rep, 5, 10, or 15 min IOA, respectively, followed by 10 min of reperfusion (Rep) prior to the beginning of the coronary occlusion. Classical PC resulted in a highly significant reduction of infarct size compared to control hearts (***P<0.001). 15 IOA no Rep had no influence on infarct size, however if the IOA was followed by a 10-min reperfusion period of the lower limb (15 IOA 10 Rep) infarct size was significantly reduced (***P<0.001). This reduction was comparable to that seen in PC hearts. Shortening the IOA period to 10 min, however, followed by a 10-min reperfusion period (10 IOA 10 Rep) still significantly reduced the infarct size (*P<0.05) whereas in the 5 IOA 10 Rep group the reduction showed only a trend for a reduced infarct size. The linear trend in the loss of infarct size dependent on the duration of the IOA period length was highly significant (P<0.001).

 
Fig. 5 presents the infarct size data for PKC-inhibition by 5 mg/kg chelerythrine. The vehicle, 5% glucose, for chelerythrine had no influence on infarct size neither in classically nor remotely preconditioned hearts (Table 2). Chelerythrine blocked protection obtained from classical PC (56±5%, P<0.001 vs. classical PC without chelerythrine). In control hearts chelerythrine had no influence on the infarct size (66±6%). Blocking PKC in hearts receiving 15 min IOA with 10 min reperfusion completely prevented the protection obtained from remote preconditioning (68±4% vs. 18±3% without chelerythrine, P<0.001). Blocking the neurogenic pathway by hexamethonium failed to block the protection from 15 IOA 10 Rep (14±4%).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Remote preconditioning of the heart can be blocked by PKC inhibition. Infarct size normalized as a % of the ischemic (risk) zone in in situ rat hearts. For comparison the control, classical PC, and 15 IOA 10 Rep groups are shown again on this figure. Chelerythrine (5 mg/kg; Chel) was infused intravenously over a 2-min period starting immediately after the equilibration period. Three min after the infusion stop the infarct-inducing ischemia (Chel+control), the classical PC protocol (Chel+classical PC), or the IOA protocol (Chel+15 IOA 10 Rep) started. Chelerythrine had no influence on infarct size in control hearts whereas it could block protection obtained from classical PC (***P<0.001 vs. classical PC without chelerythrine). Chelerythrine in 15 IOA 10 Rep hearts completely blocks protection which is similar to the observation of the classical PC hearts (***P<0.001 vs. 15 IOA 10 Rep without chelerythrine). This suggests a PKC-dependent signal transduction pathway of remote preconditioning. Hexamethonium (20 mg/kg) was administered for 3 min, 15 min prior to the IOA protocol. Ganglion blockade by hexamethonium could not block the protection from 15 IOA 10 Rep, which excludes a neurogenic pathway for remote preconditioning.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The present study demonstrates that remote preconditioning by this newly established rat model of infrarenal occlusion of the aorta was highly protective against infarction. The protection obtained was equivalent to that seen with classical ischemic preconditioning. This protection shares PKC as a common signal in the signal transduction pathways of classical and remote preconditioning.

The data presented here show a time-dependent reduction in infarct size dependent on the duration of the infrarenal occlusion of the aorta. Fifteen minutes IOA reduced infarct size to an amount which is comparable to that seen in classical preconditioned hearts. Shorter periods of IOA still decreased infarct size, but part of the protection was lost. From classical preconditioning it is known that a single ischemic stimulus has to be of at least 3 min duration to induce preconditioning [21]. Longer ischemic periods below the threshold of infarction or repetitions of ischemia–reperfusion to precondition the heart do not reduce the infarct size any further. Thus classical and remote preconditioning are somewhat different regarding the power of protection. We hypothesize that during ischemia of the lower limb a protecting factor is produced in the ischemic trigger organ. The power of protection seems to be due to the amount of protecting factor produced or/and released. This assumption is supported by the observation that with shorter periods of IOA part of the protection is lost. This is supported by the finding of Birnbaum et al. [12] who showed in a remote preconditioning model with a reduced blood supply of the lower limb there is only protection of the heart if the gastrocnemius muscle was rapidly stimulated during the remote ischemia. This protecting substance then needs a circulation time to move from the lower limb to the heart, since we found that a reperfusion period has to be necessarily interspaced between IOA and the induction of the infarction of the heart. We did not test different durations of the reperfusion period. Reperfusion of the aorta at the same time as occlusion of the coronary artery, however, does not protect the heart. This important finding clearly demonstrates that the ‘protecting substance’ has to reach the myocardial tissue prior to the heart being subjected to the infarct inducing ischemia. Dickson et al. [13,24] support the hypothesis of a humoral factor which protects the heart at a distance from infarction. They collected the effluate of isolated preconditioned rabbit hearts, which then itself could protect virgin hearts from infarction. They could show that the protecting factor binds to a hydrophobic matrix, the protecting substances, however, were neither adenosine nor norepinephrine, which is contrary to the observation made by Pell et al. [25] who found a blockade of remote preconditioning by 8-(p-sulphophenyl)theophylline, an adenosine receptor antagonist. In an in vivo rabbit model Dickson et al. found that whole blood transfusion between preconditioned and virgin animals can protect the latter from infarction [26]. It has to be evaluated if this supposed protecting substance is similar in rat and rabbit.

In the rat model of remote preconditioning presented here neuronal factors can be ruled out since ganglion blockade by hexamethonium did not influence the reduction in infarct size afforded by remote preconditioning. Gho [11] and Schoemaker [9] found in another model of remote preconditioning by brief mesenterical ischemia or intramesenteric bradykinin infusion, however, controversial data, since they could prevent the protection of the heart by pretreatment with the ganglion blocker hexamethonium [9,11]. The drop in the heart rate and the mean arterial pressure with initiation of the infusion of hexamethonium can be ruled out for conflicting results since Gho [11] already excluded that the hemodynamic effects of hexamethonium do influence the infarct size. The depression of the hemodynamic parameters by hexamethonium seen here were comparable to the ones observed by Gho [11] and Schoemaker [9]. Moreover, the necessity of a reperfusion period between IOA and the coronary occlusion presented here supports the hypothesis that mainly humoral factors are responsible for the protection at a distance.

Lactate was measured in the venous blood of the IOA animals to verify metabolically the ischemia of the lower limb. Lactate increased several-fold compared to baseline values. This rise in lactate, however, was compensated within 3 to 5 min of reperfusion by the buffering capacity of the blood. This short lactate peak might be one of the signals for the heart to promote preconditioning. As shown, however, a 10-min reperfusion period between IOA and the infarction of the heart has to be interspaced. Since the rise and the decline of lactate is very fast it is unlikely that lactate is responsible for the observed protection. Moreover, in fasted animals, Doenst et al. [27] showed that adding lactate to the perfusate of isolated hearts neither promotes nor blocks the protection obtained from fasting alone. Lactate not as a trigger but as a mediator of remote preconditioning is also unlikely since several studies showed that lactate did not increase but decrease in classical preconditioning [28].

Another possibility to trigger the protection of remote preconditioning could be hypothesized to be a stretch stimulus since in the model used here the infrarenal occlusion of the aorta (IOA) caused a short rise in the mean arterial pressure (MAP). It is known that brief periods—as long as 10 min—of stretch can precondition the heart [29]. We observed a 13% increase in the mean arterial pressure immediately after the initiation of the IOA (Fig. 2) which decreased to baseline values within the next 2 min. Thus the duration of stretch is much shorter and more moderate than the one used experimentally by an external stimulus to induce preconditioning [29]. After releasing the clip from the aorta the MAP decreased at about one third and returned to baseline values within 4 min of reperfusion. To sum up: hearts which received IOA without reperfusion, IOA with only reperfusion at the time of initiation of the ischemia on the heart, or 15 min IOA with reperfusion but PKC-blockade showed no reduction in infarct size despite an increase in the MAP. Thus these data rule out a pressure-derived effect of IOA. In a model of isolated perfused rabbit hearts which excludes a pressure-induced protective effect, Dickson et al. [13] found the effluent of prior preconditioned hearts to be protective in virgin isolated hearts.

We observed a significant drop in the heart rate during the whole duration of infrarenal occlusion and the beginning of the reperfusion of the aorta. Interestingly this heart rate drop could be blocked by PKC-inhibition. The initial rise of the blood pressure beginning with the initiation of IOA could activate the baroreceptors thus resulting in a decreased heart rate by the baroreceptor reflex. With the initiation of the infarction of the heart we could not detect a difference in heart rate any more between the groups. Since the infarction of the heart is a strong circulation depressive stimulus the baroreceptors either could be reset independently if the animals were prior preconditioned or not or could be activated also in the non-preconditioned group. The baroreflex however had no influence on the infarct size since all IOA-hearts had a comparable drop in the heart rate but only the IOA hearts with reperfusion were protected.

It is noteworthy, that in our hands remote preconditioning in the rat model was not successful when using mesenterical [9–11] or renal [8,11,25] ischemia as a protecting trigger. The passager ligation of the femoral artery in combination with rapid stimulation of the gastrocnemius muscle [12] or limb ischemia induced by placing a thin elastic tourniquet around the hind extremity [19] was not successful either. One reason for our failure using already published methods of remote preconditioning could be species differences since in most experimental settings rabbits were used. In rats, too, renal ischemia for remote preconditioning was used by different groups. However one group could show that inducing remote preconditioning by renal ischemia was only successful if the body core temperature was decreased to 30–31 °C whereas at 36–37 °C they saw no protection [11]. We therefore developed a new model of remote preconditioning by infrarenal occlusion of the aorta which is reliable to use, reproducible by different investigators and apt to investigate protecting trigger transmitters of remote preconditioning, since the venous blood of the lower limb can be easily obtained by puncture of the inferior vena cava.

Classical ischemic preconditioning can be pharmacologically mimicked by stimulating ₁-adrenergic-, adenosine-, bradykinin-, opioid-, and other G_q-protein coupled receptors. So far similarities on the receptor level have been investigated for ₁-adrenergic- [19], adenosine- [25], bradykinin- [9], and opioid-receptors [20] in remote preconditioning. ATP-sensitive potassium channels are other important signaling elements of classical preconditioning. However, it is not clear yet if they serve as triggers, mediators, or end-effectors [30]. Classical and remote preconditioning also have the ATP-sensitive potassium channels as signal transduction element in common, since the channel blockers glibenclamide [31] and sodium 5-hydroxydecanoate [25] can abolish the protection obtained from remote preconditioning.

To our knowledge so far nothing is known about the role of protein kinase C (PKC) in remote preconditioning. It is very likely that PKC is a key enzyme in the signaling mechanism of classical preconditioning, since activators of PKC mimic protection and inhibitors of PKC can block the protection obtained from ischemic or pharmacological preconditioning on a level upstream of PKC [32]. As already shown by other groups, we found that the highly selective PKC-inhibitor chelerythrine can block protection obtained from classical ischemic preconditioning without any influence on infarct size of control hearts. For the first time we could show that the powerful protection of remote preconditioning by 15 min IOA followed by 10 min of reperfusion could be completely blocked by chelerythrine. Also the functional recovery of the heart after 60 and 120 min of reperfusion was reversed by PKC-inhibition in the classically and in the remotely preconditioned hearts. Thus PKC determines this part of the signal transduction pathway both in classical and remote preconditioning.

Does remote preconditioning occur in humans? This is the most important question from a clinical point of view as it could have therapeutical implications. It is known that exercise in humans can precondition the heart [33]. It could be possible that physical activity prior to a coronary occlusion has two effects: first it would accelerate the progression of myocardial ischemia because of higher metabolic demand due to physical activity, but secondary it might attenuate the degree of myocardial cell damage through remote preconditioning. An indication for the presence of remote preconditioning in humans are the recent findings of Günaydin et al. [14] who showed that during coronary artery bypass surgery a tourniquet wrapped around an upper extremity to induce brief periods of ischemia resulted in an attenuated increase of lactate dehydrogenase suggesting a reduction in myocyte injury.

In summary, a new model of remote preconditioning by infrarenal occlusion of the aorta was developed which is very apt to investigate factors which trigger preconditioning on the heart. The protection obtained by this model is as effective and powerful as classical preconditioning of the heart. This protection can be completely abolished by the PKC-inhibitor chelerythrine. Whether other signal transduction elements downstream of PKC are also involved in the process of remote preconditioning has not been investigated so far. Since a recently published paper showed that remote preconditioning also occurs in humans this phenomenon could have clinical implications of perhaps greater relevance than classical ischemic preconditioning. Procedures that increase oxygen consumption or lactate production, including exercise, could become important determining factors in the protection of the heart.

Time for primary review 28 days.

	Acknowledgements

 
Part of this study was presented at the Annual Meeting of the American College of Cardiology 2001 and at the German Society of Cardiology 2002. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (We 1955/2-1 and SFB 320/B4). Part of this work was sponsored by a grant of the University of Technology, Dresden (MedDrive).

	References

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