Abstract

Aims

Since mitogen-activated protein kinases (MAPKs) were found to be implicated in the signalling of ischaemic preconditioning (IPC), we tested the hypothesis of a contribution of these protein kinases to remote preconditioning (RPC).

Methods and results

To determine the role of p38, ERK1/2, and JNK1/2 MAPKs in mediating cardiac protection, an in vivo model of myocardial infarction was applied in male Wistar rats. RPC or IPC was induced by occlusion of the superior mesenteric artery or the left coronary artery, respectively. Infarct size (IS) was determined based on 2,3,5-triphenyltetrazolium chloride staining. Phosphorylation of the various MAPKs was analysed by immunoblotting in samples of the small intestine and myocardium obtained after IPC or RPC procedures. The MAPK inhibitors SB203580 (p38), PD98059 (ERK1/2), and SP600125 (JNK1/2) were administered to assess the potential significance of MAPK signalling in RPC. Both preconditioning stimuli decreased myocardial IS significantly after a lethal period of ischaemia. Each of the applied MAPK inhibitors was capable of abrogating the RPC-induced cardioprotection. Western blot analysis of myocardial samples revealed an increase in phosphorylated amounts of ERK1/2 and JNK1 after IPC, whereas phosphorylation of p38 protein was decreased significantly. Likewise, RPC resulted in a considerable increase in phosphorylation of ERK1/2 and JNK1/2 proteins in the small intestine, whereas it did not alter the MAPK phosphorylation state in the myocardium.

Conclusion

All investigated MAPK pathways appear to be involved in RPC-induced cardioprotection; however, they do not contribute to the alterations that define the preconditioned state of the myocardium prior to the infarction.

Introduction

Ischaemia/reperfusion injury of the heart frequently represents a sequel to an underlying cardiovascular disease. An enormous interest has therefore arisen in mechanisms capable of limiting myocardial damage. The extensively investigated ischaemic preconditioning (IPC) is a powerful innate protection induced by local brief ischaemic episodes that initiates tolerance to a subsequent, more sustained ischaemia.1 Since IPC is not applicable in a clinical setting, a variant preconditioning stimulus—remote ischaemic preconditioning (RPC)—has received ever increasing attention. RPC additionally evokes protection in virgin tissues subsequent to brief ischaemia in remote organs.2 In non-ischaemic myocardial regions, RPC-induced protection has been achieved by ischaemia/reperfusion in a remote coronary vascular area3 or by brief ischaemic episodes in non-cardiac tissues such as the small intestine4 or limb skeletal muscle.5 Thus, RPC of the remote target organ differs substantially from IPC in that it does not provoke intracellular acidification or ATP depletion.6 Although the mechanisms that bring about RPC are incompletely understood, some similarities to IPC have been discovered in the conveyance of external signals to intracellular targets that ultimately lead to protection. IPC as well as RPC can be elicited via opioid, bradykinin, and adenosine receptors.1 Recently, calcitonin gene-related peptide (CGRP) was found to be released by IPC and RPC. Consecutively, application of CGRP antagonists abolished myocardial protection induced by IPC as well as RPC.7,8 For the most part, it is unknown whether the commonalties between the triggering of RPC and IPC are also reflected in common patterns of intracellular signalling and effector activation. Known parallels in intracellular signalling include the protein kinase C (PKC) pathway that has been identified to be effective in initiating IPC and RPC.1,9 In rats, the PKCε isoform presumably represents a key mediator of myocardial protection both in IPC and RPC.10,11 Moreover, recent studies established a direct connection between PKCs and the mitogen-activated protein kinases (MAPKs). Thus, it was observed in preconditioned murine cardiac mitochondria that MAPKs form signal modules with the PKC subtype ε that subsequently inactivate pro-apoptotic proteins.12

These MAPKs consist of four highly conserved subfamilies, the extracellular signal regulated kinases (ERK1/2, also referred to as p44/p42), the jun-NH2-terminal kinases 1/2 (JNK1/2, also designated as stress activated protein kinases or p54/p46), p38 MAPK, and ERK5.9,13 Whereas little is known about the latter protein kinase, the other MAPKs have been found to control a plethora of (patho)physiological processes.13 In the heart, MAPKs are involved in the proliferation of cardiomyocytes and the development of cardiac hypertrophy.14,15 Activation of the various MAPKs in IPC and ischaemia/reperfusion has been affirmed in a multitude of studies, however, their respective contribution towards myocardial protection remains a matter of debate.1,9

In general, MAPKs are activated via upstream MAPK kinases (MAPKK) that phosphorylate at a threonin (Thr) and a tyrosine (Tyr) residue located in the Thr-X-Tyr motif of the activation loop.13 Phosphorylation of p38 MAPK during ischaemia has been reported to occur in the myocardial tissue of rats,16 dogs,17 and pigs18in vivo. Studies using the specific p38 MAPK inhibitor SB20358019 have revealed a contribution to the protection by IPC in rat,20 rabbit,21 dog,17 and pig22 hearts. These findings are contradicted by other observations suggesting a potential detrimental effect of p38 MAPK activation in ischaemia or IPC of the heart.23,24

Similar to p38, the ERK1/2 MAPKs were also found to be phosphorylated during ischaemic episodes in myocardial samples taken from rat.25 Recently, it turned out that activation of ERK1/2 by IPC follows a biphasic course, a first phase occurring during ischaemia followed by a second phase during reperfusion.26,27 Interestingly, the first phase of activation seems to be required to initiate the activation during reperfusion.25 An involvement of this pathway in mediating IPC was further indicated by several studies applying specific inhibitors of the ERK1/2 pathway. Thus, for example the MAPKKs MEK1/MEK2 inhibitor PD98059 completely abolished the protective effect of IPC in isolated perfused rat hearts27 as well as in rabbits,28 rats,26 and pigs29in vivo.

JNK1/2 proteins have also been observed to become phosphorylated in pig18 or rat heart samples30 subjected to ischaemia. However, only a few studies have examined the role of JNK1/2 in IPC. By application of the protein-synthesis inhibitor and p38/JNK activator anisomycin, a reduction of myocardial infarct size (IS) was noticed in rabbits31 and pigs32in vivo. Consistently with this, the c-Jun and JNK1 inhibitor curcumin abolished the cardioprotective effects of preconditioning in isolated rat hearts.30 The contribution of JNKs to IPC was further confirmed by the observation that actinomycin-D disrupts IPC by inhibiting the phosphorylation of JNK1/2.33

In contrast to the implicit but controversially discussed role of MAPKs in IPC, nothing is known about MAPKs in eliciting RPC. Since RPC, because of its unique mode of action, affords an opportunity to examine protection locally separated from ischaemia, the present study was designed to characterize the precise roles of p38, ERK1/2, and JNK1/2 in RPC by using an in vivo infarction model of the rat heart.

Methods

Animals and surgical preparation

Experimental procedures were conducted free of external stress conforming to the guidelines approved by the ‘Ministerium für Landwirtschaft, Umwelt und ländliche Räume des Landes Schleswig-Holstein’, Germany and 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). Adult male Wistar rats weighing between 280 and 300 g (Charles-River, Sulzfeld, Germany) were anaesthetized (pentobarbital 50 mg kg−1 ip), tracheotomized, and ventilated with room air enriched with oxygen at 50 breaths/min (rodent ventilator 994500, TSE, Bad Homburg, Germany) to maintain oxygen tension within physiological ranges. The left jugular vein was cannulated to compensate for loss of fluid or to inject drugs. The left carotid artery was cannulated for measurement of blood pressure (BP) and heart rate (HR) by placing a pressure transducer (Isotec, Hugo-Sachs Electronic, Freiburg, Germany) that was again connected to a computer. Body temperature of animals was continuously measured and maintained at 37.0–37.8°C using a heating pad. Anaesthesia was maintained throughout the experiments using pentobarbital (150 µg min−1 kg−1).

As was previously described8,11 for the induction of RPC, the abdominal cavity was opened and the superior mesenteric artery was mobilized. A loose suture was placed around the vessel to ease subsequent mesenteric artery occlusion (MAO) by an atraumatic vessel clip (Micro Serrefines, FST, Heidelberg, Germany). Myocardial ischaemic preconditioning and infarction was provoked by coronary artery occlusion (CAO). This was performed in situ by lateral thoracotomy and incision of the pericardium, before a 6-0 prolene suture (Resorba, Nuremberg, Germany) was looped under the left descending coronary artery.

Experimental protocols

As depicted in Figure 1, animals were randomly assigned to six experimental procedures (n = 5–6 per group). The RPC group was subjected to 15 min MAO followed by 15 min of reperfusion, whereas the IPC group underwent two cycles of CAO for 5 min followed by 10 min of reperfusion. Solutions of the MAPK inhibitors SB203580, PD98059, and SP600125 (Merck Biosciences, Darmstadt, Germany) were freshly prepared prior to the experiments using polyethylene glycol (33 µl kg−1 b.w.; Sigma, Taufkirchen, Germany) as solubilizer. Substances were applied at the beginning of the experimental procedures at dosages reported to be effective (SB203580 1 mg kg−1, PD98059 0.3 mg kg−1 or SP600125 0.1 mg kg−1).19,34,35 Additionally, five to six animals were used for further immunoblotting analysis of MAPK activation as indicated in Figure 1. Here, myocardial samples were obtained directly after completing the RPC or IPC procedures and snap frozen in liquid nitrogen. Rat hearts subjected to myocardial infarction underwent 30 min of CAO and a subsequent 180 min of reperfusion.

Figure 1

Experimental procedures. Remote preconditioning (RPC) was induced by mesentery artery occlusion (MAO) and ischaemic preconditioning (IPC) by coronary artery occlusion (CAO). Inhibitors (I) used: SB203580, PD98059, and SP600125.

Figure 1

Experimental procedures. Remote preconditioning (RPC) was induced by mesentery artery occlusion (MAO) and ischaemic preconditioning (IPC) by coronary artery occlusion (CAO). Inhibitors (I) used: SB203580, PD98059, and SP600125.

Determination of myocardial infarct size

After termination of reperfusion, the coronary artery was reoccluded and Chinese ink was injected in situ into the left ventricle. Perfused myocardial tissue stained black and the area at risk (AAR) was delineated. Hearts were quickly excised, and the atria and right ventricles were removed. Left ventricles including the septum were cut into slices (1 mm) before being incubated in 2,3,5-triphenyltetrazoliumchloride (TTC; 1% in 0.1 M phosphate buffer, pH 7.4) for 30 min at room temperature. In this way, the viable tissue stained red so that the area of IS could be delineated as pale. After weighing the slices, areas of the left ventricle, AAR, and IS were gauged on both sides of a single slice each by computer-assisted planimetry.

Protein extraction and immunoblotting analysis

Proteins from whole tissue samples were extracted as described elsewhere8 using a modified homogenization buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton, 1 mM DTT, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin (Sigma). Protein concentrations were determined by the Lowry method (Merck). Equal amounts of proteins were separated on 10% SDS–polyacrylamide gels before being transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Eschborn, Germany). Adequate transfer of proteins was controlled by Coomassie Blue staining and equal protein loading was verified by probing the membrane with actin antibodies (Santa Cruz Biotechnology, Heidelberg, Germany). Detection of phosphorylated or total MAP kinase proteins was accomplished by overnight incubation of membranes with antibodies raised against p-p38Thr180/Tyr182 (Cell Signaling Technology, Beverly, MA, USA), p38 (Santa Cruz), p-ERK1/2Thr202/Tyr204, ERK1/2 (both Cell Signaling Technology), p-SAP/JNKThr183/Tyr185 (Stressgen, Ann Arbor, MI, USA), and SAP/JNK (Cell Signaling). After thorough washing, membranes were subjected to the appropriate horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgGs (Dako, Hamburg, Germany) for 1.5 h at room temperature. Bands were visualized by enhanced chemiluminescence (SuperSignal West Pico substrate, Perbio Science, Bonn, Germany) using a high resolution CCD camera (ChemiDoc system and Quantity One software, Bio-Rad, Munich, Germany). Immunoblots were carried out in triplicate as independent experiments.

Statistical analysis

Data are presented as means ± SEM. Differences in IS and in the grade of MAPK phosphorylation were compared among the experimental groups by one-way ANOVA, followed by Newman–Keul’s multiple comparison test. Haemodynamic data were tested for differences between the groups by using a two-way ANOVA for repeated measurements. Temporal changes in haemodynamic parameters within one group were analysed by one-way ANOVA for repeated measurements followed by paired t-tests with Bonferroni’s correction when differences were found to be significant. A P-value <0.05 was considered to denote a statistically significant difference.

Results

Haemodynamics

Mean arterial blood pressures (MAP) averaged out to 95 ± 4 mmHg after completion of the preparations and declined to 82 ± 3 mmHg until termination of the experimental procedure. There was no significant influence of treatment on BP or heart frequency throughout the experiments among all groups when analysed by two-way ANOVA.

Infarct size studies

Influence of RPC and IPC on myocardial infarct size

Infarct size measured in the control group of the RPC protocol amounted to 55.1 ± 1.8% of AAR as depicted in Figure 2. Controls corresponding to the IPC group revealed an average IS of 51.8 ± 2.7% therefore ruling out that treatment with polyethylene glycol substantially influenced the extent of infarction. As expected, RPC and IPC markedly reduced the myocardial IS to 25.5 ± 1.8% and 12.7 ± 1.1% of AAR, respectively (P < 0.001 vs. all other experimental groups), thereby affirming the efficacy of both preconditioning procedures that were applied. Notably, the latter preconditioning stimulus appeared to be more efficient in this study compared with RPC with respect to cardioprotection (P < 0.005 vs. RPC).

Figure 2

Effects of RPC, IPC, and MAPK inhibitors on infarct size. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK1/2 (SP600125) were applied with (hatched square) or without (open square) subsequent RPC. n = 5–6; ***P < 0.001 vs. controls and inhibitor treated groups.

Figure 2

Effects of RPC, IPC, and MAPK inhibitors on infarct size. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK1/2 (SP600125) were applied with (hatched square) or without (open square) subsequent RPC. n = 5–6; ***P < 0.001 vs. controls and inhibitor treated groups.

Influence of MAPK inhibitors on RPC and myocardial infarct size

In general, all MAPK inhibitors applied prior to the experimental procedures sufficed to abrogate the protective effect of RPC as indicated by significantly increased ISs (Figure 2). Thus, IS was found to be increased to 43.3 ± 4.6% in response to the p38 MAPK inhibitor SB203580. Inhibition of the ERK1/2 pathway by PD98059 resulted in an average IS of 44.6 ± 4.5%. The JNK1/2 MAPK inhibitor SP600125 was sufficient to cause an IS of 56.6 ± 6.3% of AAR when applied prior to the RPC procedure. No differences in IS were obtained when inhibitors were applied without preconditioning. Indeed, pretreatment of animals with SB203580 tended to decrease IS but the apparent difference was found to be not significant when compared with controls. In detail, the application of SB203580, PD98059, or SP600125 produced an average IS of 44.2 ± 4.2%, 52.7 ± 3.1%, or 49.4 ± 5.4% of AAR, respectively. When ISs were compared between groups merely receiving the MAPK inhibitors and groups undergoing RPC with preceding infusions of the inhibitors, no significant differences were observed throughout. In the whole study, AARs did not differ among the experimental groups.

Analysis of MAPK activity

Phosphorylation profile of MAPKs after RPC

In order to determine the effect of RPC on the MAPK pathways, the degree of dual phosphorylation was assayed by western blot analyses in the left ventricle and small intestine and compared with the amounts of total MAPK proteins. The specificity of antibody detection was ensured by co-migration of standardized recombinant marker proteins. Immunoblotting produced specific signals representing either phosphorylated or total amounts of MAPKs at the predicted molecular weights of 38 kDa (p38), 44/42 kDa (ERK1/2), and 54/46 kDa (JNK1/2), respectively (Figure 3A). Actin that was used as a housekeeping gene did not differ in the various experimental groups. Notably, this protein was expressed at lower levels in the heart than it was in the duodenum. Regarding p38 MAPK, analysis of tissue samples obtained from the small intestine after 15 min of reperfusion revealed no difference between control and RPC irrespective of whether phosphorylated or total amounts of protein were being examined (Figure 3B). To investigate an early activation of this MAP kinase during mesenteric reperfusion, additional analyses were performed at 5 and 10 min after beginning of the reperfusion (data not shown). Again, no differences in the amounts of phosphorylated and total p38 MAPK became apparent. In contrast, a marked accumulation of phosphorylated p42 and p44 proteins was found in the small intestine indicating the activation of this pathway by ischaemia after termination of RPC. Thus, p42 increased ∼2-fold, and p44 2.5-fold compared with animals without intestinal ischaemia. Similar to ERK1/2, the RPC procedure was also capable of inducing dual phosphorylation of JNK1/2 proteins localized within the intestine. The increase in phosphorylated proteins that occurred after ischaemia was ∼2-fold, that of phosphorylated p54 ∼2.2-fold. Again, the quantities of total p46 and p54 proteins were not influenced by the experimental procedure when antibodies were used recognizing total MAPK proteins. Interestingly, RPC was not efficient to induce any significant phosphorylation of the various MAP kinases in the left ventricle. Although phosphorylated p46 and p54 proteins seemed to increase weakly, the differences turned out not to be significant when compared with non-preconditioned hearts. Total amounts of these proteins did not change significantly, an observation which also applied to ERK1/2 and p38. Notably, all applied MAPK inhibitors were sufficient to impair phosphorylation of its target MAPK pathways in the small intestine when antibodies were used detecting phosphorylated MAPK only (Figure 4). Total amounts of p38, ERK1/2, and JNK1/2 proteins were not influenced by the application of the various inhibitors.

Figure 3

Effects of RPC and IPC on protein expression of MAPKs in the small intestine and the heart. Representative immunoblots (A) and densitometric analysis (B) demonstrate that either RPC or IPC were sufficient to activate ERK1/2 and JNK1/2 pathways as indicated by increased amounts of phosphorylated proteins (hatched bars) when compared with sham-treated animals. Total amounts of MAPKs (full bars) were not influenced by treatment regimen. All quantitative data are depicted as ratios to the corresponding non-preconditioned controls. n = 5–6; *P < 0.05 vs. controls.

Figure 3

Effects of RPC and IPC on protein expression of MAPKs in the small intestine and the heart. Representative immunoblots (A) and densitometric analysis (B) demonstrate that either RPC or IPC were sufficient to activate ERK1/2 and JNK1/2 pathways as indicated by increased amounts of phosphorylated proteins (hatched bars) when compared with sham-treated animals. Total amounts of MAPKs (full bars) were not influenced by treatment regimen. All quantitative data are depicted as ratios to the corresponding non-preconditioned controls. n = 5–6; *P < 0.05 vs. controls.

Figure 4

Effects of the MAPK inhibitors SB203580, PD98059, and SP600125 on the expression of total and phosphorylated fractions of p38, ERK1/2, and JNK1/2 in samples obtained from the small intestine of animals undergoing sham operation (con) or RPC procedure (RPC).

Figure 4

Effects of the MAPK inhibitors SB203580, PD98059, and SP600125 on the expression of total and phosphorylated fractions of p38, ERK1/2, and JNK1/2 in samples obtained from the small intestine of animals undergoing sham operation (con) or RPC procedure (RPC).

Phosphorylation profile of MAPKs after IPC

In contrast to RPC, the short-term ischaemia that was used to induce IPC was effective to elevate the phosphorylation state of the MAPK proteins in the heart. In detail, the amounts of phosphorylated p44 protein were found to be increased by ∼1.8-fold, that of phosphorylated p42 1.9-fold after termination of cardiac ischaemia. Regarding the phosphorylation of JNK1/2, only JNK2 turned out to be elevated significantly (1.4-fold). Interestingly, ischaemia induced by CAO resulted in a significantly reduced activity of p38 MAPK when compared with controls. Overall, the two cycles of 5 min ischaemia in the left ventricle appeared to be a less efficacious activator of MAPK phosphorylation compared with the 15 min MAO in the small intestine.

Discussion

In the present study, we demonstrated that the p38, ERK1/2, and JNK1/2 pathways are involved and functionally significant in bringing about myocardial protection induced by RPC in vivo. However, RPC appears not to directly influence MAPK proteins located in the protected tissue, e.g. the myocardium. As such, the mechanisms of RPC contrast to those of IPC, for which there is substantial evidence that various MAPK pathways participate in mediating IPC. This interpretation is further supported by the present study since phosphorylation of myocardial ERK1/2 and JNK1 increased during two brief cycles of non-lethal ischaemia. In contrast, the phosphorylated p38 MAPK existed in the non-ischaemic myocardium to the extent that IPC as well as the application of SB203580 reduced the amounts of the phosphorylated protein (Figures 3 and 4). This finding adds to the controversial debate concerning the role of p38 MAPK in IPC and myocardial damage.16–18,20–24 On the one hand, the time of detection of phosphorylated p38 in this study could be important, since Marais et al.36 had observed that phosphorylation of the p38 protein disappeared during repeated preconditioning episodes. On the other hand, an alternative explanation is provided by previous data revealing a more unfavourable role for p38 in IPC and myocardial protection. Thus, it was reported that p38 phosphorylation did not change or even decreased during ischaemia.9,24,26,37 Moreover, inhibition of this pathway during ischaemia/reperfusion by SB203580 resulted in a cardioprotective effect in another study.23 More concise results were obtained from the analysis of ERK1/2 proteins. As stated above, the observed increase in phosphorylated p42 and p44 proteins complies with recent observations showing activation during ischaemia/reperfusion as well as mediation of IPC in a manner sensitive to ERK1/2 inhibitors.25–27 A comparatively less distinctive phosphorylation after IPC was observed for the JNK1/2 proteins of which only JNK1 was found to be significant. This observation suggests a contribution of this pathway to IPC, although the precise role of JNK1/2 in IPC needs to be evaluated further.

In sharp contrast to IPC, none of the examined MAPKs were found to be altered in their phosphorylated fractions in the myocardium after RPC. Since the myocardial tissue had not been exposed to ischaemia and reoxygenation, it may be postulated that the generation of oxygen radicals in the course of preconditioning is minimal when compared with IPC. This may indeed explain the lack of activation of myocardial MAPKs since free radicals are known to be capable of activating protein kinases.9 In fact, it was reported previously that RPC might depend on the generation of free radicals in a model of femoral artery occlusion although the increase in levels of free radicals was observed as late as 2 h after termination of the RPC procedure.38 In addition, a comparatively long procedure of RPC induction (four cycles of repetitive 10 min ischaemia/reperfusion) was applied in that study that might have also triggered an additional generation of free radicals. Further explanation that myocardial MAPKs are not phosphorylated by RPC is provided by studies examining gene alterations at levels of mRNA by cDNA microarray techniques in remotely preconditioned organs.39 Thus, in the heart, mRNA levels of MAPK were not influenced by MAO-induced RPC, whereas mediators known to be involved in RPC (e.g. CGRP) were activated as reflected by a 2-fold increase in mRNA encoding for the CGRP receptor component protein. Exclusively in the kidney, a 2-fold upregulation of MAPKAP-2 mRNA, a substrate for p38, became apparent after 24 h.39 Sun et al.40 investigated the approach of inducing RPC in the brain by limb ischaemia. Interestingly, in that study, p38 was found to be the most important trigger for transducing RPC-induced protection to the brain. Immunoblotting revealed an increase in phosphorylated p38 in hippocampal tissues after 6 h post RPC. Furthermore, the p38 MAPK inhibitor SB203580 abrogated the RPC-induced ischaemic tolerance of the brain. These observations parallel the findings of our study.

The abrogation of RPC by each of the applied MAPK inhibitors indicates that RPC clearly depends on the activity of MAPKs despite these MAP kinases will not be activated by RPC in the heart. Apparently, ischaemia occurring remotely from the protected organ is essential for bringing up RPC. This ischaemia might in turn induce activation of local MAPKs as it would emerge directly during IPC. This explains the increased phosphorylation of MAPKs observable in the small intestine as a direct consequence of ischaemia and is likely to be associated with free radical generation. This pathway of activation is consistent with the protocol of this study since all myocardial MAPKs (except p38) were found to be increased markedly in its phosphorylated amounts after two cycles of 5 min myocardial ischaemia. However, the signal cascade following MAPK activation during IPC and RPC still awaits further characterization but is complicated by the vast array of signalling pathways of MAPKs.

It should be noted that SB203580 and SP600125 altered the state of phosphorylation of p38 and JNK1/2, respectively. These inhibitors act by interfering with the catalytic centre or the ATP-binding site of the respective protein kinases and would therefore not be expected to influence their phosphorylation state. The observed attenuation of phosphorylation may therefore indicate secondary reactions to the direct effects of the inhibitors or point to the existence of additional drug targets located upstream of the investigated kinases. Such responses to the inhibition of these protein kinases are regularly observed in in vivo studies25 and may even contribute to the inhibitory efficacy of the pharmacological intervention. Specifically, the JNK1/2 inhibitor SP600125 is known for its activity on additional protein kinases, such as the protein kinase D1 or the maternal embryonic leucine zipper kinase.35 Although this unselectivity limits the significance of obtained results, there is up to date no alternative to the frequently used substance for in vivo studies. Hence, the application of SP600125 in our study is not able to ultimately rule out a contribution of protein kinases additional to JNK1/2 in RPC.

The suppression of RPC-induced protection by any of the applied inhibitors indicates an essential role of the activity of the respective MAPK. This does not necessarily mean that MAPKs will be activated in the course of RPC. In the case of p38 MAPK, we did not observe activation neither in the small intestine nor in myocardial tissue. However, the existence of p38 in a highly phosphorylated state in non-ischaemic organs indicated a high basal activity that may constitute the target of SB203580. Thus, p38 MAPK may not be considered as a direct mediator of RPC nevertheless its activity seems to be required for the underlying signalling. Similar considerations may apply to the significance of the further investigated MAPKs in the myocardium where no activation could be detected after RPC. Again, this absence of activation will not exclude a potential protective function of these MAPKs in the myocardium because their basal activity or their activation may become essential in the course of ischaemia and reperfusion in this organ. Indeed, ERK1/2 has been characterized as a protective MAPK pathway whose activation in the early phase of reperfusion is enabled by precedent IPC.27 In our study, the applied MAPK inhibitors given prior to RPC will still attenuate a potential activation of MAPKs in the heart during lethal ischaemia and reperfusion. Since the activity profiles of the MAPKs during these experimental phases were not analysed, the significance of myocardial MAP kinases for RPC-induced protection cannot be ruled out ultimately.

This study provides evidence that MAPKs are indispensable for bringing about RPC albeit possibly without participation of MAPK located in the target organ. In contrast, IPC in this study induced activation of myocardial ERK1/2 and JNK1/2, thereby providing a possible explanation for the higher efficacy of IPC, as reflected by a significantly lower IS compared with RPC. In previous studies, RPC and IPC have been reported to culminate in a similar extent of myocardial protection even under application of an RPC protocol similar (15 min MAO and 10 min reperfusion) to the one employed in the present study,41 but our analysis of MAPKs points to a distinction between both modes of preconditioning, evident at the level of signal transduction. In the heart, it may be that the signal cascade resulting in protection proceeds via remote ischaemia-induced transmitter release to activation of PKCε, where the latter is known to have a functionally significant role in protecting the myocardium.10–12 This further component of signalling might not be executed in RPC due to the failure of activation of myocardial MAPK as occurring otherwise during IPC.

Taken together, this study provides new insight into the complex processes affording cardiac protection in RPC. Broadening the knowledge about this variant of preconditioning shall ease the transition from experimental model to clinical application, especially in view of recent observations demonstrating that RPC still appears to be functional during lethal cardiac ischaemia.42

Acknowledgements

The authors wish to thank Cornelia Magnussen for excellent technical assistance.

Conflict of interest: none declared.

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