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Cardiovascular Research 2002 55(3):690-700; doi:10.1016/S0008-6363(02)00319-X
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Copyright © 2002, European Society of Cardiology

p38 MAP kinase is a mediator of ischemic preconditioning in pigs

Rainer Schulza, Sergej Belosjorowa, Petra Gresa, Johanna Jansena, Martin C Michelb and Gerd Heuscha,*

aAbteilungen für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstrasse 55, 45122 Essen, Germany
bNieren- und Hochdruckkrankheiten, Zentrum für Innere Medizin Universitätsklinikum Essen, 45122 Essen, Germany

gerd.heusch{at}uni-essen.de

* Corresponding author. Tel.: +49-201-723-4480; fax: +49-201-723-4481

Received 12 October 2001; accepted 12 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: The role of p38MAPK in ischemic preconditioning (IP) is still equivocal, insofar as the p38MAPK-inhibitor SB203580 abolished IP in rats, rabbits and dogs, but not in pigs. Blockade of p38MAPK prior to the sustained ischemia also generated contradictory findings, insofar as p38MAPK acted as trigger in dogs but as mediator in rats. We have now tested whether the two structurally unrelated p38MAPK-inhibitors, BIX-645 and SB203580, abolished infarct size (IS) reduction by IP in pigs and whether their effects depended on the time of administration. Methods: Sixty-five enflurane-anesthetized pigs underwent 90 min low-flow ischemia and 120 min reperfusion without or with one preceding cycle of 10 min preconditioning ischemia and 15 min reperfusion. Pigs received BIX-645 (1 mg/kg, i.v.) or SB203580 (10 µM, i.c.) prior to either IP or the sustained ischemia. Results: IS (% TTC-staining) was reduced by IP [4.8±3.1(S.E.M.), P<0.05] compared to placebo (25.8±5.5). BIX-645 or SB203580 per se had no effect on IS (23.5±5.2 and 21.8±4.4, respectively). IS reduction by IP was abolished by BIX-645 (26.2±6.4 or 25.5±4.7) and SB203580 (19.9±4.3 or 16.7±4.7), given either prior to IP or the sustained ischemia, respectively. The supernatant of homogenized myocardial biopsies taken during the sustained ischemia from preconditioned pigs receiving either BIX-645 or SB203580 inhibited the anisomycin-stimulated ATF-2 phosphorylation in cultured Rat1 fibroblasts. This in vitro inhibition of ATF-2 phosphorylation correlated to the actual IS. Conclusion: The attenuation of the IS-reducing effect of IP depends on the effectiveness of blockade of p38MAPK activity. p38MAPK is a mediator of IP in pigs.

KEYWORDS Ischemia; Preconditioning; Reperfusion; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
The role of p38 MAP kinase in ischemia–reperfusion in general and in ischemic preconditioning in particular is still controversial. An increase in p38 MAP kinase phosphorylation during ischemia per se has been described in several [1–3] but not all studies (for review see [4,5]). The p38 MAP kinase phosphorylation appeared to be a variable phenomenon and not related to the severity of ischemia [2]. In isolated rat [6,7] and rabbit [8] hearts, SB202190 or SB203580 blocked the ischemia-induced p38 MAP kinase phosphorylation [6] or the ischemia-induced increase in p38 MAP kinase activity [7,8] and reduced infarct size. Also, in pigs [1] and dogs [3], which had increased p38 MAP kinase phosphorylation during ischemia, intramyocardial [1] or intracoronary [3] infusion of SB203580 decreased infarct size.

With ischemic preconditioning, p38 MAP kinase phosphorylation during the subsequent sustained ischemia has been reported to be either increased [9–13], unchanged [2,8] or decreased [3,14]. In pigs, intramyocardial infusion of SB203580 did not affect the infarct size reduction achieved by ischemic preconditioning [1], but it effectively blocked protection in cell models of ischemic preconditioning [11,15,16] and abolished the infarct size reduction by ischemic preconditioning in rats [6,13,17], rabbits [18] and dogs [3]. Also, the importance of timing of p38 MAP kinase inhibition for the loss of ischemic preconditioning's protection and therefore its role as trigger or mediator of ischemic preconditioning (IP) remains controversial. In isolated rat hearts [7], SB202190 given just prior to the sustained ischemia did not affect infarct size reduction by IP, suggesting a role of p38 MAP kinase as a trigger. In vivo, SB203580 given just prior to the sustained ischemia was effective in preventing protection in rats [13,17] but not in dogs [3], while on the contrary, administration just during the preconditioning phase was effective in dogs but not in rats.

Explanations for these controversial findings might relate to different species (pigs vs. dogs, rabbits or rats) and experimental models (no-flow vs. low-flow, in vitro vs. in vivo), mode of application (intramyocardial, intracoronary, intravenous) and the selectivity and potency of different inhibitors. We therefore used two different, structurally unrelated p38 MAP kinase inhibitors (SB203580, BIX-645). The importance of timing of administration was addressed by either starting the administration of the respective antagonists prior to the preconditioning cycle or just prior to the sustained ischemia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
The experimental protocols employed in the present study were approved by the Bioethical Committee of the district of Düsseldorf, and they adhere to the guiding principles of the American Physiological Society.

2.1 Experimental preparation
Sixty-five enflurane-anesthetized Göttinger minipigs were instrumented for the measurement of left ventricular pressure and wall thickness [2]. Rectal temperature was monitored and kept between 37 and 38 °C by use of a heated surgical table and drapes. The left anterior descending (LAD) coronary artery and vein were cannulated, and the artery was perfused by an extracorporeal circuit, including a roller pump. The extracorporeal circuit included sidearms for the intracoronary injection of microspheres and of SB203580 or dimethylsulfoxide (DMSO). Systolic wall thickening was calculated and regional myocardial blood flow was measured with microspheres [2]. To quantify infarct size, six transverse myocardial slices from each heart were incubated in a triphenyl tetrazolium chloride solution [2].

2.2 p38-MAP kinase phosphorylation assay
Myocardial biopsies taken at baseline and 8 min of the sustained index ischemia were weighed, diluted with sample buffer (1:60, 2% sodium dodecyl sulfate, 50 mM dithiothreitol, 10% glycerol, 0.1% bromphenol blue, 62.5 mM Tris, pH 6.8 at 25 °C) and homogenized. The homogenates were boiled for 5 min, cooled on ice, and centrifuged at 14 000 g for 5 min at 4 °C. Aliquots (20 µl) of the supernatants from each experiment were loaded in parallel on two 10% PAGE–SDS gels. The proteins were separated by electrophoresis (25 µA, for 2 h at 4 °C), and the separated proteins were transferred to nitrocellulose membranes by electroblotting (40 V, overnight, at 4 °C). The membranes were blocked with Tris-buffered saline (TBS: 20 mM Tris, 120 mM NaCl at 25 °C) containing 5% non-fat dry milk for 90 min and washed four times with TBS containing 0.05% Tween 20 (T–TBS) for 10 min. The resulting blots were incubated for 2 h either with an antiserum recognizing total p38 or with an antiserum specific for the dually phosphorylated form of p38 (New England Biolabs., Beverly, MA, USA). The blots were then washed four times with 80 ml of washing buffer (150 mM NaCl, 0.1% Tween-20, 50 mM Tris, pH 7.4 at 25°) and were then incubated for 1 h with a secondary antibody (antirabbit immunoglobulin linked to horseradish peroxidase: Phototope-HRP detection kit; New England Biolabs.). After four more washes with buffer, detection was performed by enhanced chemoluminiscence. The resulting autoradiographs were analyzed by quantitative two-dimensional densitometry, using commercially available software (Herolab, Wiesloch, Germany). The two-dimensional band intensity of phosphorylated MAP kinase was expressed relative to that of total MAP kinase, as assessed with the parallel blot prepared simultaneously. The ratio for the sample taken at baseline was set as 100%, and the value at 5 min of the sustained index ischemia from the same experiment was then expressed as percent of baseline.

2.3 p38 MAP kinase activity assay
Rat-1 fibroblasts (Pfizer Central Research, Sandwich, Kent, UK) were cultured in the absence of serum for 24 h. Myocardial biopsies (diluted in 10 volumes of cold isotonic homogenization buffer (mM: imidazol acetate 50, Mg acetate 10, KH2PO4 4, EDTA 2; µM: N-acetylcysteine 50, sulfur 12.5; pH 7.6 [19])) were taken at 8 and 85 min of the sustained ischemia from the area at risk of pigs receiving placebo, BIX-645 (Boehringer Ingelheim, Ridgefield, CT, USA) or SB203580 and homogenized (Branson digital sonifier model 450D, Schwäbisch Gmünd, Germany). For reference, different concentrations of BIX-645 (Fig. 1) or SB203580 were added in vitro. Cells were stimulated with anisomycin (50 µg/ml) in the presence of fresh medium at 37 °C for 10 min. Reactions were terminated by aspiration of the culture medium, followed by two washes with ice-cold, Ca-free phosphate-buffered saline, and then addition of 500 µl cell lysis buffer (plus 1 mM phenylmethansulfonylfluoride). The cells were scraped off the dishes immediately, sonicated four times for 5 s on ice and centrifuged at 14 000 g for 10 min at 4 °C. Prior to measurement of ATF-2 phosphorylation at Thr71 by Western blotting using a phospho-ATF2 antibody (New England Biolabs.). p38 MAP kinase was immunoprecipitated by specific antibodies using a commercially available kit (p38 MAP kinase assay kit, Cell Signaling Technology, Beverly, MA, USA).


Figure 1
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  		Fig. 1 Inhibition of the anisomycin-stimulated ATF2-phosphorylation (y axis) in Rat1 cells in vitro by BIX-645 (x axis). BIX-645 dose-dependently inhibited the anisomycin-stimulated ATF2-phosphorylation.

 
2.4 Experimental protocols (Fig. 2)
2.4.1 Group 1 (placebo+90 min ischemia, n = 8)
After control measurements of systemic hemodynamics, regional myocardial function and blood flow, coronary blood flow was decreased by reducing the pump speed of the extracorporeal circuit to reduce anterior systolic wall thickening by >90%. This level of hypoperfusion was maintained for 90 min. Measurements were repeated at 5 and 85 min ischemia. Following 90 min ischemia, the myocardium was reperfused for 120 min to facilitate the identification of necrotic tissue.


Figure 2
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  		Fig. 2 Scheme of the experimental protocols.

 
2.4.2 Group 2 (ischemic preconditioning (IP)+90 min ischemia, n = 8)
After control measurements, the myocardium was subjected to 10 min of preconditioning ischemia with a >90% reduction in anterior systolic wall thickening and then reperfused for 15 min. After reperfusion, coronary blood flow was once again reduced to the same level as during the preconditioning ischemia. Thereafter, the protocol was identical to that of group 1.

2.4.3 Group 3 (BIX-645+90 min ischemia, n = 7)
Following control measurements, the p38 MAP kinase inhibitor BIX-645 (1 mg/kg body weight in 40 ml saline: PEG400 1:1 solution) was infused intravenously over 3 min starting 30 min prior to the sustained ischemia. Thereafter, the protocol of group 3 was identical to that of group 1.

2.4.4 Group 4 (SB203580+90 min ischemia, n = 8)
After control measurements, the p38 MAP kinase inhibitor SB 203580 (10 µM solved in 0.1% DMSO) was continuously infused into the LAD starting 30 min prior to and lasting until the end of the sustained ischemia. Thereafter, the protocol of group 4 was identical to that of group 1.

2.4.5 Group 5 (BIX-645+IP+90 min ischemia, n = 7)
After control measurements, pigs received BIX-645 (1 mg/kg). Thereafter, the protocol of group 5 was identical to that of group 2.

2.4.6 Group 6 (SB 203580+IP+90 min ischemia, n = 7)
After control measurements, SB203580 (10 µM) was continuously infused into the LAD starting 30 min prior to the preconditioning ischemia and lasting until the end of the sustained ischemia. Otherwise, the protocol of group 6 was identical to that of group 2.

2.4.7 Group 7 (IP+ BIX-645+90 min ischemia, n = 6)
The protocol of group 7 was identical to that of group 2, except that pigs received BIX-645 (1 mg/kg) over 3 min starting 10 min prior to the sustained ischemia.

2.4.8 Group 8 (IP+SB203580+90 min ischemia, n = 6)
The protocol of group 8 was identical to that of group 2, except that the continuous infusion of SB203580 (10 µM) started 10 min prior to and lasted until the end of the sustained ischemia.

2.4.9 Group 9 (DMSO+90 min ischemia, n = 4)
After control measurements, DMSO (0.1%) was continuously infused into the LAD starting 30 min prior to and lasting until the end of the sustained ischemia. Thereafter, the protocol of group 9 was identical to that of group 1.

2.4.10 Group 10 (DMSO+IP+90 min ischemia, n = 4)
After control measurements, DMSO (0.1%) was continuously infused into the LAD starting 30 min prior to the preconditioning ischemia and lasting until the end of the sustained ischemia. Otherwise, the protocol of group 10 was identical to that of group 2.

2.5 Data analysis and statistics
All data are reported as mean values±S.E.M. Statistical analysis of systemic hemodynamics and regional myocardial function comprised 2-way ANOVA for repeated measures and Fisher's LSD (least square differences) tests when significant overall effects were detected using SYSTAT software (SPSS Science, Chicago, IL, USA). The area at risk, infarct size and subendocardial blood flow as well as p38 MAP kinase phosphorylation at 8 min sustained ischemia were compared by one-way ANOVA. The actual infarct size in preconditioned pigs receiving BIX-645 or SB203580 was expressed as fraction of expected infarct size [2], the latter being derived from the infarct size–subendocardial blood flow relationship of pigs receiving either BIX-645 or SB203580 undergoing 90 min ischemia and 120 min reperfusion and the actual blood flow of the individual preconditioned pig. Infarct size was then related to the inhibition of anisomycin-stimulated ATF-2 phoshorylation in vitro using linear regression analysis. A P value of <0.05 was accepted as indicating a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 Systemic hemodynamics and regional myocardial function
Heart rate remained constant throughout the experimental protocol in each group of pigs and was not different among groups (Table 1). Left ventricular peak pressure was significantly decreased at 5 min ischemia and remained reduced until the end of ischemia in all groups, except groups 1, 7 and 10 (Table1). However, left ventricular peak pressure during ischemia was not significantly different among the 10 groups. The reduction of coronary blood flow reduced mean coronary arterial pressure and anterior systolic wall thickening in all groups to a similar extent (P<0.05 vs. baseline; Table 1).


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  		Table 1
 
3.2 Area at risk and infarct size
The area at risk (AAR%) and subendocardial blood flow were similar among all groups (Table 2). Infarct size was reduced by IP (Fig. 3). DMSO did not alter infarct size per se and did also not interfere with IP's protection. BIX-645 or SB203580 per se had no effect on infarct size, but abolished the infarct size-limiting effect of IP, when given either before the preconditioning cycle or just prior to the sustained ischemia (Fig. 3).


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  		Table 2
 

Figure 3
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  		Fig. 3 Infarct size was reduced by ischemic preconditioning (group 2) compared to placebo (group 1). Administration of BIX-645 (group 3) or SB203580 (group 4) had no effect on infarct size per se, but abolished the infarct size-limiting effect of ischemic preconditioning (groups 5 and 6). A similar effect was obtained when BIX-645 or SB203580 were given just prior to the sustained ischemia (groups 7 and 8). DMSO did neither alter infarct size per se (group 9) nor did it interfere with ischemic preconditioning's protection (group 10).

 
3.3 p38 MAP kinase phosphorylation
In the placebo group, p38 MAP kinase phosphorylation was increased at 8 min of the sustained ischemia (group 1, Fig. 4), and this increased p38 MAP kinase phosphorylation was not significantly altered by ischemic preconditioning (group 2). As expected, neither BIX-645 nor SB203580 altered p38 MAP kinase phosphorylation (groups 3 and 4). Combination of IP with either BIX-645 or SB203580, independent of whether pigs received the compounds prior to (groups 5 and 6) or following the preconditioning ischemia (groups 7 and 8), attenuated the increase in p38 MAP kinase phosphorylation.


Figure 4
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  		Fig. 4 In the placebo group, p38 MAP kinase phosphorylation was increased (group 1). Ischemic preconditioning did not alter p38 MAP kinase phosphorylation (group 2). Neither BIX-645 nor SB203580 decreased p38 MAP kinase phosphorylation (groups 3 and 4). Combination of ischemic preconditioning with either BIX-645 or SB203580, independent on whether pigs received the compounds prior to (groups 5 and 6) or following the preconditioning ischemia (groups 7 and 8), attenuated p38 MAP kinase phosphorylation; *, P<0.05 vs. baseline.

 
3.4 Rat1 fibroblasts bioassay
Anisomycin induced a strong p38 MAP kinase activation resulting in increased ATF2-phosphorylation. Supernatant from myocardial biopsies of hearts undergoing ischemia–reperfusion without or with a preceding IP episode inhibited the anisomycin-induced ATF2-phosphorylation by 4±2% and 7±2%, respectively. Phosphorylation of ATF2 was inhibited by the supernatant of myocardial biopsies taken at 8 min of sustained ischemia from pigs undergoing the preconditioning protocol and prior administration of BIX-645 or SB203580 (groups 5 and 6). The inhibition was more pronounced with BIX-645 (69±8%) than with SB203580 (37±9%), respectively. The inhibition of anisomycin-stimulated ATF2-phosphorylation correlated to infarct size, when expressed as a percent of the expected value (Fig. 5). In pigs receiving BIX-645 or SB203580 10 min prior to the sustained ischemia (groups 7 and 8), inhibition of ATF2-phosphorylation averaged 39±17 and 30±7% at 8 min of the sustained ischemia, respectively. Whereas the inhibition of ATF2-phosphorylation in pigs receiving BIX-645 increased to 51±19% with prolongation of ischemia to 85 min, it remained unaltered in pigs receiving SB203580 (33±8%). There was no significant correlation between the inhibition of anisomycin-stimulated ATF2-phosphorylation at 8 min of the sustained ischemia and infarct size (y = 0.0092x+0.3576, r = 0.34, P = NS, data not shown), but such correlation was apparent at 85 min of sustained ischemia (Fig. 6).


Figure 5
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  		Fig. 5 Data are derived from pigs receiving BIX-645 or SB203580 prior to the preconditioning ischemia (groups 5 and 6). The inhibition of the anisomycin-stimulated ATF2-phosphorylation in Rat1 cells in vitro by the supernatant of myocardial biopsies taken at 8 min of sustained ischemia correlated to the actual infarct size; the actual infarct size in preconditioned pigs receiving BIX-645 or SB203580 was expressed as fraction of expected infarct size, the latter being derived from the infarct size-subendocardial blood flow relationship of pigs receiving either BIX-645 (group 3) or SB203580 (group 4) undergoing 90 min ischemia and 120 min reperfusion and the actual blood flow of the individual preconditioned pig.

 

Figure 6
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  		Fig. 6 Data are derived from pigs receiving BIX-645 or SB203580 just prior to the sustained ischemia (groups 7 and 8). The inhibition of the anisomycin-stimulated ATF2-phosphorylation in Rat1 cells in vitro by the supernatant of myocardial biopsies taken at 85 min of sustained ischemia correlated to the actual infarct size; the actual infarct size in preconditioned pigs receiving BIX-645 or SB203580 was expressed as fraction of expected infarct size, the latter being derived from the infarct size–subendocardial blood flow relationship of pigs receiving either BIX-645 (group 3) or SB203580 (group 4) undergoing 90 min ischemia and 120 min reperfusion and the actual blood flow of the individual preconditioned pig.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
In the present study the infarct size reduction by IP was abolished by both BIX-645 and SB203508, which did not alter infarct size per se. The loss of protection by IP was independent of the time of blocker administration but closely correlated to the blockade of p38 MAP kinase activity in an in vitro bioassay.

4.1 Critique of methods
The strengths and limitations of the experimental preparation have been discussed in detail before [2].

In the present study, p38 MAP kinase phosphorylation was determined during normoperfusion and the sustained ischemia, thereby not permitting a distinction between a trigger or mediator role of p38 MAP kinase in IP. As in our previous publication [2], IP did not significantly alter the extent of p38 MAP kinase phosphorylation. While neither BIX-645 nor SB203580 [20,21] altered p38 MAP kinase phosphorylation, the combination of IP with either compound attenuated p38 MAP kinase phosphorylation (groups 5–8). Although we have no explanation for the inhibition of p38 MAP kinase phosphorylation by combining BIX-645 or SB203580 with IP, this observation in vivo supports the in vitro data, in that the extent of inhibition of stimulated p38 MAP kinase activity is associated with loss of the protection otherwise achieved by IP.

We did not determine ATF-2 phosphorylation in myocardial biopsies in vivo, as ATF-2 is phosphorylated by both p38 and JNK MAP kinases [22–25] and the contribution of both kinases to ATF-2 phosphorylation in vivo has not yet been analyzed in detail.

We also did not assess p38 MAP kinase activity in vivo using an in gel assay. Since SB203580 is a reversible inhibitor of p38 MAP kinase activity [20,21], the immunoprecipitation procedure washes SB203580 off [1,7], resulting in a renewed catalytic activity of p38 MAP kinase. Therefore, arbitrary concentrations of SB203580 must be added to p38 MAP kinase activity assays [1]. Whether such arbitrary inhibitor concentrations truly reflect the in vivo concentrations, however, is unknown. We therefore refrained from determining p38 MAP kinase phosphorylation and activity in myocardial biopsies, but instead developed an in vitro p38 MAP kinase activity bioassay and correlated the observed inhibition of p38 MAP kinase activity in vitro to the actual infarct size in vivo.

In Rat1 cells, anisomycin stimulates p38 MAP kinase activity, and this effect is largely attenuated (70±12%) by 20 µM SB203580 [26]. While the supernatant from myocardial biopsies of placebo and preconditioned hearts only minimally affected the anisomycin-stimulated p38 MAP kinase activity, the supernatant from myocardial biopsies of pigs receiving BIX-645 or SB203580 inhibited p38 MAP kinase activity. Although all pigs received the same dose of BIX-645 (1 mg/kg, i.v.) or SB203580 (10 µM, i.c.), the in vitro inhibition had a sizable interindividual variability, which, however, correlated to the interindividual variation in loss of IP's protection (Figs. 3 and 4Go), and probably relates to the interindividually different drug delivery into the myocardium.

In the present study, a second p38 MAP kinase inhibitor—BIX-645—was used as an alternative to SB203580; BIX-645 indeed effectively inhibited the anisomycin-stimulated p38 MAP kinase activity in Rat1 cells in vitro (Fig. 1). The fact that both BIX-645 and SB203580 attenuated stimulated p38 MAP kinase activity in vitro and also attenuated infarct size reduction by IP in vivo highlights the importance of p38 MAP kinase in mediating preconditioning's protection; it appears unlikely that two structurally unrelated substances elicit the same unspecific effects.

4.2 p38 MAP kinase and infarct size
In isolated rat [13] and rabbit hearts [18], which had no increase in p38 MAP kinase phosphorylation, infarct size following ischemia–reperfusion remained unaltered with SB203580. In studies in rats [6,7], rabbits [8] and dogs [3], in which p38 MAP kinase phosphorylation and/or activity were increased during ischemia, SB202190 or SB203580 reduced infarct size.

SB203580 effectively blocked protection in cell models of ischemic preconditioning [11,15,16], and abolished the infarct size reduction of ischemic preconditioning in rats [10,13,17], rabbits [18] and dogs in vivo [3].

In the present study, p38 MAP kinase phosphorylation was also increased during the sustained ischemia. In contrast to the above studies, infarct size following ischemia–reperfusion was not significantly reduced in pigs receiving BIX-645 or SB203580, although infarct size tended to be somewhat lower with SB203580. It appears unlikely that such an effect of SB203580 was related to inhibition of p38 MAP kinase activity, since the inhibition of p38 MAP kinase activity was more pronounced with BIX-645 (see Rat1 cells in vitro). BIX-645 and SB203580, however, abolished the infarct size reduction by ischemic preconditioning.

To our best knowledge there is only one other study investigating the effect of p38 MAP kinase in ischemia–reperfusion in pigs [1] which came to almost the opposite conclusions, in that blockade of p38 MAP kinase reduced infarct size per se but did not influence the cardioprotection achieved by IP. In this particular study using an intramyocardial infusion of SB203580, the amount of infarcted tissue around an implanted needle was reduced by SB203580 in pigs undergoing ischemia–reperfusion as compared to an area around a second needle supplying buffer solution only. Local application of SB203580, however, did not interfere with the protection achieved by IP. Using such an experimental approach, the local drug concentration cannot be controlled so that a nonspecific effect of SB203580 cannot be excluded. Most importantly, SB203580 at high concentrations can activate ERK kinases [26,27], which in the same model have been proven to be involved in the cardioprotection achieved by IP [28]. Furthermore, the above model lacks the ability to quantify infarct size, since the area at risk cannot be determined.

The importance of timing of p38 MAP kinase inhibition for the loss of IP's protection and therefore its role as trigger or mediator of IP has been addressed in previous studies by modifying the time of application of p38 MAP kinase inhibitors; i.e. infusion of inhibitors prior to the preconditioning (trigger phase) or sustained (mediator phase) ischemia [3,7,13,17]. Using the same approach with infusion of SB203580 or BIX-645 prior to the preconditioning or the sustained ischemia, our data support the notion that the activation of p38 MAP kinase acts as a mediator rather than a trigger of IP's protection. However, the role of p38 MAP kinase as trigger or mediator of ischemic preconditioning remains controversial [3,7,13,17]. The conclusion that p38 MAP kinase is a mediator fits well with the current ideas on the signal cascade of ischemic preconditioning. Activated by endogenous triggers such as adenosine, bradykinin, opioids or free radicals, both protein kinase C and protein tyrosine kinases act as mediators in IP's protection [29–31]. At least some of the protein tyrosine kinases belong to the group of p38 MAP kinase kinases [4], and indeed p38 MAP kinase activity is inhibited by genistein, an inhibitor of protein tyrosine kinases [12].

4.3 Study limitations
In the present study, we did not assess the activity of the different p38 MAP kinase isoforms. p38 {alpha}- and β-MAP kinases are expressed within the heart [32] and appear to mediate opposing biological functions (for review see [4]); i.e. in neonatal rat cardiomyocytes, p38{alpha} mediates apoptosis whereas p38β is antiapoptotic [33], and the anti-ischemic effects of SB203580 are mediated by p38{alpha} only [34]. Thus, part of the controversies on the involvement of p38 MAP kinase in IP's protection might relate to differences in the involvement of p38 MAP kinase isoforms among different species and the selectivity and potency of inhibitors at a certain dose to block specific isoforms.

Only one concentration of SB203580 and BIX-645 was used in the present study. Since we did not acquire dose–response curves, it cannot completely be ruled out that different concentrations of the compounds would have altered the effect on infarct size development per se and IP's protection.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
The attenuation of the infarct-size-reducing effect of ischemic preconditioning depends on the effectiveness of blockade of p38 MAP kinase activity. p38 MAP kinase is a mediator of ischemic preconditioning in pigs.

Time for primary review 24 days.


   Acknowledgements
 
This study was supported by a grant from the DFG (He 1320/9-1 and 9-2). Boehringer Ingelheim (Ridgefield, CT, USA) provided BIX-645 and partial financial support.


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

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  2. Behrends M., Schulz R., Post H., et al. Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs. Am J Physiol Heart Circ Physiol (2000) 279:H1111–H1119.[Abstract/Free Full Text]
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  7. Schneider S., Chen W., Hou J., Steenbergen C., Murphy E. Inhibition of p38 MAPK {alpha}/β reduces ischemic injury and does not block protective effects of preconditioning. Am J Physiol Heart Circ Physiol (2001) 280:H499–H508.[Abstract/Free Full Text]
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