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1 Abteilung für Pathophysiologie, 2 Abteilung für Nieren- und Hochdruckkrankheiten, Zentrum für Innere Medizin des Universitätsklinikums Essen, 45122 Essen, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 ± 12.4 (SD)% (group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n = 14) reduced infarct size to 6.2 ± 5.1% (P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.
p38; extracellular signal-regulated kinase; c-Jun NH2-terminal kinase; Western blot; myocardial ischemia-reperfusion
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ONE OR SEVERAL SHORT EPISODES of myocardial ischemia and reperfusion delay the infarct development resulting from a subsequent, prolonged ischemic insult (17). The infarct size-limiting effect of ischemic preconditioning (IP) is impressive, but its underlying mechanisms are still incompletely understood. Current concepts of IP propose that activation of G protein-coupled receptors results in the activation of intracellular protein kinases that ultimately activate the end effector, possibly the ATP-sensitive K+ channel (14) or the cytoskeleton (13). The proposed sequence of intracellular events is that activated phospholipase C or D degrades phosphatidylinositol 4,5-bisphosphate, thus producing diacylglycerol, which then activates specific protein kinase C (PKC) isoforms. There is also experimental evidence for the activation of a protein tyrosine kinase (PTK) pathway, either parallel to (12, 29) or downstream of PKC (2), which may vary among species. Although the exact pathway is not yet established, activation of PKC can result in activation of the mitogen-activated protein kinase (MAPK) cascade (11). Each subfamily of the MAPK family, p38 (16, 18, 31), extracellular signal-regulated kinase (ERK) (21, 28), or c-Jun NH2-terminal kinase (JNK) (3, 20) has been suggested to play a role in the cardioprotection achieved by IP.
Evidence for the role of MAPK in IP is derived from investigations in which pharmacological inhibitors were used in isolated rabbit cardiomyocytes (1, 31), rat myoblasts (18), buffer-perfused rat hearts (16), and recently in two preliminary studies in pig (28) and dog hearts in situ (23). However, pharmacological inhibitors and activators may have nonspecific effects that could account for their effects on infarct size. A new approach to overcome the limitations of pharmacological inhibitors is the use of transfected cells with dominant negative mutants of the respective MAPK. However, this technique so far is restricted to cultured cardiomyocytes.
An alternative approach to elucidate the role of MAPK in IP is to assess variations of MAPK activation in the time course of a preconditioning protocol. In a preliminary study in anesthetized pigs, activities of p38, ERK, and JNK were increased after short episodes of ischemia or ischemia-reperfusion (3). Several studies measured MAPK activation after a preconditioning procedure and infarct size reduction by IP. However, these studies did not assess MAPK activation and infarct size reduction by IP in the same hearts but used either different models (31) or different subgroups within a given experimental protocol (15, 16, 18). Two recent studies by Ping et al. (20, 21) measured the activities of MAPK after IP in vivo using in-gel kinase assays: in conscious rabbits increased ERK (21) and JNK (20) activities were measured after an IP protocol that has been shown to induce late protection against myocardial stunning and infarction (6, 22). Again, in both studies, infarct size reduction by IP was not assessed in the same animals.
In anesthetized pigs, increased activity of JNK after intramyocardial anisomycin and okadaic acid infusion reduced infarct size, once more suggesting a role for MAPK in cardioprotection (4). To the best of our knowledge, no study so far has quantitatively correlated IP-induced MAPK activation to infarct size reduction within a given animal. However, such correlation appears mandatory to support a causal role for MAPK activation in the cardioprotection by IP, because the reduction of infarct size as well as the activation of all kinases associated with IP exhibit a high interindividual variability.
The present study tested, using an established porcine model of IP, the hypothesis that 1) IP activates MAPK and 2) their activation is correlated to the reduction of infarct size by IP.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
Experimental Model
A total of 39 Göttinger miniswine (20-40 kg) of both sexes were initially sedated using ketamine hydrochloride (1 g im) and then anesthetized with thiopental (Trapanal; 500 mg iv). Through a midline cervical incision, the trachea was intubated for connection to a respirator (Dräger, Lübeck, Germany). Anesthesia was then maintained using enflurane (1-1.5%) with an oxygen-nitrous oxide mixture (40-60%). Arterial blood gases were monitored frequently in the initial stages of the preparation until stable and then periodically throughout the study (Radiometer, Copenhagen, Denmark). Rectal temperature was monitored and kept between 37 and 38°C by use of a heated surgical table and drapes.Through the cervical incision, both common carotid arteries and internal jugular veins were isolated. The arteries were cannulated with two polyethylene catheters: one for the measurement of arterial pressure and the other to supply blood to the extracorporeal circuit. The jugular veins were cannulated for volume replacement with warmed 0.9% NaCl and for the return of blood to the animal from the coronary venous line.
A left lateral thoracotomy was performed in the fourth intercostal space, and the pericardium was opened. A micromanometer (P7, Konigsberg Instruments, Pasadena, CA) was placed in the left ventricle through the apex together with a saline-filled polyethylene catheter (used to calibrate the micromanometer in situ). Ultrasonic dimension gauges were implanted in the left ventricular (LV) myocardium to measure the thickness of the anterior and posterior (control) walls.
In five pigs, a suture was passed around the left anterior descending coronary artery (LAD) distal to its first diagonal branch to enable complete occlusion of the vessel. In all other pigs (n = 34), the proximal LAD was cannulated in <30 s and then perfused from an extracorporeal circuit. Before coronary cannulation, the pigs were anticoagulated with 20,000 IU heparin sodium; additional doses of 10,000 IU were given every 2 h. The system included a roller pump, windkessel, and a side port for the injection of radiolabeled microspheres. Coronary arterial pressure was measured from the sidearm of a polyethylene "T" connector (Cole-Parmer, Chicago, IL) used as catheter tip with an external transducer (Bell and Howell type 4-327I, Pasadena, CA). The completed preparation was allowed to stabilize for at least 30 min before baseline measurements were made.
Regional Myocardial Function
End diastole was defined as the point when the first derivative of LV pressure (LV dP/dt) started its rapid upstroke after crossing the zero line. Regional end systole was defined as the point of maximal wall thickness within 20 ms before peak negative LV dP/dt. Systolic wall thickening was calculated as the percentage of end-diastolic wall thickness.Regional Myocardial Blood Flow
Radiolabeled microspheres (15 µm diameter, 141Ce, 114In, 103Ru, 95Nb, and 46Sc; NEN-DuPont, Boston, MA) were injected into the coronary perfusion circuit (1-2 × 105 suspended in 1 ml saline) to determine the regional myocardial blood flow and its distribution throughout the LAD coronary artery perfusion bed (model 5912, Gammaszint BF 5300 Packard). This procedure for the determination of blood flow has been validated extensively (26). The tissue was divided into transmural thirds of approximately equal thickness, and blood flow to the subendocardium within the area at risk (AAR) is reported.Biopsies
Transmural drill biopsies (about 4-7 mg wt) were taken from the LAD perfusion bed. A 1.5-mm diameter bit was used in conjunction with a modified dental drill. The sample was rapidly expelled into a stainless steel mortar cooled with liquid nitrogen. The frozen biopsy was stored in liquid nitrogen until the subsequent analysis. Holes in the myocardium resulting from the biopsies were closed using a shallow purse-string suture.MAPK Assays
The tissue samples were weighed, diluted with sample buffer (1:60, 2% SDS, 50 mM dithiothreitol, 10% glycerol, 0.1% bromphenol blue, and 62.5 mM Tris at 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 and 120 mM NaCl at 25°C) containing 5% nonfat dry milk for 90 min and washed four times with TBS containing 0.05% Tween 20 (TTBS) for 10 min. The resulting blots were incubated for 2 h either with an antiserum recognizing total p38, ERK, or JNK or with an antiserum specific for the dually phosphorylated forms of p38 and JNK or the tyrosine phosphorylated form of ERK (New England Biolabs, Beverly, MA). The blots were then washed four times with 80 ml of washing buffer (150 mM NaCl, 0.1% Tween 20, and 50 mM Tris at pH 7.4 at 25°C) and were then incubated for 1 h with a secondary antibody (anti-rabbit immunoglobin linked to horseradish peroxidase: phototope-horseradish peroxidase detection kit; New England Biolabs). After four more washes with buffer, detection was performed by enhanced chemoluminescence. The resulting autoradiographs were analyzed by quantitative two-dimensional densitometry using commercially available software (Herolab, Wiesloch, Germany). The two-dimensional band intensity of phosphorylated MAPK was expressed relative to that of total MAPK, as assessed with the parallel blot prepared simultaneously. The ratio for the sample taken at baseline was set as 100%, and values for all other pairs of samples from the same experiment were then expressed as a percentage of the baseline.To test for the specificity of the antisera against the phosphorylated forms of MAPK, two samples each were pretreated with 4 IU of intestinal alkaline phosphatase (Glaxo Wellcome, Greenford, UK) for 30 min at 37°C before gel electrophoresis. Such pretreatment resulted in almost complete loss of the bands in the blots of phosphorylated p38, ERK, and JNK MAPK.
Morphology
At the end of each study, the heart was removed and sectioned from base to apex into five to seven transverse slices in a plane parallel to the atrioventricular groove. The tissue slices were immersed in a 0.09 M sodium phosphate buffer (pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC; Sigma-Aldrich Chemie, Deisenhofen, Germany) and 8% Dextran for 20 min at 37°C to identify infarcted tissue. The amount of infarcted tissue is expressed as a percentage of the LV AAR, as determined by the radioactive microspheres technique.Experimental Protocols
In five animals, the time course of the activation of MAPK during complete coronary occlusion was investigated. The distal LAD was occluded after collection of baseline samples, and biopsies were taken every 2 min from the central ischemic area, starting at 4 min until 12 min of coronary occlusion. Coronary occlusion resulted in an increase in p38 and ERK phosphorylation in each of the five pigs, reaching a maximum at 6-8 min (Fig. 1). Because of the limited number of experiments that were designed to determine the optimal time for biopsy sampling during the subsequent hypoperfusion protocol, no statistical analysis was performed.
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Coronary hypoperfusion. In group 1 (control group, n = 14), systemic hemodynamics, regional myocardial dimensions, and blood flow were measured at baseline and, thereafter, two tissue samples were taken. Blood flow to the LAD was then reduced to a level sufficient to induce regional akinesis. At 5 and 85 min ischemia, measurements of systemic hemodynamics, regional myocardial dimensions, and blood flow were repeated. Biopsies were taken at 8 and 88 min ischemia after the respective functional and blood flow measurements. After 90 min ischemia, the myocardium was reperfused for 120 min to facilitate TTC staining.
In group 2 (IP group, n = 14), after baseline measurements and sampling of two tissue samples were done, LAD blood flow was decreased for 10 min to a level sufficient to induce regional akinesis. At the end of the preconditioning ischemia, systemic hemodynamics, regional dimensions, and blood flow were measured again and further tissue samples were taken. At the end of the subsequent 15-min reperfusion period at a constant mean coronary arterial pressure, measurements and tissue sampling were repeated. Thereafter, the protocol was identical to that of group 1. Additionally, six pigs served as a time-matched control group to ensure stability of the model and to exclude MAPK phosphorylation in the absence of ischemia (group 3). Biopsies were taken at 0, 10, 25, 33, and 113 min of normoperfusion, i.e., at the times corresponding to those of the IP protocol.Data Analysis and Statistics
Systemic hemodynamic and regional dimension data were recorded on an eight-channel recorder (Gould MK 200A, Cleveland, OH) and stored directly to the hard disk of an AT-type computer. Hemodynamic and functional parameters were digitized and recorded over a 20-s period during each microsphere injection (approximately 33 consecutive beats over at least 2 complete respiratory cycles) using CORDAT II software (27). Parameters reported are heart rate, LV peak pressure, maximum LV dP/dt (LV dP/dtmax), mean coronary arterial pressure, mean coronary blood flow, and anterior systolic wall thickening. Calculation of these parameters was done on a beat-to-beat basis, and data were then averaged.Statistical analysis was performed using SYSTAT software (Urbana, IL).
Data on hemodynamics and regional myocardial function are reported as
mean values ± SD and were compared using a two-way analysis of
variance, accounting for the different times throughout the protocol
and the three groups of pigs. When significant differences were
detected, individual mean values were compared using Bonferroni post
hoc tests. AAR, infarct size, and subendocardial blood flow at 5 min
ischemia were compared between groups 1 and 2 using an unpaired t-test. Linear regression analyses between
subendocardial blood flow at 5 min ischemia in the LV AAR and infarct
size (expressed as a percentage of the LV AAR) were compared by
analysis of covariance (ANCOVA). Infarct size reduction by IP
was calculated for each pig of group 2 as the difference
between the actually measured infarct size and the expected infarct
size, as determined from the regression line of group 1 (y = 
380x + 38, n = 14, r = 0.79) and the actually measured
subendocardial blood flow measured at 5 min ischemia in group
2. The infarct size reduction is expressed as the percentage of
expected infarct size. Data on phosphorylation of p38, ERK, p46 JNK,
and p54 JNK are reported as median values, and the 95% confidence
interval is given. Comparisons between groups and different time points
were performed using the two-sample Kolmogorov-Smirnov test. A
P value <0.05 was accepted as indicating a significant difference.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Systemic Hemodynamics and Regional Myocardial Function
Reduction of coronary blood flow reduced mean coronary arterial pressure and anterior systolic wall thickening in groups 1 and 2 to a similar extent (Table 1). In group 3, systemic hemodynamics and regional dimensions did not change.
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AAR and Infarct Size
The percentage of AAR was similar in both groups 1 and 2 (group 1: 42.8 ± 5.6%, group 2: 41.2 ± 8.1%). Infarct size was smaller in group 2 (6.2 ± 5.1 vs. 18.3 ± 12.4%, P < 0.05), although subendocardial blood flow tended to be lower in group 2 (0.04 ± 0.02 ml · min
1 · g1) than in
group 1 (0.05 ± 0.03 ml · min1 · g1; not
significant). Also, infarct size for any given subendocardial blood
flow at 5 min ischemia was significantly reduced in group 2 compared with group 1 (regression lines: group 1,
y = 380x + 38, r = 0.79; group 2, y = 74x + 9, r = 0.31; P < 0.05, ANCOVA; Fig.
2).
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p38 Phosphorylation
Phosphorylated p38 was quantified as a percentage of total p38 determined from a parallel plot of the same sample. Specificity of antibody detection was ascertained as comigration with authentic standard. In group 1, p38 phosphorylation increased significantly within 8 min ischemia and remained elevated at 88 min ischemia (Table 2, Fig. 3), although p38 phosphorylation did not increase in all pigs above baseline at 8 and 88 min ischemia. In group 2, 10 min IP increased p38 phosphorylation, followed by a modest decrease in phosphorylation during the subsequent 15 min reperfusion. During the sustained ischemia, p38 phosphorylation was still increased on the average to a similar extent as in group 1, but again not in all animals. p38 phosphorylation was not increased during the time course of group 3.
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ERK Phosphorylation
Quantification and specificity checks were performed as described above for p38. Within 8 min ischemia, ERK phosphorylation increased significantly in group 1 but decreased in the further course of ischemia (Table 2 and Fig. 3). ERK phosphorylation did not increase in all pigs above baseline at 8 and 88 min ischemia. In group 2, 10 min IP increased ERK phosphorylation significantly. Within the 15-min reperfusion period after the IP, ERK phosphorylation decreased but it increased again with the start of the sustained ischemia. The increase and subsequent slight decrease in ERK phosphorylation during the sustained ischemia in group 2 was similar to that in group 1. Also in group 2, ERK phosphorylation was not increased in all pigs above baseline at 8 and 88 min ischemia. ERK phosphorylation was not increased during the time of the time-matched control study.JNK Phosphorylation
Quantification and specificity checks were performed as described above for p38. The signal for phosphorylated JNK found in our Western blots was of lower quality than that of the p38 and ERK blots. Therefore, JNK phosphorylation could only reliably be measured in seven experiments of group 1, seven experiments of group 2, and five (p46 JNK) and six (p54 JNK) experiments of group 3. In group 1, p54 JNK phosphorylation increased significantly, although inconsistently within 8 min ischemia, and was still elevated at 88 min ischemia (Table 2), whereas the increase in p46 phosphorylation occurred during late ischemia. In group 2, 10 min IP increased p54 JNK but not p46 JNK phosphorylation. p46 JNK and p54 JNK phosphorylation were both increased during the 15-min reperfusion period after the IP. During the sustained ischemia, p46 JNK and p54 JNK phosphorylation were similar in groups 1 and 2. Neither p46 JNK nor p54 JNK phosphorylation were increased during the time course of the time-matched control study.No correlation was found between calculated infarct size reduction and
the increase in phosphorylation of p38, ERK, or JNK at the end of the
IP, at the end of preconditioning reperfusion (RepIP), or at 8 min
sustained ischemia (Fig. 4). There was
also no correlation between ischemic blood flow during the sustained ischemia and the phosphorylation of p38, ERK, p46 JNK, or p54 JNK in
the two groups (Fig. 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, the infarct size reduction achieved by IP did not correlate to the phosphorylation of p38, ERK, and p46 JNK and p54 JNK MAPK. Also, control and preconditioned hearts did not differ in the extent of MAPK phosphorylation during the sustained ischemia.
Critique of Methods
The present experiments were performed in pigs because infarct development in this species, as a result of the sparsity of the innate collateral circulation, most closely resembles that observed in humans. Moreover, pigs have a heart large enough to permit multiple, sequential biopsies.Cannulation of the LAD permitted controlled reduction of coronary blood flow. Cannulation of the LAD resulted in a large AAR (on the average 42% of the LV mass) and a small infarct size when expressed as a percentage of the AAR (18% in group 1). However, infarct size expressed as the percentage of LV in the present study averaged 7.8 ± 5.4% in group 1 and was thus comparable to that in a previous study using pigs with a total occlusion of only one distal branch of the LAD (25). Low-flow hypoperfusion in conjunction with the measurement of regional myocardial blood flow using microspheres permitted us to establish the relationship between infarct size and ischemic subendocardial blood flow, which is a more sensitive end point of cardiomyocyte protection than infarct size per se. Furthermore, with the low-flow hypoperfusion approach, a potential correlation between the severity of ischemia and the activation of MAPK could be analyzed.
A potential limitation of the present study, using the heparinized pig preparation with sequential biopsies, is the small sample size of a given biopsy. Nevertheless, the Western blots for p38 and ERK were of good quality (Figs. 1 and 3). The Western blots for JNK revealed consistently weaker bands for the phosphorylated p46 JNK isoform but were of acceptable quality that allowed quantitative densitometry (Fig. 3).
MAPK activation can be assessed by immunoblotting with epitope-specific antiphosphotyrosine-antibodies to assess MAPK phosphorylation (present study). Alternatively, MAPK activity can be assessed by phosphorylation of specific substrates. It is possible that MAPK phosphorylation is not a fully quantitative indicator of MAPK activity, whereas enzyme substrates may not be fully specific for a certain MAPK. Therefore, phosphorylation and enzyme activity assays should ideally be combined, but this again was not possible in the present study due to the small sample size of the biopsies.
MAPK and Ischemia-Reperfusion
Cellular stresses, including ischemia-reperfusion, activate p38 and JNK (5, 8, 10), whereas the activation of ERK by ischemia-reperfusion is still controversial (3, 5, 19, 21, 28). In the present study, the phosphorylation of all MAPK was increased by ischemia. However, a high interindividual variability in the activation of MAPK was observed (in some pigs activation of MAPK was even lacking) and also a correlation of activation to ischemic blood flow was lacking.The role of MAPK in the signal transduction of IP was addressed in several studies. Inhibition of p38 using SB-203580 in rat heart myoblasts (18), rabbit cardiomyocytes (1, 31), and in preliminary experiments in dog hearts in situ (23) abolished the protection achieved by IP. Inhibition of ERK using the MEK inhibitor PD-98059 revealed conflicting results; whereas PD-98059 did not abolish the protective effects of IP in rat heart myoblasts (18), it completely abolished the protective effects of IP in preliminary studies in porcine hearts in situ (28). However, apart from the inhibition of p38 and MEK by SB-203580 and PD-98059, respectively, both substances inhibit cyclooxygenase-1 and -2, and SB-203580 also inhibits thromboxane synthase (7) and JNK at higher concentrations (9). Therefore, the specificity of these inhibitors is somewhat questionable.
The data on MAPK activation in IP are not conclusive. Several investigations reported an increased (3, 16, 18), a transient activation (20), or even a lack of (1) activation of p38 after IP. During the sustained ischemia, both higher (1, 31) and lower (18) p38 activation have been reported in preconditioned compared with control myocardium. ERK phosphorylation was found to be increased (3, 21) or unchanged (18) after IP. An increased activity of p46 JNK and p55 JNK after 10 min ischemia and 30 min reperfusion has been described in a preliminary study using a porcine model of regional ischemia (3). In conscious rabbits, p46 JNK was increased with IP only in the cytosolic fraction, whereas p54 JNK increased only in the nuclear fraction (20). The consequences of such increased or attenuated MAPK activation remained unaddressed in most studies. The present study was the first to attempt to quantitatively correlate the infarct size reduction by IP with the activation of MAPK in the same animal. When looking at average data, phosphorylation of p38, ERK, p46 JNK, and p54 JNK was indeed increased during IP; however, in some preconditioned animals, MAPK phosphorylation did not increase at all. There was also no correlation between the increase of MAPK phosphorylation during IP and protection by IP. Furthermore, during the sustained ischemia there was no difference in MAPK phosphorylation between preconditioned and control hearts, and once again no correlation between MAPK phosphorylation during the sustained ischemia and protection by IP existed. The present data provide, therefore, no evidence for a causal role of MAPK phosphorylation in IP.
However, the present findings certainly do not rule out a role of MAPK
activation in IP. Multiple isoforms of p38 have been identified; some
of them may contribute to cardioprotection, whereas activation of other
isoenzymes could be detrimental (30). The absence of a
consistent increase in total p38 phosphorylation does not, therefore,
completely rule out the possibility that a specific isoform of p38 may
be activated by ischemia and subsequently be responsible for the
mediation of cardioprotection achieved by IP. Evidence for an
isoform-specific action is derived from a preliminary study, which
found an inhibition of p38
in a surrogate model of IP
(24). However, isoform-specific antibodies or
isoform-specific blockers to test such hypothesis in vivo are currently
unavailable. The limited power of biochemical assays to detect the
activation of specific isoforms and the limited specificity of
activator and inhibitor drugs may, therefore, explain some of the
observed differences. It is also tempting to explain the conflicting
results on MAPK activation with differences in signal transduction
between species and models, whereas IP is a universal phenomenon in all species studied so far. In conclusion, currently it is not possible to
decide whether activation of MAPK plays a causal role in IP or just
represents an epiphenomenon.
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ACKNOWLEDGEMENTS |
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We thank Petra Gres for excellent technical support.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (He 1320/9-1 and 9-2).
Address for reprint requests and other correspondence: G. Heusch, Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstrasse 55, 45122 Essen, Federal Republic of Germany (E-mail: gerd.heusch{at}uni-essen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 February 2000; accepted in final form 5 April 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) and at 8 min
sustained ischemia (I8, 
) in group 2.
