INTRODUCTION

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MITOGEN-ACTIVATED PROTEIN (MAP) kinases and MAP kinase (MAPK)-activated protein (MAPKAP) kinase 2 have recently been shown to play a role in mediating intracellular signal transduction events associated with ischemia and reperfusion (5, 21). In mammalian cells, the mitogenic signal is transmitted from the cytoplasm into the nucleus by the nuclear translocation of p42/p44^MAPK isoforms [extracellular signal-regulated kinases (ERK1 and ERK2)] (17). Although the kinase cascades have been well characterized for prokaryotic systems, their precise role in mammalian systems is far from clear. Three distinct mammalian MAP kinases, each with apparently unique signaling pathways, have been identified: the ERK group (p42/p44^MAPK), the stress-activated protein kinase (SAPK) group [also known as c-Jun NH₂-terminal kinases (JNK)], and the p38 MAP kinase (a mammalian homolog of HOG1).

The newly discovered p38 MAP kinase (13) has a very specific cellular target: MAPKAP kinase 2 (12). Unlike p42/p44^MAPK, which are readily activated by growth signals via a Ras-dependent signal transduction pathway (9), the activation of JNKs and p38^MAPK is potentiated by diverse stresses and proinflammatory cytokines (6). However, the JNK and p38^MAPK cascades appear to be involved in distinct cellular functions, because they possess different cellular targets and locate on different signaling pathways. For example, JNK kinases activate c-Jun, whereas p38^MAPK stimulates MAPKAP kinase 2 (25, 27). On activation by upstream kinases, p38 MAP kinase phosphorylates and activates MAPKAP kinase 2, which in turn leads to the phosphorylation of heat shock protein (HSP) 27 (8). The precise mechanism of p38 MAP kinase activation is not known, but its activation appears to be regulated by dual phosphorylation on threonine and tyrosine within the motif Thr-Gly-Tyr (26). Recently, the nucleus has been shown to be a target for the signal transduction of p38 MAP kinases (27).

The finding that stress induced by repeated periods of brief ischemia and reperfusion renders the heart tolerant to subsequent lethal ischemia has stimulated general interest among scientists investigating the intracellular signaling pathway that leads to myocardial adaptation to stress. Stress mediated by repeated ischemia and reperfusion has been shown to trigger a tyrosine kinase-dependent signaling pathway leading to the activation of MAP kinases and MAPKAP kinase 2 (5, 21). More recently, we have shown that both oxidative and heat stresses rapidly activated p38^MAPK and MAPKAP kinase 2, leading to the phosphorylation of HSP 27 (34). To further define the role of MAPKAP kinase 2 in the ischemia-reperfusion-mediated stress signaling pathway, we used a specific blocker for p38^MAPK before ischemia and reperfusion. The results of our study demonstrated that ischemia-reperfusion resulted in the translocation of p38^MAPK into the cytoplasm and that the beneficial effects of myocardial adaptation to stress by repeated ischemia and reperfusion were abolished by inhibiting p38^MAPK with simultaneous inhibition of MAPKAP kinase 2, suggesting a role of the p38^MAPK-MAPKAP kinase 2 signaling pathway in myocardial adaptation to stress.

	MATERIALS AND METHODS

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Isolated perfused heart preparation. All animals received care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health [DHEW Publication No. (NIH) 80-23, Revised 1978]. Fifty-four Sprague Dawley rats weighing ~300 g were anesthetized with pentobarbital sodium (80 mg/kg ip). After intravenous administration of heparin (500 IU/kg), the chests were opened and the hearts were rapidly excised and mounted on a nonrecirculating Langendorff perfusion apparatus (14). Retrograde perfusion was established at a pressure of 100 cmH₂O with an oxygenated normothermic Krebs-Henseleit bicarbonate (KHB) buffer with the following ion concentrations (in mM): 118.0 NaCl, 24.0 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.7 CaCl₂, and 10.0 glucose. The KHB buffer had been previously equilibrated with 95% O₂-5% CO₂, pH 7.4 at 37°C. The hearts were randomly divided into five groups. In group I, isolated hearts were perfused with KHB buffer for 1 h, followed by 30 min of ischemia and 120 min of reperfusion. In group II, isolated perfused rat hearts were subjected to ischemic stress adaptation in the form of repeated ischemia and reperfusion by the induction of global ischemia for 5 min, followed by 10 min of reperfusion, with repetition of the process four times as described previously (3). Group III treatment was the same as that for group II but was followed by 30 min of ischemia and 120 min of reperfusion. In group IV, isolated hearts were preperfused with 5 µM SB-203580 for 15 min, followed by ischemic stress adaptation and then 30 min of ischemia and 120 min of reperfusion, as described for group III. Group V treatment was the same as that for group IV except that SB-203580 was replaced with 100 µM genistein. A schematic diagram of the protocol is shown in Fig. 1. Experiments were terminated at various points, and hearts were processed to evaluate nuclear translocation of p38 MAP kinase by immunohistochemistry and confocal microscopy. Western blot analysis was used to evaluate p38 MAP kinase.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Experimental protocol for groups I-V. S, stabilization; 15 P, 15-min perfusion with Krebs-Henseleit bicarbonate (KHB) buffer; 30 I, 30-min ischemia (I); 120 R, 120-min reperfusion (R); 15 SB, 15-min preperfusion in presence of SB-203580; 15 G, 15-min preperfusion in presence of genistein. Preconditioning consisted of 4 cycles of ischemia-reperfusion (I/R).

Evaluation of myocardial functions. To evaluate myocardial performance, the Langendorff preparation was switched to the working mode after the preconditioning protocol (10). Control experiments were performed without subjecting the hearts to preconditioning. Aortic flow was measured with a calibrated rotameter. Coronary flow rate was measured via a timed collection of the coronary perfusate that dripped from the heart. After a 10-min aerobic perfusion of the heart, the aortic inflow line was clamped at a point close to the origin of the aortic cannula. Reperfusion was initiated by unclamping the aortic line. Before ischemia and during reperfusion, heart rate and coronary and aortic flow rates were registered. Left ventricular developed pressure (LVDP), defined as the difference between LV systolic and end-diastolic pressures, and the first derivative of LVDP (LV dP/dt), were also recorded.

Western blot analysis of p38 MAP kinase. It has been well documented that three groups of MAP kinases are activated by distinct upstream kinases through phosphorylation of both threonine and tyrosine in a regulatory Thr-Xaa-Tyr site found on each kinase and that protein phosphorylation of these kinases reflects an accurate indicator of their activation. To quantify the protein phosphorylation of p38 MAP kinase, heart tissues were homogenized and suspended (5 mg/ml) in sample buffer (10 mM HEPES, pH 7.3, 11.5% sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM diisopropyl fluorophosphate, 0.7 mg/ml pepstatin A, 10 mg/ml leupeptin, and 2 mg/ml aprotinin). Proteins were then solubilized with the addition of the same amount of 2× Laemmli solution [9% SDS (wt/vol), 6% -mercaptoethanol (vol/vol), 10% glycerol (vol/vol), and a trace amount of bromphenol blue dye in 0.196 M Tris · HCl (pH 6.7)]. The cellular proteins (50-µl samples) were electrophoresed through 10% SDS-PAGE and then transferred to Immobilon-P membranes (Millipore) using a semidry transfer system (Bio-Rad). Prestained protein standards (Bio-Rad) were run in each gel. The blots were blocked in Tris-buffered saline/Tween-20 (20 mM Tris base, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) supplemented with 5% BSA for 1 h, incubated with 1:1,000 diluted primary rabbit antibodies specifically against Tyr-182-phosphorylated p38 MAP kinase (NEB) for 2 h, and then incubated with 1:10,000 diluted secondary antibodies of horseradish peroxidase-conjugated anti-rabbit IgG (Boehringer Mannheim) for 1 h at room temperature. After three washes of 5 min each, blots were treated with enhanced chemiluminescence (Amersham) reagents and the phosphorylated MAP kinases were detected by autoradiography for variable lengths of time (15 s to 3 min) with Kodak X-Omat film.

Protein phosphorylation assay. To evaluate enzymatic activities of tissue MAPKAP kinase 2, heart tissues were homogenized and suspended in sample buffer. After centrifugation at 5,000 g for 10 min, the kinase activity in the resulting supernatant was examined with an in vitro protein phosphorylation assay using commercially available recombinant human HSP 27 (rHSP 27; StressGen Biotechnologies) as the specific substrate for MAPKAP kinase 2 (34). The reaction was initiated by the addition of an equal volume (30 µl) of freshly prepared phosphorylation reaction mixture containing 30 mM HEPES (pH 7.3), 20 mM MgCl₂, 2 mM EGTA, 10 µM sodium orthovanadate, 5 µM okadaic acid, 4 mM dithiothreitol, 30 µM H-7, 0.4 mM [-³²P]ATP (10⁵ cpm/pmol), and 0.5 µg rHSP 27 to 30 µl of tissue supernatant. The in vitro phosphorylation reaction was carried out at 30°C for 10 min and stopped by the addition of 60 µl 2× Laemmli solution. Proteins were then separated on 11% SDS-PAGE, and the induced protein phosphorylation of rHSP 27 was detected by autoradiography.

Immunofluorescence microscopy. Myocellular localization of p38 MAP kinase and its translocation after cyclic ischemia-reperfusion was detected using immunofluorescence staining. In these experiments hearts were harvested and perfused as described in Isolated perfused heart preparations. Specimens were obtained at baseline, after ischemia, and after preconditioning followed by ischemia-reperfusion protocol. Ventricular tissue was excised from beating hearts, blotted, embedded in OCT compound, rapidly frozen in dry ice-cooled 2-methylbutane, and stored at 70°C until used. Transverse 6-µm cryosections were prepared with a cryostat (2800 Frigocut E, Reichert-Jung) and collected on poly-L-lysine-coated slides. Sections were fixed for 10 min in a 70% acetone-30% methanol mixture at 20°C and air dried. After three washes in PBS (3 min each), normal goat serum (10% in PBS) was applied as a blocking agent and briefly rinsed with PBS. Sections from each experimental group were then incubated with anti-p38 antibody at 1:50 dilution (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at room temperature. After three washes in PBS (3 min each), the primary antibody was labeled with Cy3-conjugated goat anti-rabbit IgG (Jackson Laboratory) and the nuclei stained with bisbenzimide (0.0001% wt/vol) for 1 h. Excess secondary antibody was removed by three washes with PBS (3 min each) followed by one wash for 3 min with 0.1% Triton X-100 in PBS. Slides were mounted with a glycerol-based antiquenching medium (o-phenylenediamine-HCl) and stored at 4°C. Sections were viewed with a Leica microscope equipped with fluorescence optics and photographed with a Spectronix charge-coupled device camera.

Statistical analysis. For statistical analysis, a two-way ANOVA followed by Scheffé's test was first carried out using the Primer computer program (McGraw-Hill) to test for any differences between groups. If differences were established, the values were compared using Student's t-test for paired data. The values are expressed as means ± SE. The results were considered significant for P < 0.05.

	RESULTS

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Myocardial adaptation to ischemic stress by repeated ischemia and reperfusion and its inhibition by genistein and SB-203580. As expected, hearts subjected to repeated ischemia and reperfusion demonstrated significantly improved postischemic ventricular recovery compared with control hearts. Neither genistein nor SB-203580 altered heart rate during ischemia and reperfusion (Table 1). Aortic flow was drastically reduced during reperfusion in both control and adapted groups. However, the degree of reduction was significantly smaller in the adapted group. Coronary flow was not affected by ischemic adaptation (Table 1). For both groups, LVDP and LV dP/dt were lower during reperfusion compared with baseline, but these values were significantly higher in the adapted hearts. Ischemic adaptation reduced the myocardial infarct size, which was reversed by either genistein or SB-203580.

Stimulation of p38 MAP kinase during ischemic stress adaptation and its inhibition by SB-203580 or genistein. To understand the regulation of p38 MAP kinase in myocardium, kinase activity was evaluated by using Western blot to detect the induced protein phosphorylation as described in MATERIALS AND METHODS. As shown in Fig. 2, the antibodies specific against the tyrosine-phosphorylated p38 MAP kinase detected two proteins with molecular masses of ~38 kDa. The antibodies against p38 MAP kinase also probed two protein bands in rat myocardium (unpublished observation). Western blot analysis showed that tissue p38 MAP kinase became phosphorylated and, hence, activated by preconditioning (Fig. 2, lane 2) and/or ischemic stress (Fig. 2, lane 3). To regulate tissue p38 MAP kinase activity, SB-203580, a specific kinase inhibitor for p38 MAP kinase with no apparent effect on other protein kinases, was used as described in MATERIALS AND METHODS. The inhibitors genistein and SB-203580 blocked the induced protein phosphorylation of p38 MAP kinase in response to the stimulus (ischemia) (Fig. 2, lanes 4 and 5) and completely inhibited the p38 MAP kinase activation to stimulate MAPKAP kinase 2 (Fig. 3, lanes 4 and 5). Basal phosphorylation of p38 MAP kinase was not blocked. The reason for the inhibition of p38 MAP kinase phosphorylation by SB-203580 was not clear. These data indicate that p38 MAP kinase signal pathway is activated in response to preconditioning and ischemic stress in isolated rat hearts.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Phosphorylation and activation of myocardial p38 mitogen-activated protein (MAP) kinase. Tissue proteins were prepared from hearts at end of each experiment, separated on 10% SDS-PAGE, and transferred to Immobilon-P membranes. Phosphorylated p38 MAP kinase (phos. p38; right) was detected by Western blotting using specific antibodies and enhanced chemiluminescence method, as described in MATERIALS AND METHODS. Molecular mass standards (kDa) are shown at left. Results are representative of 3 similar experiments per group. Lane 1: control (group I). Lane 2: preconditioned (group II). Lane 3: preconditioned followed by 30-min ischemia and 120-min reperfusion (group III). Lane 4: 15-min preperfusion in presence of SB-203580 followed by preconditioning, 30-min ischemia, and 120-min reperfusion (group IV). Lane 5: 15-min preperfusion in presence of genistein followed by preconditioning, 30-min ischemia, and 120-min reperfusion (group V).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Enzymatic activity assay of myocardial MAP kinase-activated protein (MAPKAP) kinase 2. MAPKAP kinase 2 activity was evaluated using an in vitro protein phosphorylation assay with recombinant heat shock protein (HSP) 27 as specific substrate, as described in MATERIALS AND METHODS. Phosphorylated proteins were separated on 11% SDS-PAGE, and autoradiograph is shown. Molecular mass standards (kDa) are shown at left. Position of phosphorylated HSP 27 is indicated at right. Arrow at right indicates a myocardial protein that was phosphorylated in vitro and used as an internal marker to monitor equal protein amounts in each reaction. Results are representative of 3 similar experiments per group. Lane 1: control (group I). Lane 2: preconditioned (group II). Lane 3: preconditioned followed by 30-min ischemia and 120-min reperfusion (group III). Lane 4: 15-min preperfusion in presence of SB-203580 followed by preconditioning, 30-min ischemia, and 120-min reperfusion (group IV). Lane 5: 15-min preperfusion in presence of genistein followed by preconditioning, 30-min ischemia, and 120-min reperfusion (group V).

Activation and regulation of MAPKAP kinase 2 in myocardium. MAPKAP kinase 2 has been demonstrated to be a specific cellular substrate for p38 MAP kinase and is responsible for the induced protein phosphorylation of small heat shock proteins (HSP 25 and HSP 27). Enzymatic activity of tissue MAPKAP kinase 2 was examined with an in vitro protein phosphorylation assay with the use of rHSP 27 as a specific substrate as described in MATERIALS AND METHODS, and the resultant autoradiograph is shown in Fig. 3. Preconditioning and/or ischemic stress of isolated rat hearts induced the tissue MAPKAP kinase 2 activation (Fig. 3, lanes 2 and 3), which resulted from the p38 MAP kinase activation (Fig. 2, lanes 2 and 3). The induced activation of MAPKAP kinase 2 was completely inhibited by genistein or SB-203580 (Fig. 3, lanes 4 and 5). These results indicate that preconditioning and/or ischemic stress stimulate myocardial MAPKAP kinase 2 and that the induced activation of MAPKAP kinase 2 can be blocked by inhibition of tissue p38 MAP kinase with genistein or SB-203580.

Translocation of p38 MAP kinase by ischemia-reperfusion. p38 MAP kinase distribution was not different between hearts subjected to 5 min of ischemia and normally perfused control hearts (not shown). p38 MAP kinase staining in myocytes was diffuse in the cytoplasm and bright in perinuclear regions (Fig. 4A, ×40 magnification). Some interstitial cells appear to contain p38 MAP kinase. After preconditioning with four cycles of ischemia and reperfusion (Fig. 4B, ×63 magnification), there is increased p38 staining within the nuclei of both myocytes and certain interstitial cells (histiocytes). Within myocytes, a striated cytoplasmic stain pattern begins to develop. An intercellular junction appears as a bright arc (Fig. 4B, top).

View larger version (140K): [in this window] [in a new window]   Fig. 4.   Immunofluorescence microscopy demonstrating myocellular localization of p38 MAP kinase. Hearts were preconditioned with 4 cyclic episodes of I/R, followed by 30 min of ischemia and up to 120 min of reperfusion, as described in MATERIALS AND METHODS. A: representative sections from 2 different hearts frozen immediately after 5-min ischemia. Diffused cytoplasmic stain and bright perinuclear stain appear in myocytes (nuclei appear blue, whereas p38 MAP kinase appears red). Some interstitial cells show same stain pattern. B: representative sections from 2 different hearts frozen immediately after preconditioning protocol. Nuclear stain in myocytes is not symmetrical. There is an increase of stain in intranuclear region. Cytoplasmic stain shows striated pattern. Interstitial cells show very intense stain. C: representative section from 2 different hearts frozen immediately after preconditioning followed by 30-min ischemia. Perinuclear and intranuclear stains are still very intense but have started to diffuse outward. Cytoplasmic striations are more defined than in A and B. D: representative section from 2 different hearts frozen immediately after preconditioning followed by 30-min ischemia and 120-min reperfusion. Nuclear stain has decreased, whereas cytoplasmic striations are still very strong.

	DISCUSSION

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The results of our study documented that ischemic preconditioning potentiates a tyrosine kinase-dependent signal transduction resulting in the phosphorylation of p38 MAP kinase. Preconditioning caused increased phosphorylation of p38 MAP kinase, which was blocked by inhibiting tyrosine kinase. p38 MAP kinase appears to be localized in the perinuclear region. It moves to the nucleus after preconditioning stimulus and translocates to cytoplasm after ischemia-reperfusion. Increased p38 MAP kinase phosphorylation is associated with the enhancement of MAPKAP kinase 2 activity, which was inhibited by both genistein and SB-203580.

Although the activation of p38 MAP kinase, like other members of the MAP kinase family, requires dual phosphorylation, the substrate specificity of p38 MAP kinase is quite different from that of JNK or ERK subgroups of MAP kinases. Thus, unlike other MAP kinases, p38 MAP kinase activates the MAPKAP kinase 2 (34). It is speculated that p38 MAP kinase signaling has a distinct function in the cell, and this was supported by the recent findings that proinflammatory cytokines lead to the activation of p38 MAP kinase, which in turn results in the phosphorylation of HSP 27 (16, 28). Recently, two MAP kinase kinases (MKK3 and MKK4) have been discovered, the former being specific for p38 MAP kinase, whereas the latter can activate both p38 and JNK MAP kinases (7).

Recent years have witnessed the development of a novel concept for myocardial preservation based on the fact that the enhancement of the endogenous cellular defense system provides each cell with new protein synthesis and, thereby, the means to protect itself when it is more susceptible. Using this concept, a number of investigators have shown that stress induced by repeated nonlethal ischemia and reperfusion can delay the onset of further irreversible injury (24) or even reduce the subsequent postischemic ventricular dysfunction (32) and incidence of arrhythmias (15). Our laboratory has demonstrated that repeated ischemia distinguished from a single ischemic insult can reduce subsequent ischemia-reperfusion injury (11) and postischemic ventricular fibrillation (31). Such myocardial preservation by repeated short-term reversible ischemia led to the development of the concept of stress adaptation. With the use of this concept, a number of studies from different laboratories developed approaches to simulate ischemic preconditioning. For example, heat shock was used to adapt the heart against ischemic injury (18, 19). In addition, oxidative stress was also used to induce myocardial adaptation to ischemia (20, 22). Interestingly, irrespective of the type of approach used to induce stress in all the studies, all the stresses including ischemic stress have been found to enhance the defense system of the heart as evidenced by increased heat shock proteins and antioxidant enzymes (4).

Much evidence exists to support the notion that mammalian hearts can be adapted to stress to better tolerate subsequent lethal ischemia, but the precise signal transduction pathway remains highly speculative. Recently, a role of protein kinase C in ischemic preconditioning has been suggested. Short-term ischemia as well as ischemia followed by reperfusion was previously shown to translocate and activate protein kinase C (23). Furthermore, both ₁-receptor stimulation and Ca²⁺ can translocate and activate protein kinase C (2, 30). Given the fact that both ₁-receptor activation and intracellular Ca²⁺ overloading are the manifestations of ischemia-reperfusion injury, it was not surprising when ischemic preconditioning consisting of repeated ischemia and reperfusion was also found to translocate and activate protein kinase C. Recent findings from our laboratory that MAP kinase-activated protein kinase (MAPKAP kinase 2) is present in abundance in the heart, compared with significantly lower levels of protein kinase C, led us to speculate on the importance of MAPKAP kinase 2 in stress adaptation (5). This finding receives further support from the evidence that rapid activation of MAPKAP kinase 2 occurs by stress, including heat stress, oxidative stress, and ischemia-reperfusion (34). Most importantly, it is MAPKAP kinase 2, and not protein kinase C, that can phosphorylate small heat shock proteins (HSP 25 and 27), which are also activated by stress adaptation (5, 34). To the best of our knowledge, this study provides the first evidence that ischemic adaptation results in a tyrosine kinase signaling that is directly linked with p38 MAP kinase and MAPKAP kinase 2.

First isolated as an in vitro substrate for ERKs, MAPKAP kinase 2, a serine/threonine kinase, has been shown to be phosphorylated and activated by MAP kinases both in vivo and in vitro (29). It is a 370-amino acid protein containing a highly conserved catalytic domain of proline-rich NH₂ termini and a COOH-terminal region containing a MAP kinase phosphorylation site at Thr-334 (35). The physiological functions of MAPKAP kinase 2 remain largely unknown. This kinase is largely expressed in myocardium and skeletal muscle (34). A number of recent studies have indicated that MAPKAP kinase 2 may play a significant role in stress-activated signal transduction potentiated by diverse environmental stresses and proinflammatory cytokines as well as a variety of mitogens (12, 28). In addition, phorbol ester and heat shock as well as oxidative stress can also activate MAPKAP kinase 2 in cardiac myoblast cells (34).

The locations of endogenous p38 MAP kinase in mammalian hearts have not been previously examined. Our studies on ventricular tissue indicate that p38 MAP kinase is present in both myocytes and interstitial histiocytes but not fibroblasts. These cell types may prescribe the mechanisms of ischemic preconditioning. Although the small size of the interstitial cells precludes definite subcellular localization, in the myocyte p38 appears to migrate into the perinuclear membranes, especially near the nuclear pole caps. Before translocation, the cytosolic stain is punctate but not readily identifiable within any one compartment. These basal locations are similar to the findings of Raingeaud (26), who overexpressed a FLAG-tagged p38 in Chinese hamster ovary (CHO) cells. Before stimulation, FLAG-tagged p38 was detected at the cell surface, cytoplasm, and nucleus. Unlike the basal perinuclear presence seen in myocytes, in CHO cells perinuclear staining was increased after ultraviolet irradiation.

Our experiments indicate that an ischemic stress period of >5 min is required to induce appreciable translocation. Similar suggestions were made by Bogoyevitch et al. (1), who found that either ischemic durations of >10 min or a 20-min period of reperfusion after 10 min of ischemia was required to activate p38 kinase activity in rat hearts. However, in contrast to this immunoblot study, our immunofluorescence images suggest that, during the sustained ischemic period after preconditioning, translocation appears diminished (Fig. 4, C and D), suggesting a reversal of translocation that became more evident during continued reperfusion. p38 MAP kinase translocation into the nucleus and to cytosolic striations was clearly detectable after four cyclic episodes of 5-min ischemia and 10-min reperfusion, but not after a single 5-min ischemic episode. The antibody we have used recognizes the COOH-terminal region of p38 MAP kinases, and thus the dual phosphorylation status of the translocated p38 in the different cellular compartments remains indeterminate. There are two possibilities: either ischemic stress activates p38 MAP kinase, which then translocates to the nucleus, or, alternatively, postischemic signaling escorts the p38 MAP kinase to a nuclear assemblage.

The cardiac stress signaling mediated by p38 MAP kinase may involve several compartments within myocytes and other selected ventricular cells. Like other MAP kinases (JNK/SAPK1, ERK1, ERK2) (33), p38 MAP kinase also translocates to the nucleus and cytosolic compartments. These compartments presumably contain phosphorylation targets such as transcription factors and kinases downstream of p38 MAP kinase. The cross-strial translocation may be particularly interesting in the context of functional preconditioning induced by p38 because the well-characterized downstream kinase (MAPKAP kinase 2) and its cognate target (HSP 27) have been implicated in regulating cytoplasmic actin (34). Our data also suggest that p38 MAP kinase is probably deactivated, albeit at different rates from each compartment.

In summary, the facts that ischemic stress adaptation is mediated by a distinct signal transduction pathway involving tyrosine kinase-phospholipase D-MAPKAP kinase 2 (21), that p38 is tyrosine phosphorylated in response to stress (34), and that cardiac cells stimulated by oxidative stress or phorbol ester can rapidly activate p38 MAP kinase and phosphorylation of HSP 27 (34) strongly suggest a crucial role of p38 MAP kinase MAPKAP kinase 2 in myocardial adaptation to ischemic stress.