Differential Activation of Mitogen-activated Protein Kinases in Ischemic and Anesthetic Preconditioning

This study was conducted in accordance with the guidelines of the Animal Care and Use Committee of the University of Zurich, Zurich, Switzerland.

Male Wistar rats (250 g) were heparinized (500 U intraperitoneally) and 20 min later were decapitated. The hearts were rapidly removed and perfused in a noncirculating Langendorff apparatus with Krebs-Henseleit buffer (155 mm Na⁺, 5.6 mm K⁺, 138 mm Cl⁻, 2.1 mm Ca²⁺, 1.2 mm PO⁴³⁻, 25 mm HCO³⁻, 0.56 mm Mg²⁺, 11 mm glucose, and 13 mm sucrose) gassed with 95% O²–5% CO²and maintained at a pH of 7.4 and a temperature of 37°C. Perfusion pressure was set to 80 mmHg. A water-filled balloon was inserted through the mitral valve into the left ventricle and inflated to set an end-diastolic pressure of 0–5 mmHg during the initial equilibration. The volume of the balloon was not changed thereafter. The distal end of the catheter was connected to a performance analyzer (Plugsys Modular System; Hugo Sachs, March-Hugstetten, Germany) by means of a pressure transducer. Perfusion pressure, epicardial electrocardiogram, and coronary flow (Transit Time Flowmeter type 700; Hugo Sachs) were simultaneously recorded on the same performance analyzer. All recorded data were digitized and processed on a personal computer using the software IsoHeart (Hugo-Sachs).

Untreated hearts were subjected to 40 min of global ischemia and 30 min of reperfusion (fig. 2) or, in some experiments, to 180 min of reperfusion (determination of cellular injury). APC was induced by administration of 15 min of isoflurane at 2.1% (vol/vol; 1.5 minimum alveolar concentration [MAC] isoflurane in rats) at 37°C until 10 min before sustained test ischemia. Buffer solution was equilibrated with isoflurane using an Isotec 3 vaporizer (Datex-Ohmeda, Tewksbury, MA) with an air bubbler. The administered vapor concentration of isoflurane was continuously controlled by the infrared gas analyzer Capnomac Ultima (Datex-Ohmeda). The applied concentration of isoflurane was also measured in the buffer solution using a gas chromatograph (Perkin-Elmer, Norwalk, CT; 0.52 ± 0.04 mm). IPC was induced by three cycles of ischemia interspersed by 5 min of reperfusion. IPC and APC were bracketed with the blockers, which were administered from 3 min before until 3 min after the IPC- and APC-preconditioning stimulus, respectively, as depicted in figure 2. The following blockers were used: PD98059 (Calbiochem, San Diego, CA) at 20 μm and SB203580 (CD Biosciences, Nottingham, United Kingdom) at 10 μm. 13,18The drugs were dissolved in dimethyl sulfoxide, with a final concentration of less than 0.10% reached in the perfusate. In some experiments, PD98059 was administered from 5 min before induction of prolonged test ischemia until the end of reperfusion. Separate experiments were performed to show that dimethyl sulfoxide alone did not affect the results of the experiments. For each experimental group, seven hearts were prepared, and functional parameters were recorded.

To determine the effects of the preconditioning stimulus on activation of ERK1/2 and p38 MAPK, separate experiments were conducted. For this purpose, tissue was collected shortly after the administration of the preconditioning stimulus (short-term protocols without ischemia–reperfusion as opposed to the long-term protocols in which the tissue was collected after completion of sustained test ischemia [40 min] and reperfusion [30 min]). Five hearts of each experimental group were prepared for immunohistochemical analyses, and five hearts of each experimental group were prepared for Western blot analyses. Additional groups of rat hearts were treated with the blockers chelerythrine (10 μm; Biomol, Plymouth Meeting, PA), a specific PKC inhibitor, and 5-hydroxydecanoate (500 μm; Biomol), a specific mitochondrial K^ATPchannel blocker, directly dissolved in Krebs-Henseleit solution and used for Western blot analyses (long-term protocols;fig. 2). Doses of isoflurane and blockers selected were those known to induce or inhibit preconditioning, respectively. 2Separate experiments served to determine cellular injury in APC-treated hearts in the presence and absence of PD98059 during 40 min of ischemia and 180 min of reperfusion as compared to time-matched controls (n = 5 for each group).

Tissue preparation was performed as previously described. 2Briefly, the samples were crushed in liquid nitrogen, and lysis buffer, 1 m dithiothreitol solution, and protease inhibitor cocktail (Complete Mini Protease Inhibitor Cocktail; Roche, Basle, Switzerland) were added. After homogenization and heating (7 min at 100°C in a water bath), the samples were centrifuged for 5 min at 20,000 g , and the supernatants were used for Western blotting. Gel electrophoresis was performed on a 10% SDS polyacrylamide gel. The samples were electroblotted overnight at 4°C onto a nitrocellulose membrane (Osmotics Inc., Westborough, MA). Primary antibodies were diluted in 5% nonfat dry milk in Tris buffered saline and incubated for 2.5 h. The following antibodies were used: monoclonal mouse antibody against total actin (Chemicon, Temecula, CA), phospho-ERK1/2 (Thr202/Tyr204), polyclonal rabbit antibody (Cell Signaling Technology, Beverly, MA), phospho-p38 MAPK (Thr180/Tyr182), and polyclonal rabbit antibody (Cell Signaling Technology). The membrane was washed in Tris buffered saline-tween 20 and incubated in horseradish-peroxidase–labeled secondary antibody (goat antirabbit and goat antimouse immunoglobulin G horseradish peroxidase (Perbio Science, Bonn, Germany) for 1 h. The membrane was then incubated with chemiluminescence substrate (Super Signal; Perbio Science) and exposed to x-ray film (New RX; Fuji, Tokio, Japan). Quantitative analysis of the band density was performed using simultaneously blotted actin density to correct for protein content (MCID Imaging Inc., Fonthill, Ontario, Canada).

Activation of ERK1/2 and p38 MAPK and myofibrillar translocation of heat-shock protein 27 (Hsp27) were visualized by immunofluorescence staining in response to preconditioning. 19Left ventricular tissue samples were placed in optimal cutting temperature medium (Tissue-Tek; Sakura Finetek Inc., Torrance, CA), frozen in liquid nitrogen, and stored at −70°C. Cryosections (5 μm) were prepared with a cryostat (Cryo-star HM 560M; Microtom, Kalamazoo, MI) and collected on gelatin-precoated slides. All sections were fixed for 10 min in 0.5% paraformaldehyde at room temperature, rinsed with phosphate-buffered saline, and incubated in 10% normal goat serum for 30 min to block nonspecific binding. Sections were incubated for 1 h at room temperature with primary antibodies. Rabbit polyclonal antibodies against phospho-ERK1/2, phospho-p38 MAPK, and Hsp27 (Cell Signaling Inc.) diluted in phosphate-buffered saline (1:50 for ERK1/2 and p38 MAPK, 1:100 for Hsp27) containing 4% normal goat serum were added. Phospho-ERK1/2 and phospho-p38 MAPK antibodies were combined with guinea pig polyclonal m-dystrophin (DYS12) antibodies (against the N-terminal portion of the molecule 20; 1:1000) as a sarcolemmal marker and mouse monoclonal α-N-cadherin antibodies (Sigma, St. Louis, MO) as a marker for intercalated discs. Hsp27 antibodies were combined with mouse monoclonal α-myomesin antibodies as a marker of the contractile apparatus (gift from Hans M. Eppenberger, Ph.D., Professor of Cell Biology, Department of Cell Biology, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland; 1:5). All sections were then washed with phosphate-buffered saline and incubated for 1 h with a mixture of secondary antibodies conjugated to Alexa Fluor 555 goat α-rabbit, Alexa Fluor 488 goat α-mouse, or Alexa Fluor 488 goat α-guinea pig (Molecular Probes, Eugene, OR; 1:500) and, in some experiments, with 4′,6-diamino-2-phenylindole (DAPI, 10 ng/ml; Sigma) as a marker of nuclei in phosphate-buffered saline at room temperature. Sections were protected with cover slips using DAKO mounting medium (DAKO Corporation, Carpinteria, CA) and analyzed by epifluorescence microscopy using an upright microscope (Axioplan 2; Zeiss, Jena, Germany) with appropriate filter blocks for the detection of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, and ultraviolet fluorescence. In addition, confocal images were obtained with an LSM 5 confocal microscope (Zeiss) using the appropriate laser lines and filter blocks (fluorescein isothiocyanate: excitation 488 nm, emission 525 nm; tetramethylrhodamine isothiocyanate: excitation 540 nm, emission 570 nm; ultraviolet: excitation 360 nm, emission 461 nm). Randomly chosen fields at 400× magnification from sections of samples of each group were examined for colocalization of phospho-ERK1/2 and phospho-p38 MAPK with dystrophin (sarcolemma), N-cadherin (intercalated discs), or DAPI (nuclei) without previous knowledge of the treatment. Colocalization was measured and quantified by using the software Imaris/Colocalization version 2.1.2 (Bitplane Inc., Zurich, Switzerland). This software generates a map of locations at which multichannel images have a common event as defined by a signal of a certain intensity found in two or more channels at the same spatial location. The number of pixels of these events was calculated and served to express the degree of colocalization as an x-fold increase in the various groups as compared to the control group. Also, the presence or absence of Hsp27 translocation to myofibrillar structures was determined for the various treatment groups.

Hearts for these experiments (five in each group) underwent 40 min of test ischemia and 180 min of reperfusion or time-matched perfusion. For the last 15 min of perfusion, they were perfused with Krebs-Henseleit solution containing 5 μm propidium iodide. After removing right ventricular and atrial tissue, they were embedded in optimal cutting temperature medium (Tissue-Tek) and frozen in liquid nitrogen. Twenty longitudinal cryosections (5 μm) covering the entire left ventricle were prepared, fixed in 4% paraformaldehyde for 20 min at room temperature, and stained with DAPI. The number of propidium iodide-positive nuclei per section was determined microscopically at 200× magnification using a grid and expressed as percentage of the total number of DAPI-positive nuclei.

Functional parameters at identical time points were compared for the groups by unpaired t  tests. Repeated-measures analysis of variance was used to evaluate differences over time between groups for the hemodynamic parameters. The Bonferroni correction was used to correct for multiple comparisons. P < 0.05 was considered to be statistically significant. Data are expressed as mean ± SD. StatView Version 4.5 (Abacus Concepts, Berkeley, CA) was used for the statistical analysis.

Western blot analyses for the dual phosphorylation of ERK1/2 and p38 MAPK served to determine the activity profile of ERK1/2 and p38 MAPK in IPC and APC 10 min after the administration of the preconditioning stimulus (short-term protocols) and at the end of the ischemia–reperfusion injury (long-term protocols) (fig. 3; n = 5 for each group). ERK1/2 and p38 MAPK activities were exclusively and markedly increased shortly after IPC but not after APC when compared to nonpreconditioned hearts (fig. 3A). The specific blockers PD98059 and SB203580 abolished the activation of ERK1/2 and p38 MAPK in IPC. In the tissues collected from hearts after ischemia–reperfusion injury, IPC as well as APC markedly enhanced activation of ERK1/2 when compared to nonpreconditioned hearts, albeit in IPC more profoundly than in APC (figs. 3B and C). However, p38 MAPK activity was only markedly enhanced in IPC but not APC after ischemia–reperfusion when compared to nonpreconditioned hearts. Interestingly, although coadministration of PD98059 and SB203580 during the triggering process of preconditioning clearly abolished the increase in ERK1/2 and p38 MAPK activities after ischemia–reperfusion injury in IPC, coadministration of these blockers during APC did not modify postischemic ERK1/2 and p38 MAPK responses. These results demonstrate that ERK1/2 and p38 MAPK are strongly activated during the triggering phase of IPC but not APC and that the two types of preconditioning (ischemic vs.  anesthetic) exhibit a differential activity profile of MAPKs after ischemia–reperfusion injury.

Because postischemically enhanced ERK1/2 activity in APC was not inhibited by administration of PD98059 during the triggering phase of APC, separate experiments were performed to evaluate whether activation of PKC or opening of mitochondrial K^ATPchannels would be upstream signaling steps of the observed postischemic ERK1/2 enhancement. Administration of chelerythrine (10 μm) or 5-hydroxydecanoate (500 μm) diminished ERK1/2 activation in IPC and APC to the level observed in nonpreconditioned hearts (figs. 3B and C). These results demonstrate that enhanced activation of ERK1/2 after ischemia–reperfusion is dependent on PKC activation as well as on opening of mitochondrial K^ATPchannels during the triggering phase in both types of preconditioning.

Administration of isoflurane at 1.5 MAC over 15 min or administration of three ischemic episodes of 5 min before a sustained test ischemia of 40 min significantly improved postischemic functional recovery when compared to untreated hearts (fig. 4; n = 7 for each group). Coadministration of PD98059 (20 μm), a specific mitogen-activated ERK-activating kinase 1–ERK1/2 inhibitor, abolished postischemic functional improvement in hearts preconditioned with ischemia but not in hearts preconditioned with isoflurane (figs. 4A and B). Similarly, coadministration of SB203580 (10 μm), a specific p38 MAPK inhibitor, annihilated the protective effects by IPC but not APC (figs. 4C and D). Administration of the blockers alone did not change postischemic functional recovery in nonpreconditioned hearts. These experiments provide evidence that ERK1/2 and p38 MAPK play a pivotal role in the triggering process of IPC but that these stress protein kinases do not actively participate in the triggering process of APC.

Because postischemically enhanced ERK1/2 activity closely paralleled functional recovery in IPC and APC, we further tested whether inhibition of ERK1/2 during ischemia–reperfusion would affect cardioprotection in IPC- and APC-treated hearts. For this purpose, separate experiments were performed with PD98059 administered from shortly before test ischemia until the end of reperfusion (n = 7 for each group). Administration of PD98059 during ischemia–reperfusion markedly reduced functional recovery in IPC and APC but did not further deteriorate functional recovery in nonpreconditioned hearts (figs. 4E and F). A summary of preischemic and postischemic hemodynamic variables in all treatment groups is presented in table 1.

To further elucidate the effects of ERK1/2 inhibition during ischemia–reperfusion in APC-treated hearts, the percentage of propidium iodide–positive nuclei was determined in the left ventricle and compared to APC-treated hearts and time-matched controls after 180 min of reperfusion (table 2). Propidium enters through necrotic membranes and accumulates in the nuclei 21,22and thus can serve as an indicator for myocardial injury. Importantly, administration of PD98059 during ischemia–reperfusion in APC-treated hearts significantly increased the percentage of damaged nuclei and cells, as compared to APC (table 2). Collectively, ERK1/2-dependent postischemic cellular and functional recovery in APC provides evidence for ERK1/2 acting as a mediator in APC, whereas in IPC, ERK1/2 and p38 MAPK may represent independent and/or alternate signaling pathways with trigger and mediator properties.

ERK1/2 was weakly constitutively activated in intercalated discs (colocalization with N-cadherin) and nuclei (colocalization with DAPI) in control hearts (fig. 5A). However, IPC markedly enhanced ERK1/2 activity in intercalated discs and in nuclei. These features were abolished by administration of PD98059. Notably, APC did not activate ERK1/2 compared to control hearts (fig. 5B). Similarly, p38 MAPK was weakly constitutively activated in the sarcolemma (colocalization with dystrophin) but markedly enhanced after IPC (fig. 6A). In addition, there was a pronounced perinuclear and intranuclear enhancement of p38 MAPK (colocalization with DAPI) in IPC but not APC. These features were absent after administration of SB203580 (fig. 6B). In accordance with this notion, Hsp27, an effector target of the p38 MAPK signaling pathway, was characteristically accumulated within myofibrillar structures exclusively after IPC but not APC (fig. 6C).

There are several salient findings in this study. First, APC-induced cardioprotection, in contrast to IPC-induced cardioprotection, does not require activation of the stress protein kinases ERK1/2 and p38 MAPK at the time of triggering, i.e. , during the administration of the preconditioning stimulus itself. Second, IPC and APC exhibit a differential activity profile of these key protein kinases not only during the triggering phase but also after ischemia–reperfusion. Although ERK1/2 and p38 MAPK are markedly increased shortly after IPC but not APC, ERK1/2 but not p38 MAPK activity is enhanced in APC after ischemia–reperfusion. Conversely, IPC concomitantly enhances ERK1/2 and p38 MAPK activities after ischemia–reperfusion. In accordance with these observations, increased activity of ERK1/2 and p38 MAPK can be exclusively visualized in subcellular compartments, primarily the nuclei, the sarcolemma, and the intercalated discs after IPC but not APC. Both types of preconditioning exhibit postischemic enhancement of ERK1/2 activity, which is dependent on PKC and mitochondrial K^ATPchannel activation and closely correlates with functional recovery. Finally, effective prevention of cellular damage in APC-treated hearts is dependent on intact enhanced ERK1/2 activity during ischemia–reperfusion, emphasizing the role of ERK1/2 as a mediator in APC. Collectively, although IPC and APC may have many signaling steps in common, the findings of the current study reveal fundamental differences in cellular signaling with respect to MAPKs between IPC and APC.

Previous studies on signal transduction in APC identified PKC as one important integral component of the signaling cascade. 2,23,24However, a complex network of other protein kinases, particularly the stress-related MAPKs, has been increasingly implicated in triggering and mediating cardiac preconditioning (fig. 1). 12MAPKs, similar to PKC, are G protein–coupled and receptor-activated intermediate signal transducers within the cytoplasm, ultimately phosphorylating transcriptional factors and effector proteins. 25,26They are activated in a multitude of cellular events, such as inflammation, proliferation, differentiation, and cell death and survival. The findings of the current study are compatible with the general conceptual paradigm that MAPKs are activated during ischemia–reperfusion phenomena and provide novel mechanistic insight into the significance of these stress protein kinases in two distinct types of cardiac preconditioning. Although MAPKs may orchestrate cytoprotection as triggers and mediators in IPC, they are devoid of triggering effects in APC, but they may have mediator effects.

Considerable controversy surrounds the precise role of MAPKs in cardioprotection by preconditioning. 12,25In particular, whether activation of MAPKs is obligatory at the time of IPC to convey effective protection against sustained ischemia is still under debate. SB203580, a specific p38 MAPK inhibitor, effectively blocked cytoprotection in myocyte models of IPC 27,28and annihilated infarct size limitation by IPC in rats, 14,29rabbits, 27dogs, 30and pigs. 31In contrast, another pig study was unable to block infarct size reduction by intracoronary infusion of SB203580 in IPC. 32Similarly, several groups of investigators demonstrated PD98059-dependent inhibition of IPC in isolated myocytes 33and in in vivo  rat and pig models, 14,34whereas others rejected the dependence of IPC-induced protection on ERK1/2 activation. 18In this context, two recent studies merit a more detailed discussion. Fryer et al.  14evaluated the significance of ERK1/2 in ischemia- and opioid-induced cardioprotection using an in vivo  rat model of regional ischemia. This study determined functional recovery and infarct size after test ischemia of 30 min and reperfusion of 2 h. IPC was induced by 5 min of coronary occlusion and 5 min of subsequent reperfusion and a specific δ¹-opioid agonist (TAN-67) was administered 15 min before test ischemia. PD98059 was applied as a bolus 10 min before IPC and 5 min before TAN-67. In this study, postischemic functional recovery and infarct size reduction were clearly abrogated by PD98059, implying ERK1/2 dependence of IPC- and opioid-induced cardioprotection. Interestingly, and consistent with our findings, ERK1/2 activity was not only increased immediately after the IPC stimulus but further enhanced on reperfusion. Conversely, Mocanu et al.  18recently reported the results of a study assessing the role of phosphatidyl inositol 3-OH kinase (PI3 kinase) or ERK1/2 in an in situ  rat model of regional ischemia. Two cycles of 5 min of ischemia with 10 min of reperfusion were used as an ischemic preconditioning stimulus, and hearts were subjected to 35 min of regional ischemia followed by 120 min of reperfusion. PD98059 and the PI3 kinase inhibitor wortmannin were infused during the preconditioning process. Although PD98059 inhibited ERK1/2 activation during IPC, PD98059 did not affect infarct size, whereas wortmannin blocked the infarct reduction by IPC. No functional parameters were obtained in this study. These results are in contrast to those by Fryer et al.  14and to our own results. However, the differences could be attributed to several sources, including the infarct model (in situ  vs.  isolated perfused model), the variable nature of the ischemic injury (regional vs.  global), the presence of multiple isoforms of individual MAPKs exhibiting opposing functions, or simply differences in dosing and timing of the blockers used. Taken together, our data confirm the results of previous studies that activation of ERK1/2 and p38 MAPK are required at the time of IPC to convey effective subsequent protection against prolonged ischemia. As a novel finding, they further demonstrate that protection conferred by APC is independent of MAPK-mediated triggering.

In striking contrast to the lack of activation and significance of MAPKs during the triggering process in APC, ERK1/2 is markedly enhanced after ischemia–reperfusion in APC. Importantly, improved postischemic functional recovery closely paralleled enhanced ERK1/2 activation in APC as well as IPC. This is supported by the following findings. First, PD98059 administered during APC affects neither functional recovery nor postischemic ERK1/2 enhancement in APC, whereas chelerythrine and 5-hydroxydecanoate, previously reported effective blockers of functional recovery in APC, 2diminish ERK1/2 activity after ischemia–reperfusion in APC to the level observed in nonpreconditioned hearts. Second, PD98059, chelerythrine, and 5-hydroxydecanoate, effective blockers of IPC, 2,14inhibit postischemic enhancement of ERK1/2 in IPC. Third, inhibition of ERK1/2 activity during ischemia–reperfusion diminishes postischemic functional improvement by IPC and APC. Also, inhibition of ERK1/2 during ischemia–reperfusion increases the number of propidium iodide-positive cells, a marker of myocardial injury, 21,22in APC-treated hearts.

Given these relations, it seems that postischemic enhancement of ERK1/2 activity represents an important common mediator of cytoprotection in both types of preconditioning. In fact, members of MAPKs, particularly ERK1/2, have been implicated in cell survival signaling. In a model of ischemia–reperfusion in intact hearts, ERK1/2 activation was shown to attenuate apoptosis during ischemia–reperfusion. 35In support of this conclusion is the observation that transgenic mice overexpressing mitogen-activated ERK-activating kinase 1, an upstream kinase of ERK1/2, develop hypertrophic hearts with enhanced contractile performance and exhibit partial resistance to apoptosis induced by ischemia–reperfusion. 36Conversely, Ras-overexpressing mice with activation of a much broader spectrum of MAPKs develop cardiomyopathy and die prematurely. 37Little is known about how increased ERK1/2 activity results in cellular protection. Possible downstream mechanisms conferring cytoprotection include activation of cyclooxygenase 2, phosphorylation of multiple transcriptional factors (c-myc, STAT, UBF), and inhibition of the caspase cascade. 25Another mechanism by which ERK1/2 may elicit protection was recently reported by Baines et al.  38This group showed increased mitochondrial formation of functional ERK1/2-PKC modules as a result of PKC overexpression in transfected rabbit cardiomyocytes, ultimately inducing phosphorylation and inactivation of Bad, a proapoptotic protein. Using proteomic analysis, ERK and p38 MAPK were also found to form signaling complexes with PKC in the murine myocardium. 39Interestingly, ERK1/2 activity was significantly enhanced immediately after IPC but not APC in our experiments. This, coupled with our previous observation of a marked PKC translocation to mitochondria on IPC, 2supports the potential role of ERK1/2–PKC interactions as functional modules in IPC but not APC. Our immunohistochemical analyses revealed enhanced MAPK activity in intercalated discs, the sarcolemma, and the nuclei immediately after IPC. This has two important implications. First, MAPKs may accelerate and reinforce PKC-induced decline in intercellular communication, 17contributing to reduced hypercontraction and cellular damage during ischemia and at the initiation of reperfusion in IPC. 40Second, increased nuclear MAPK activity after IPC, as observed in our experiments, may point to the potential role of MAPKs in the genesis of a delayed type of protection, which is thought to require novel gene expression. In support of this concept, Fryer et al.  15recently demonstrated that opioid-induced delayed cardioprotection requires activation of p38 MAPK and ERK1/2. Punn et al.  41demonstrated that sustained activation of ERK1/2 occurring during reoxygenation after sublethal simulated ischemia in cardiomyocytes is required for the development of a delayed protection. Finally, Carroll et al.  16reported that delayed cardioprotection in a human cardiomyocyte-derived cell line involves p38 MAPK activation. Intriguingly, isoflurane-induced preconditioning in an in vivo  dog model was unable to induce delayed protection, which is consistent with the observed lack of MAPK activation during the triggering phase of APC in our study. Although these observations are not a proof of any causal relation, they are nevertheless hypothesis-generating.

Hsp27 is a terminal substrate of the p38 MAPK (terminal phosphorylation via  MAPK-activated protein kinase 2) and was reported to confer cytoskeletal protection when in the phosphorylated state. 28It is typically translocated on stimulation and associates with sarcomeric and cytoskeletal proteins, thereby preventing misfolding of proteins and preserving cellular integrity. 42In accordance with our Western blot analyses, the results of the immunohistochemical staining show that IPC but not APC elicits activation and translocation of Hsp27.

The following specific comments should be added: (1) The results of our study are largely dependent on the effectiveness and specificity of the pharmacologically inhibiting agents PD98059 and SB203580. However, the absolute specificity and potential toxicity of these drugs are difficult to appreciate, given the fact that the mammalian genome encodes more than 1,000 kinases. (2) SB203580 is believed not to interfere with the dual phosphorylation of p38 MAPK but to inhibit the catalytic activity of the phosphorylated kinase. 43However, in accordance with the recent results of another research group, phosphorylation of p38 MAPK was inhibited in our experiments by this blocker. 44(3) We previously unraveled PKCδ and PKCε as integral components in IPC and APC using the same experimental setup. The current studies demonstrate the lack of significance of p38 MAPK and ERK1/2 as triggers in APC but not IPC. Nonetheless, we cannot exclude the involvement of other kinases in the complex signaling cascade of APC. Specifically, we did not evaluate the role of c-Jun N-terminal kinase, another MAPK, in the current study (fig. 1). (4) Activation of MAPKs at higher doses of isoflurane (> 1.5 MAC) cannot be ruled out. In fact, activation of ERK1/2 by isoflurane was previously reported in vascular tissue. 45However, it should be noted that the administered dose of isoflurane in the current study was at the upper level of clinically applicable doses. (5) Preconditioning induced with ischemia has limited potential because of the inevitable reduction in contractile function (“stunning”) and the depletion of intracellular energy stores. Therefore, unraveling the detailed signaling pathways in the different types of preconditioning is important and may help to comprehend their short- and long-term beneficial and detrimental consequences in the myocardium.

In conclusion, the results of the current study show that MAPKs are differentially regulated in IPC- and APC-treated isolated perfused rat hearts exposed to global ischemia and highlight the possibility that these differences may generate distinct types of cardioprotection. Moreover, for the first time, this study identified ERK1/2 as a potential mediator and downstream target of PKC and mitochondrial K^ATPchannel activation in APC. The observations presented provide a rationale for dissecting the precise roles of individual kinases in the development of early and the putative delayed protection in APC.

The authors thank Marina Uecker (Ph.D. Student, Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland) for helpful assistance with some of the staining procedures.