INTRODUCTION

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ISCHEMIC PRECONDITIONING is the phenomenon whereby a brief episode of ischemia and reperfusion protects the heart against infarction from a subsequent ischemic insult (29). Preconditioning occurs in two phases: an early phase, also known as acute preconditioning, in which protection lasts up to 1-2 h following preconditioning, and a second phase, known as the second window of protection, in which protection reappears 24-72 h following preconditioning. Although a variety of mediators and effectors have been proposed to be essential for conferring preconditioning, including the adenosine receptor (6, 7, 21), protein kinase C (2, 32), and the ATP-sensitive K⁺ channel (3, 8), the precise signaling pathways mediating the protective effect remain to be fully elucidated and have been under intense investigation.

Over the last few years, a number of studies in both whole hearts and isolated cardiomyocytes have described the activation of members of the mitogen-activated protein kinase (MAPK) family of signaling proteins during ischemia and ischemia-reperfusion (9, 27, 35, 36, 41). All of the MAPKs are proline-directed, serine/threonine-protein kinases and are activated by dual phosphorylation on tyrosine and threonine residues by upstream kinases. The family consists of three members, the extracellular signal-regulated kinases 1 and 2 (ERK1/2; p42/p44 MAPK), c-Jun NH₂-terminal kinases 1 and 2 (JNK1/2), and p38 MAPK. Whereas ERK1/2 are predominantly activated by growth factors, the JNKs and p38 MAPKs are generally activated by stresses such as ultraviolet light, inflammatory cytokines, heat shock, and ischemia-reperfusion and thus form a subfamily known as stress-activated protein kinases (SAPKs). Because preconditioning is characterized by a brief period of ischemia and reperfusion, members of the MAPK family are ideal candidate components of the signaling pathway(s) leading to myocardial adaptation. Indeed, a number of studies in rabbits (37), rats (26), and isolated cells (1, 30) have suggested that p38 MAPK mediates the protection conferred by acute preconditioning. In contrast, although the initial trigger(s) is likely to be consistent between acute and delayed protection, there have been relatively few studies investigating the signaling pathways responsible for conferring delayed preconditioning.

Therefore, the aim of this study was to determine whether members of the MAPK family (ERK1/2, p38 MAPK, and JNK) are activated during the preconditioning protocol and to ascertain their role using an in vitro model of delayed preconditioning (12). We show that both ERK1/2 and p38 MAPK are transiently phosphorylated during both minimal metabolic preconditioning (MMPC) and simulated reperfusion, an effect blocked by the MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data presented here suggests that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials. Tissue culture products were obtained from Life Technologies (Paisley, Scotland). Phospho-specific antibodies against ERK1/2 and p38 MAPK, recognizing the dually phosphorylated forms of these kinases, were obtained from New England Biolabs (Hitchin, UK). Antibodies to ERK2 and p38 MAPK were obtained from Santa Cruz (Wembley, UK). Anti-mouse and anti-rabbit-HRP conjugated secondary antibodies were obtained from Dako (Cambridge, UK). Ro31-8220, SB-203580, and PD-98059 were obtained from Calbiochem (Nottingham, UK), and stock solutions (1-50 mM) were prepared in DMSO. Prestained molecular weight markers, enhanced chemiluminescence blotting reagents, Hybond-C nitrocellulose, and Hyperfilm were from Amersham International (Amersham, UK). SDS-PAGE equipment was from Bio-Rad (Hemel Hempstead, UK), whereas acrylamide was obtained from National Diagnostics (Hull, UK). General laboratory reagents were from Sigma (Poole, UK).

Cell culture. Primary cultures of cardiomyocytes were prepared from neonatal rats as previously described (12). Briefly, hearts were removed from Sprague-Dawley rats, and ventricles were trisected and dispersed in a series of incubations at 37°C in nominally calcium-free, HEPES-buffered salt solution containing pancreatin (0.6 mg/ml; Life Technologies) and type II collagenase (0.5 mg/ml; Worthington Biochemicals, Reading, UK). The dispersed cells were preplated for 30 min to minimize fibroblast contamination thus giving a cardiomyocyte-rich culture. Myocytes were plated onto gelatin-coated plates at a final density of 7 × 10⁵ per well on 12-well plates for cell viability studies or 4 × 10⁶ per well on 60-mm dishes for signaling experiments in 4:1 DMEM/medium 199 (M199) supplemented with 10% horse serum, 5% fetal calf serum, and 1% penicillin-streptomycin. After 24 h in culture, cells were transferred to low-serum maintenance media (4:1 DMEM/M199 containing 1% fetal calf serum and 1% penicillin-streptomycin) for 24 h before experimentation. The investigation conforms with the principles outlined in the Declaration of Helsinki.

Delayed preconditioning protocol. MMPC was induced by treating myocytes for 2 h at 37°C with a modified Krebs buffer (in mM: 137 NaCl, 3.8 KCl, 0.49 MgCl₂, 0.9 CaCl₂, and 4 HEPES) supplemented with 10 mM 2-deoxyglucose and 20 mM sodium lactate (pH 6.8) to stimulate the extracellular milieu of myocardial ischemia. After preconditioning, the buffer was then washed off, and the cells were "reperfused" with maintenance media for 24 h. To investigate the role of PKC, ERK1/2, and p38 MAPK in delayed preconditioning, the specific inhibitors of PKC (Ro31-8220), MEK (PD-98059), and p38 kinase inhibitors (SB-203580) were employed. Cardiomyocytes were treated with a final concentration of 1 µM Ro31-8220, 10 µM SB-203580, or 50 µM PD-98059 for 30 min before preconditioning, during the preconditioning period, and, after 2 h of simulated reperfusion. These doses of inhibitors have previously been shown to inhibit PKC (13), MEK (14), and p38 MAPK (11), respectively. Inhibitors were then washed off, and cardiomyocytes were returned to maintenance media.

Determination of cardiomyocyte injury. Cell injury following simulated lethal ischemia was determined by creatine phosphokinase (CPK) efflux (membrane damage) and methyl thiazoyltetrazolium (MTT) metabolism (cell viability). Cell membrane damage was assessed following 3 h of simulated reperfusion by measuring CPK activity released into the medium. CPK activity was determined using a commercially available kit (Boehringer Mannheim) according to the manufacturer's instructions. Cell viability was determined after 24 h of simulated reperfusion using MTT assay. Cells were washed with warm PBS and incubated with 0.5 mg/ml MTT in PBS for 20 min at 37°C. The reaction was stopped by the addition of an equal volume of solubilization solution (0.1 N HCl, 10% Triton X-100 in isopropanol), and the absorbance of the blue formazan derivative read at 570 nm.

SDS-PAGE and Western blot analysis. After treatment, cardiomyocytes were rinsed with ice-cold PBS and harvested in 2× concentrated SDS-PAGE sample buffer. Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Equal protein loading between samples was verified by densitometry of gels stained with Coomassie brilliant blue and by poststaining nitrocellulose filters with Ponceau S following transfer. After transfer, membranes were blocked for 2 h with 5% nonfat milk in Tris-buffered saline (pH7.6) containing 0.1% Triton (TBST) and probed overnight at 4°C with either phospho-specific ERK1/2 or p38 MAPK antibodies at 1:1,000 dilution. Membranes were washed three times for 5 min in TBST containing 0.1% nonfat milk (TBSTM) before addition of swine anti-rabbit-HRP conjugated (phospho-ERK1/2 and p38 MAPK antibodies) or rabbit anti-mouse-HRP conjugated secondary antibody (1:2,500 dilution in TBSTM) for 2 h at room temperature. Membranes were washed three times for 5 min with TBSTM, and the antibody-antigen complexes were visualized by enhanced chemiluminescence. For signaling experiments, membranes were stripped and reprobed with antibodies recognizing nonphosphorylated ERK2 and p38 MAPK, respectively, to determine the total amount of ERK2 and p38 MAPK protein. For quantification of phosphorylation, density of the phosphorylated and nonphosphorylated proteins was determined using Scion Image software, and MAPK phosphorlyation was expressed as a percentage of the total nonphosphorylated MAPK. Results are expressed as means ± SE for the degree of fold increases in phosphorylation relative to control samples from three independent experiments.

In vitro kinase assay. To determine p38 MAPK activity, cells were lysed in lysis buffer [20 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride(PMSF)] and phosophorylated p38 MAPK immunoprecipitated using a phospho-p38 MAPK-agarose conjugated antibody overnight at 4°C. After incubation, immunoprecipitates were washed twice with kinase buffer, and kinase reaction was carried out at 30°C for 30 min in 40 µl of kinase buffer [25 mM Tris · HCl (pH 7.5), 5 mM -glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na₃VO₄, 1 mM MgCl₂] supplemented with 5 µM activating transcription factor 2 (ATF2) as the exogenous substrate and 10 µM ATP. The reaction was stopped by the addition of 20 µl 3× concentrated SDS-PAGE sample buffer, and samples were analyzed for ATF2 phosphorylation by Western blotting using an anti-phospho-ATF2 antibody (NEB).

Statistical analysis. Data are expressed as means ± SE from at least three independent experiments. Statistical analysis was performed with ANOVA followed by modified least significant difference test (Stat-View).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Activation of p38 MAPK and ERK1/2 during metabolic preconditioning and simulated reperfusion. To investigate whether members of the MAPK family are activated during the preconditioning protocol, cells were lysed at different time points either during MMPC or simulated reperfusion following MMPC (MMPC/reperfusion), and phosphorylation of p38 MAPK, JNK, and ERK1/2 was determined using antisera recognizing dually phosphorylated forms of the kinases. As shown in Fig. 1, phosphorylation of p38 MAPK on Thr¹⁸³/Tyr¹⁸⁵ was detected after 10 min (Fig. 1, A and C). ERK1/2 dual phosphorylation (Fig. 1, D and F) on Thr²⁰²/Tyr²⁰⁴ was detected after 5 min, rising to a maximum after 10 min and transiently returning toward basal levels after 40 min. In the case of ERK1/2, both isoforms (p44 and p42; Fig. 1F) were phosphorylated during MMPC, with ERK2 (42 kDa) being the predominantly phosphorylated isoform. Once phosphorylation of both ERK1/2 and p38 MAPK had returned to basal levels, no further activation of either kinase was detected for the remainder of the MMPC period (results not shown). However, no JNK phosphorylation was observed. Blots were reprobed to show total (nonphosphorylated) p38 MAPK (Fig. 1B) and ERK2 (Fig. 1E), respectively. Quantification of MAPK phosphorylation when maximal showed a 3-fold induction of p38 MAPK (Fig. 1C) and a 2.5-fold and 4-fold induction of ERK1 and ERK2 phosphorylation, respectively (Fig. 1F).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) during minimal metabolic preconditioning (MMPC) in cardiomyocytes. Myocytes were treated with MMPC buffer for up to 40 min, lysed with 2× sample buffer, resolved by 10% SDS-PAGE, and analyzed by Western blotting using antibodies recognizing dually phosphorylated p38 MAPK (A) and ERK1/2 (D). pan p38 MAPK (B) and pan-ERK-2 (E) antibodies were used to determine the total amount of p38 MAPK and ERK2, respectively. Results are representative of three independent experiments. C and F show the quantification of p38 MAPK and ERK1/2 phosphorylation during MMPC. Results are expressed as degree of fold increases in phosphorylation compared with time 0 control and represent the means ± SE from 3 experiments. *P < 0.05; **P < 0.01 compared with time 0 (control).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of p38 MAPK and ERK1/2 during reperfusion following MMPC in cardiomyocytes. Myocytes were treated with MMPC buffer and reperfused for up to 40 min, lysed with 2× sample buffer, resolved by 10% SDS-PAGE, and analyzed by Western blotting using antibodies recognizing dually phosphorylated p38 MAPK (A) and ERK1/2 (D). pan-p38 MAPK (B) and pan-ERK-2 (E) antibodies were used to determine the total amount of p38 MAPK and ERK2, respectively. Results are representative of three independent experiments. C and F show the quantification of p38 MAPK and ERK1/2 phosphorylation during reperfusion following MMPC. Results are expressed as degree of fold increases in phosphorylation compared with time 0 control and represent the means ± SE from 3 experiments. *P < 0.05; **P < 0.01 compared with time 0 (control).

Activation of ERK1/2 and p38 MAPK during LSI. Figure 3 shows that phosphorylation of both p38 MAPK and ERK1/2 was also detected during the initial stages of LSI. As shown in Fig. 3, A and C, phosphorylation of p38 MAPK was detected after 5 min of LSI, being maximal after 10 min, and then transiently returning to control levels by 40 min. A similar time course of phosphorylation of both ERK1 and ERK2 isoforms (Fig. 3, D and F) was also observed with maximal phosphorylation of both isoforms occurring after 10 min. No further phosphorylation was observed for the remainder of LSI. As found during MMPC, no JNK activation was detected (results not shown). Blots were reprobed for total (nonphosphorylated) p38 MAPK (Fig. 3B) and ERK2 (Fig. 3E), respectively. Quantification of MAPK phosphorylation when maximal showed a 2.5-fold induction of p38 MAPK (Fig. 3C) and 1.75-fold and 3.5-fold induction of ERK1 and ERK2 phosphorylation, respectively (Fig. 3F).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of p38 MAPK and ERK1/2 during lethal ischemia in cardiomyocytes. Myocytes were treated with lethal simulated ischemia (LSI) buffer for up to 40 min, lysed with 2× sample buffer, resolved by 10% SDS-PAGE, and analyzed by Western blotting using antibodies recognizing dually phosphorylated p38 MAPK (A) and ERK1/2 (D). pan-p38 MAPK (B) and pan-ERK-2 (E) antibodies were used to determine the total amount of p38 MAPK and ERK2, respectively. Results are representative of 3 independent experiments. C and F show the quantification of p38 MAPK and ERK1/2 phosphorylation during LSI. Results are expressed as degree of fold increases in phosphorylation compared with time 0 control and represent the means ± SE from 3 experiments. *P < 0.05; **P < 0.01 compared with time 0 (control).

Effect of preconditioning on MAPK phosphorylation during LSI. Recently, Nagarkatti and Sha'afi, (30) using an in vitro model of acute preconditioning, demonstrated that p38 MAPK activation during lethal stress was attenuated in preconditioned cells compared with cells subjected to lethal stress alone. To determine whether an analogous mechanism occurs in delayed preconditioning, cells were preconditioned for 2 h and reperfused for 24 h, and phosphorylation of both and p38 MAPK and ERK1/2 was determined after 10 min of LSI and compared with cells receiving LSI alone. Figure 4 shows that activation of both p38 MAPK (Fig. 4A) and ERK1/2 (Fig. 4D) was maximal after 10 min of LSI in both preconditioned cells (lane 3) and cells treated with LSI alone (lane 2). However, when the pattern of MAPK phosphorylation during LSI in preconditioned cells was compared with cells receiving LSI alone, no difference in the response was observed (Fig. 4, C and F). Blots were reprobed to show total (nonphosphorylated) p38 MAPK (Fig. 4B) and ERK2 (Fig. 4E), respectively.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effect of MMPC on phosphorylation of p38 MAPK and ERK1/2 during lethal ischemia. Myocytes were treated with preconditioning buffer for 2 h followed by reperfusion with maintenance media for 24 h before lethal ischemia for 10 min. After treatment, cells were lysed with 2× sample buffer, resolved by 10% SDS-PAGE, and analyzed by Western blotting using antibodies recognizing dually phosphorylated p38 MAPK (A) and ERK1/2 (D). pan p38 MAPK (B) and pan-ERK-2 (E) antibodies were used to determine the total amount of p38 MAPK and ERK2, respectively. Results are representative of 3 independent experiments. C and F show the quantification of p38 MAPK and ERK1/2 phosphorylation during LSI with or without MMPC. Results are expressed as degree of fold increases in phosphorylation compared with time 0 control and represent the means ± SE from 3 experiments.

Delayed protection is independent of p38 MAPK and/or ERK1/2 activation in cardiomyocytes. To determine the role of ERK1/2 and p38 MAPK in delayed protection, the specific MEK inhibitor PD-98059 and p38 MAPK inhibitor SB-203580 were used. However, before these inhibitors on delayed protection were examined, the ability of the inhibitors to block the pathways were determined. Myocytes were treated with the MAPK inhibitors for 30 min before and during MMPC, and ERK1/2, and p38 MAPK phosphorylation was determined. Figure 5A shows that phosphorylation of ERK1/2 was specifically blocked during MMPC by the MEK inhibitor PD-98059 (Fig. 5A, lane 4) and was specific for ERK1/2 because treatment had no effect on p38 MAPK phosphorylation (Fig. 5B, lane 4). SB-203580, a p38 MAPK inhibitor, did not block p38 MAPK phosphorylation (Fig. 5B, lane 3) but did prevent the downstream kinase effect of p38 MAPK on ATF2 phosphorylation (Fig. 5D, lane 3); SB-203580 had no effect on ERK1/2 phosphorylation (Fig. 5A, lane 3). Similar results were also obtained when the inhibitors were given during simulated reperfusion following MMPC (results not shown). Furthermore, treatment with a PKC inhibitor Ro-318220 (1 µM) did not block either ERK1/2 (Fig. 5A, lane 5) or p38 MAPK phosphorylation (Fig. 5B, lane 5), suggesting that MAPK activation is PKC independent. Pretreatment with Ro31-8220 (1 µM) before and during MMPC completely abolished delayed protection, as shown in Fig. 6, as determined by both CPK release (Fig. 6A) and MTT metabolism (Fig. 6B). Ro31-8220 alone had no effect on viability (results not shown). This indicated that protection following MMPC was PKC dependent.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Effect of inhibitors on blocking MAPK phosphorylation during preconditioning. Myocytes were treated with PD-98059 (PD; 50 µM), SB-203580 (SB; 10 µM), or Ro31-8220 (Ro; 1 µM) for 30 min before preconditioning and for 10 min into preconditioning. Cells were lysed with 2× sample buffer, resolved by 10% SDS-PAGE, and analyzed by Western blotting using antibodies recognizing dually phosphorylated ERK1/2 (A) and p38 MAPK (B). In C, pan-ERK-2 show ERK2 levels did not change over treatment period. In D, cells were treated as above ±SB-203580, lysed in buffer containing 1% Triton X-100, and phospho-p38 MAPK immunoprecipitated. Kinase reaction was carried out using activating transcription factor (ATF-2) as the exogenous substrate and incorporation of ATP into the substrate determined by Western blotting using a phospho-ATF-2 antibody.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of Ro31-8220 given during MMPC on cell survival after lethal ischemia. Cardiomyocytes were preconditioned with MMPC in the presence/absence of Ro31-8220 for 2h. Twenty four hours after MMPC, cells were subjected to LSI. In A, cell injury was determined by creatine phosphokinase (CPK) release into the medium after 3 h of simulated reperfusion following LSI. In B, cell viability was determined by MTT conversion following 24 h of simulated reperfusion in normal maintenance medium after LSI. Ro31-8220 alone had no effect on viability (results not shown). Results expressed as means ± SE from 3 independent experiments, **P < 0.01.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of PD-98059 and SB203580 given during MMPC on cell survival following lethal ischemia. Cardiomyocytes were preconditioned with MMPC in the presence/absence of either PD-98059 or SB-203580 for 2 h. Twenty four hours after MMPC, cells were subjected to LSI. In A, cell injury was determined by CPK release into the medium after 3 h of simulated reperfusion following LSI. In B cell viability was determined by MTT conversion following 24 h of simulated reperfusion in normal maintenance medium after LSI. PD-98059 and SB-203580 alone had no effect on viability (results not shown). Results expressed as means ± SE from 3 independent experiments, *P < 0.05, **P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Since the first identification of delayed preconditioning, the vast majority of studies have focused on determining the nature of the molecule(s) responsible for triggering the effect and the induction of potentially cytoprotective proteins. Nevertheless, over the last few years there has been considerable interest in the signaling pathways that mediate delayed protection. In particular, as found during acute preconditioning, the acquisition of delayed protection appears to be dependent on both tyrosine kinase (18) and PKC activation (5, 32, 34). Consistent with these studies, we (this report; Fig. 6) and others (38) have shown that delayed preconditioning utilizing glycolytic inhibition is also PKC dependent. However, during delayed preconditioning, little is known about the intermediate signaling pathways linking the receptor to the nucleus and the resultant induction of cardioprotective proteins. Members of the MAPK signaling family have been suggested to be suitable signaling intermediates because they have been shown to translocate to the nucleus during ischemia and ischemia-reperfusion and regulate a number of transcription factors (27, 28). In this report, we used a previously reported model of delayed preconditioning utilizing inhibition of glycolysis under acidotic conditions (12, 31, 38), and we demonstrated that, during the initial stages of MMPC, MMPC/reperfusion, and LSI, both ERK1/2 and p38 MAPK were transiently activated (Fig. 1). The transient nature of these time courses is consistent with studies in cardiomyocytes in culture during hypoxia and hypoxia/reoxygenation and whole hearts during ischemia and ischemia-reperfusion for both ERK1/2 (27, 33, 36) and p38 MAPK (9, 36). Furthermore, we also determined for the first time whether these kinases play a role in the protective effect.

In the case of ERK1/2, the potential role in delayed preconditioning has recently been suggested by Ping et al. (33). These investigators demonstrated that ERK1/2 were activated during the preconditioning stimulus in both isolated rabbit cardiomyocytes and rabbit hearts. In contrast to the data presented here (Fig. 5B, lane 5), activation of both ERK1/2 could be blocked by PKC inhibition. Furthermore, treatment with PD-98059 during simulated ischemia alone blocked the cytoprotective effect of PKC-transfected cardiomyocytes, suggesting a role of ERK1/2 in PKC-dependent protection against ischemia-reperfusion injury and delayed preconditioning. Although these investigators propose that ERK1/2 activation plays a protective role in delayed preconditioning, the effect of blocking ERK1/2 activation using PD-98059 during ischemic preconditioning was not investigated. Thus the role of ERK1/2 in their delayed preconditioning model remains to be fully determined. In contrast, our findings presented here demonstrate that, despite the activation of ERK1/2, PD-98059 did not abolish protection in this model (Fig. 7), indicating that delayed preconditioning following glycolytic inhibition alone is independent of ERK1/2. It is interesting to note that although MMPC was blocked by Ro31-8220 (Fig. 6), ERK1/2 activation was not blocked by the PKC inhibitor (Fig. 5B, lane 5), suggesting that PKC is not an upstream activator of either ERK1/2 or p38 MAPK in this model. Thus it appears that although PKC is protective in these two delayed preconditioning models, the protective pathways downstream of PKC differ and may potentially be due to the activation of different PKC isoforms (PKC- vs. PKC-) during these two preconditioning stimuli. Although transient activation of ERK1/2 during MMPC, reperfusion, and LSI does not appear to play a role in this model of delayed protection, we have also found that preconditioning with a similar buffer containing the O₂ scavenger sodium dithionite causes a sustained activation of ERK1/2 during reperfusion. In this case, blockade of the sustained ERK1/2 activation during reperfusion with PD-98059 does block the protection (Punn A, Mockridge JW, Farooqui S, Marber MS, and Heads RJ, unpublished observations). Therefore, the role of ERK1/2 in protection is complex, and sustained versus transient activation may elicit quite different responses.

In this study, we also investigated the potential role of p38 MAPK in delayed preconditioning in this model. However, although p38 MAPK was transiently phosphorylated, blocking its activation using SB-203580 did not abolish protection (Fig. 7). This is in contrast to studies during acute preconditioning because activation of p38 MAPK during the initial phases of acute preconditioning has been proposed by a number of investigators to mediate the protective effect (26, 30, 37) in both whole hearts and isolated cells. However, treatment with the p38 MAPK inhibitor SB-203580 during the lethal stress has also been shown to protect against injury (22, 23, 30). Thus, paradoxically, p38 MAPK appears to play both a protective role during the preconditioning stress and a detrimental role during the lethal stress. To account for this effect, Nagarkatti and Sha'afi (30) proposed that p38 MAPK activation during preconditioning results in a downregulation of this pathway during the lethal stress and thus a decrease in cell death. We investigated whether an analogous mechanism occurs during delayed preconditioning by determining whether prior p38 MAPK or ERK1/2 phosphorylation during MMPC could modulate LSI-induced p38 MAPK or ERK1/2 phosphorylation 24 h later. However, MMPC did not alter p38 MAPK or ERK1/2 phosphorylation during the lethal stress (Fig. 4).

With the protective effect observed being independent of either ERK1/2 or p38 MAPK activation in this model, an alternative protective pathway must be responsible for conferring protection. Although this pathway downstream of PKC remains to be fully elucidated, recent studies in both delayed and acute preconditioning have suggested that the activation of the nuclear factor B (NFB) may be an important component in cardioprotection (24, 39). Within the context of delayed preconditioning, NFB may provide a potential link between upstream PKC activation and the expression of the protein(s) responsible for cardioprotection. Whether activation of NFB during the preconditioning ischemia is due to a direct effect of PKC or via an intermediate signaling pathway, for example PI3-K, remains to be established. Such studies are currently underway to investigate whether NFB is activated in our model of delayed preconditioning.

Another interesting finding in this report is the differential activation of members of the SAPK subfamily. In general, studies investigating SAPK activation during ischemia-reperfusion have shown that both p38 MAPK and JNK are activated (9, 36). In contrast, our findings demonstrate that although p38 MAPK was rapidly activated during MMPC and MMPC/reperfusion, no JNK activation was detected. This was not due to the inability of neonatal cardiomyocytes to activate the JNK pathway because a variety of stresses and agonists have been shown to activate both JNK1/2 in these cells (10, 17, 19, 20, 35). Thus the data suggest that there may be a differential regulation of the SAPK signaling pathways upon MMPC/reperfusion. Investigating the nature of the mediators of MMPC/reperfusion-induced SAPK activation may provide an important insight into the specificity of these signaling cascades.

The potential limitations of studies of this nature are correlating the observed effects in isolated neonatal cardiomyocytes with those seen in adult cardiomyocytes and the intact heart. Unfortunately, direct comparison of our results with others investigating delayed preconditioning in the intact rat heart is limited due to the paucity of data available in the rat because most studies have demonstrated delayed preconditioning in the rabbit. However, correlating the mechanism underlying preconditioning in the rat and rabbit is problematical because there appears to be two different mechanisms operating in these species. For example, during acute preconditioning, PKC and tyrosine kinases have been implicated in both species, yet the timing of kinase activation necessary for protection appears to differ in the rat (15, 16, 25) and rabbit (4, 40). Nevertheless, both species utilize PKC as an integral component in preconditioning in both isolated adult cardiomyocytes and intact hearts, and therefore it appears our results, demonstrating that protection is PKC dependent in this model, are consistent with these studies. Furthermore, we have also demonstrated that signaling in neonates is, in general, comparable with that seen in the intact heart during preconditioning ischemia and reperfusion. Thus the differences seen here in the mechanism of MAPK activation and the role in protection may not be simply be due to differences in neonate versus adult cardiomyocytes but may also reflect differences in the species (rat vs. rabbit) and the models used.

In conclusion, the present study has shown that in this model of delayed preconditioning utilizing glycolytic inhibition alone, with no direct inhibition of mitochondrial respiration, although both ERK1/2 and p38 MAPK are activated during MMPC and MMPC/reperfusion, protection is independent of the activation of these kinases. This is in contrast to a model where direct inhibition of mitochondrial respiration elicits a sustained ERK1/2 activation upon reperfusion, which mediates delayed protection. Further studies are required to determine the protective mechanism underlying delayed preconditioning in this model.