FASEB J. Pierce now sold as Thermo Scientific
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by SAURIN, A. T.
Right arrow Articles by MARBER, M. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by SAURIN, A. T.
Right arrow Articles by MARBER, M. S.
(The FASEB Journal. 2000;14:2237-2246.)
© 2000 FASEB

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes

ADRIAN T. SAURIN, JODY L. MARTIN, RICHARD J. HEADS, CLAIRE FOLEY, JAMES W. MOCKRIDGE, MATT J. WRIGHT, YIBIN WANG* and MICHAEL S. MARBER1

Department of Cardiology, KCL, The Rayne Institute, St. Thomas’ Hospital London SE1 7EH, U.K.; and
* Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

1Correspondence: Department of Cardiology, KCL, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, U.K. E-mail: mike.marber{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of protein kinase C (PKC) and more recently mitogen-activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC-{delta} results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC-{delta}, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal-simulated ischemia activated only p38 (2.8±0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC-{delta} or by preconditioning (0.48±0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0±3.1%, LDH release 77.3±4.0%, and MTT bioreduction 127.1±4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild-type p38{alpha} or p38ß. Transfected p38{alpha} and ß show differential activation (P<0.001) during sustained simulated ischemia, with p38{alpha} remaining activated (1.48±0.36 vs. basal) but p38ß deactivated (0.36±0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38{alpha} (0.65±0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38{alpha}, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9±3.9% and MTT bioreduction 130.2±6.5% of control, n=8, P<0.05). Thus, inhibition of p38{alpha} activation during ischemia reduces injury and may contribute to preconditioning-induced cardioprotection in this model.—Saurin, A. T., Martin, J. L., Heads, R. J., Foley, C., Mockridge, J. W., Wright, M. J., Wang, Y., Marber, M. S. The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes.


Key Words: myocardial ischemia • cardioprotection • ischemic preconditioning • cytoprotection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE TERM ISCHEMIC preconditioning was first coined by Murry and colleagues to describe a phenomenon where brief periods of sublethal ischemia protected or ‘preconditioned’ the heart against infarction caused by a subsequent more prolonged period of coronary artery occlusion (1) . Since this initial discovery, preconditioning has become recognized as the most powerful form of cardioprotection other than reperfusion (2) . Unfortunately, the protection afforded by a brief period of ischemia is short-lived (3) and cannot be renewed (4) . In an attempt to overcome these deficiencies, the signaling pathways responsible for triggering and maintaining this powerful form of cardioprotection have become the focus of many investigators.

Early pharmacological studies delineated the mediator(s) responsible for adaptation in response to brief ischemia. These investigations in whole heart, and later in cell-based models (5) , showed that protection was dependent on the activation of a wide variety of heptahelical transmembrane receptors including adenosine type 1 and 3 (6 , 7) , bradykinin (8) , {alpha}1-adrenergic (9) , endothelin (10) , angiotensin II (11) , and delta-1 opioid receptors (12 , 13) . Although structurally diverse, these receptors have a common feature of coupling to protein kinase C (PKC) via GTP binding proteins. The importance of protein kinase C to ischemic preconditioning has been shown in a variety of studies in whole heart and in isolated ventricular cardiocytes (8 , 11 , 14 15 16) . Although it is widely accepted that PKC plays a pivotal role in ischemic preconditioning, the relevant downstream signaling molecules remain a topic of intense investigation and controversy.

Apart from PKC, the only other well-accepted component of the preconditioning signaling pathway is the ATP-dependent potassium channel. It is uncertain, however, whether this channel lies upstream or downstream of PKC (17 18 19) . Similarly, in preconditioned myocardium an increase in the activation of p38 and p42/44 mitogen-activated protein kinases (MAPK) has been linked to protection (20 , 21) . However, this is not consistent with the reduction in ischemic injury that accompanies p38-MAPK inhibition in similar models (22 23 24) . These inconsistencies only serve to further fuel the investigation of signals distal to PKC.

Neonatal rat ventricular cardiac myocytes have been used as an archetypal model to interrogate the signal transduction cascades underlying cardiac hypertrophy (25) . Hypertrophy and preconditioning have features in common, including agonists, which trigger (endothelin, norepinephrine, angiotensin II), and PKCs, which mediate the final response (25) . These similarities have caused others (26 27 28) and us (29) to characterize models of ischemic preconditioning in this cell type (5) . These and other cell-based models (13 , 30 31 32 33 34 35 36 37) have the advantages of the in vivo features of ischemic preconditioning without the disadvantages of multiple cell types, low transfection efficiency and spatial heterogeneity in depth of ischemia and quality of reperfusion (5) . Using such a model, we previously demonstrated that preconditioning is PKC dependent and can be mimicked by expression of constitutively active PKC-{delta} (29) . Our aim was to use this characterized model to investigate the interplay between preconditioning, PKC, and MAPKs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of rat ventricular cardiomyocytes
Neonatal rat ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats as described previously (29 , 38) . Briefly, cells from neonatal rat ventricles were dispersed in a series of incubations at 37°C in HEPES-buffered salt solution containing 0.5 mg/ml collagenase and 0.6 mg/ml pancreatin. Dispersed cells were then preplated for at least 30 min to minimize fibroblast contamination, and the unattached cells were replated on 6-well gelatin-coated plates at a density of ~1 million cells/well. Fibroblast contamination was less than 5%. The cardiac myocytes were cultured in 4:1, Dulbecco’s modified Eagle’s medium: M199, supplemented with 10% horse serum, 5% fetal calf serum (FCS), and 100 units/ml penicillin/streptomycin at 37°C in room air with 5% CO2 for the first 24 h. Thereafter, cells were maintained in an identical medium with a reduced serum concentration of 1% FCS. Under these conditions, in excess of 80% of cells beat spontaneously for up to 1 wk in culture. Experiments were performed after 2–4 days in culture.

cDNA constructs
The high efficiency eukaryotic expression plasmid pCAGGS was used for all PKC transfections (39) . This plasmid contains the cytomegalovirus immediate early enhancer and chicken ß-actin promoter with the first intron upstream of a multiple cloning site. It has been shown previously that this heterologous promoter is transcriptionally active in cardiac myocytes (40) . PKC mutants were constructed as described previously (29) . Two PKC isotypes were studied: 1) wild-type PKC-{delta}; 2) PKC-{delta} with a limited deletion of the inhibitory pseudosubstrate subdomain (residues 151–160). This mutant PKC isotype has been shown to code for a constitutively active functional protein (41 , 42) . All plasmids were purified by alkaline lysis of the bacterial host (DH5{alpha}), followed by polyethylene glycol precipitation.

Recombinant adenovirus vectors
Recombinant adenoviruses encoding wild-type p38{alpha}, wild-type p38ß, or dominant negative p38{alpha} driven by a cytomegalovirus promoter were generated as described previously (43 , 44) . The dominant negative p38{alpha} has a mutated phosphorylation site (TGY180–182 to AGF), rendering it resistant to phosphorylation (45) . Recombinant adenoviruses were tested for transgene expression in cardiac myocytes by reverse transcriptase-polymerase chain reaction, Western blot, or kinase assays. The concentrated recombinant adenoviruses were prepared and titered as described (44) .

Transfection of neonatal cardiomyocytes
Cardiocytes at 70–80% confluency were transfected with pCAGGS expression plasmid by an integrin targeting peptide-mediated transfection procedure described previously (46) . The peptide–Lipofectin complexes were prepared by mixing 40 µl peptide (0.1% w/v) and 0.75 µl Lipofectin (Life Technologies Ltd., Paisley, U.K.). DNA (0.01% w/v) in optimem was added to peptide–Lipofectin complex at a ratio of 2.5:1 (v/v). DNA–peptide–Lipofectin complexes were allowed to stand for 1 h at room temperature before use; 100 µl of this mix was diluted to 1 ml in optimem and added to one well of a 6-well plate. Cells were then incubated overnight at 37°C in room air supplemented with 5% CO2. Thereafter, complex/optimem was removed and replaced with maintenance medium containing 1% FCS and the cells were returned to the incubator. Cell extracts were assayed for protein 48–72 h post-transfection. By using pCAGGS-GFP as a reporter, transfection efficiency was consistently between 20 and 30%.

Cells maintained in serum-free medium were infected with adenoviruses at a multiplicity of infection of 10 plaque-forming units/cell for 1 h at 37°C in room air containing 5% CO2. Cells were then cultured in maintenance medium containing 1% FCS for an additional 48–72 h before biochemical analysis.

Ischemia model
The cells were washed once with phosphate-buffered saline (PBS) before addition of 1 ml of ischemia buffer (118 mM NaCl, 24 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.5 mM sodium.EDTA.2H2O, 20 mM sodium lactate, and 16 mM KCl, pH 6.2) pregassed with 5% CO2, 95% argon. On addition of ischemia buffer, spontaneous contraction within the monolayer ceased. Cells were then transferred to anaerobic GasPak pouches (Becton Dickinson, Sparks, Md.) and incubated at 37°C for up to 6 h. The O2 content of the atmosphere inside the pouches was <1% for the duration of the experiment as measured by an anaerobic indicator.

Measurement of enzyme release
Upon opening the ischemia chamber (reoxygenation), 200 µl samples of the ischemia buffer were gently collected for the determination of creatine kinase (CK) and lactate dehydrogenase (LDH). The next day a spectrophotometric CK and LDH enzyme assay was performed with Boehringer Mannheim (Mannheim, Germany; MPR-1) and Sigma (St. Louis, Mo.; TOX-7) assay kits, respectively.

Evaluation of cell viability
After simulated ischemia, cells were reoxygenated in maintenance medium containing 1% FCS. After 2 h, medium was aspirated and cells incubated in 500 µl PBS containing 2.5 mg 3-(4,5-dimethylthiaziazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) for 30 min at 37°C in room air containing 5% CO2. During this incubation the tetrazolium component of the dye is reduced, in metabolically active cells, to a formazan dye. Thereafter the reaction was terminated by addition of 500 µl solubilization solution (0.1 mol/l HCl, 10% triton X100, in isopropanol) and the absorbance of the lysate was recorded at 570 nm using an ELISA reader.

Western blot analysis
Cells from parallel plates were washed three times in ice-cold PBS and harvested in 1 ml of hot electrophoresis sample buffer (250 mM Tris-HCl, 4% sodium dodecyl sulfate, 10% glycerol, and 2% ß-mercaptoethanol, pH 6.8), then boiled for an additional 5 min. The cell extracts were then centrifuged for 5 min to remove insoluble material; 0.003% bromphenol blue was added and the samples were loaded on a 10% polyacrylamide gel. After 1-dimensional separation the protein was electrophoretically transferred to nitrocellulose membranes (Hybond C, Amersham, U.K.). Coomassie staining of identically loaded gels confirmed uniform protein loading.

Blots were sequentially probed with either murine monoclonal antibodies specific for ERK2 (Santa Cruz Biotechnology, Santa Cruz, Calif.) and a peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (DAKO A/S, Glostrup, Denmark) or rabbit polyclonal antibodies specific for p38, phospho-p38, or phospho-p42/44 (New England BioLabs, Hitchin, U.K.) and a peroxidase-conjugated swine anti-rabbit IgG secondary antibody (DAKO A/S). Secondary antibodies were then detected by incubation of the nitrocellulose with enhanced chemiluminescence (ECL, Amersham, Little Chalfont, U.K.) for 60 s prior to exposure to autoradiography film. The densities of all Western blot bands were analyzed using NIH Image version 1.61.

Statistical analysis
All values are expressed as mean ±SE. Data for individual treatments were collected from no more than two wells from each experimental preparation. The ‘n’ numbers under ‘results’ relate to the number of wells from which data were obtained. For each treatment, mean values were pooled to allow statistical comparisons. Statistical comparisons between groups were performed by one-way analysis of variance. All post hoc comparisons were by the Fischer protected least significant difference method. All analyses were performed using Statview version 4.0 statistical package (Abacus Concepts Inc., Berkeley, Calif.). A probability value <= 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of MAPKs during preconditioning in isolated neonatal cardiac myocytes
Preconditioning with 90 min ischemia and 30 min reoxygenation has been shown to delay cell death in response to subsequent prolonged ischemia in our model (29) . p38-MAPK activation has been reported during preconditioning-like ischemia in the intact heart (47) . It has been proposed that this activation is associated with subsequent protection against lethal ischemia (20) . Therefore, we wished to investigate whether similar activation occurred during preconditioning with simulated ischemia in our model. Cardiocytes were harvested at various time points during an acute preconditioning stimulus of 90 min ischemia and 30 min reoxygenation. Cell lysates were probed with phospho-specific antibodies for p38, p42/44, or p46/54 to quantify MAPK activation. These preliminary experiments demonstrated that although no p46/54 phosphorylation could be detected, p38 and p42/44 were transiently activated during reoxygenation after sublethal ischemia (results not shown) and that at 10 min reoxygenation both p38 and p42/44 displayed maximal activation (see Fig. 1 ).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Activation of p38 and p42/44 MAPK during reoxygenation after 90 min simulated ischemia. Myocyte cell lysates were prepared from naive cells (control) and from wells after 90 min SI and 10 min reoxygenation (ischemia). Each pair of samples is from a different cardiocyte preparation exposed independently to simulated ischemia and reoxygenation. Samples are probed with dual phospho-specific MAPK antibodies for p42/44 (Ai) and p38 (Bi) or antibodies detecting total p42 (Aii) and total p38 (Bii). Panel C represents the mean activation of p38, p42 and p44 during simulated ischemia/reoxygenation expressed as a ratio of that seen in naive cells in 5 independent experiments. *P<0.01 vs. naive control, n=5.

The specific phosphorylation of MAPKs during reoxygenation may be a consequence of PKC activation, since it had been reported previously that PKC is activated at the onset of reperfusion (48) . If this is true in our model then we should observe a comparable level of MAPK phosphorylation in cells overexpressing active PKC-{delta} even in the absence of preconditioning ischemia. Therefore, to test this hypothesis we transfected myocytes with constitutively active PKC-{delta} in an attempt to mimic the pattern of p38 and p42/44 phosphorylation seen after 90 min simulated ischemia and 10 min reoxygenation.

Effect of PKC-{delta} on MAPK activation in cardiac myocytes
To determine whether PKC-{delta} could activate MAPKs, myocytes were transfected with the eukaryotic expression plasmid pCAGGS encoding either wild-type PKC-{delta}, active PKC-{delta}, or vector alone. After 48–72 h, myocytes were either harvested, to assess basal MAPK phosphorylation, or subjected to 90 min ischemia and 10 min reoxygenation to examine preconditioning-induced MAPK activation. It is apparent from Fig. 2 that overexpression of active PKC-{delta} has no effect on basal p42/44 phosphorylation compared to controls (Fig. 2Ai ). Surprisingly, however, the increase in p42/44 phosphorylation observed during preconditioning was completely abolished in the presence of active PKC-{delta}; this is not due to a down-regulation of MAPK, as the total amounts of p42 remain constant between treatments (Fig. 2Aii ). In contrast, p38 exhibits a higher basal activation in cells expressing active PKC-{delta}; however, once again the preconditioning-induced increase in p38 phosphorylation is completely abolished (see Fig. 2Bi ).



View larger version (49K):
[in this window]
[in a new window]
  		Figure 2. Activation of p38 and p42/44 MAPK after preconditioning in myocytes overexpressing PKC-{delta}. A) Western blot probed with anti-phospho-p42/44 (i) or anti-p42 (ii). B) Western blot probed with anti-phospho-p38 (i) or anti-p38 (ii). Constituent proteins in both panels were derived from the same experimental groups. Naive cells (-) were compared to preconditioned (+) in untreated cardiocytes, cells transfected with expression plasmid containing an empty multiple cloning site, encoding wild-type PKC-{delta}, or constitutively active PKC-{delta} (see Materials and Methods). A) Maximal p42/44 MAPK activation was assessed in cell lysates harvested after 10 min reoxygenation after 90 min simulated ischemia, using dual phospho-specific p42/44 antibodies (i). Total p42 levels were detected using a monoclonal anti-p42 antibody (ii). B) p38 activation was detected using phospho specific p38 antibodies (i) and total p38 was detected using anti-p38 antibodies (ii).

These data did not support our original hypothesis that PKC-{delta} protects by causing the same activation of MAPKs that occurs with preconditioning. In contrast and paradoxically, these data suggest that PKC-{delta} inhibits MAPK activation in response to ischemia/reoxygenation. It is possible that this unexpected negative regulation may be the mechanism through with PKC-{delta} protects against lethal ischemia (29) . This alternative hypothesis is consistent with reports examining the role of p38 during ischemia in the absence of preconditioning, which show that inhibition is protective (22 23 24) . However, for PKC-{delta} to protect via this mechanism, the same inhibitory effect on MAPKs must occur during prolonged lethal ischemia. Therefore, we delineated the MAPK pathways that were activated by simulated ischemia alone.

Activation of MAPKs during prolonged simulated ischemia
To quantify the level of MAPK activation during ischemia, cardiocytes were subjected to varying duration’s of simulated ischemia before cells were harvested and constituent proteins probed with anti-phospho-p38 and -p42/44 antibodies (see Fig. 3 ). In agreement with other recent reports (22 , 49) , we found that during ischemia p38 exhibits a prolonged activation, which is maximal after 2.5 h ischemia (Fig. 3B ). In contrast, phospho-p42/44 MAPK is inhibited below basal levels for the entire duration of ischemia (Fig. 3A ). This suppression of p42/44 during ischemia eliminates the possibility that PKC-{delta} protects through negative regulation of p42/44 phosphorylation. Therefore, we wished to examine the result of PKC-{delta} overexpression on the activation of p38 during simulated ischemia.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 3. Time course of p38 and p42/44 MAPK activation during prolonged simulated ischemia in naive cardiocytes. Cardiocytes were subjected to varying duration’s of simulated ischemia (0–6 h) before cells were lysed for Western blot analysis. A) Constituent proteins were probed with dualphospho-specific p42/44 antibodies to assess p42/44 MAPK activation or anti-p42 antibodies to examine p42 levels. B) Samples identical to panel A were probed with anti-dualphospho-p38 antibodies to detect p38 activation or anti-p38 antibodies to assess total p38 levels.

Effect of active PKC-{delta} on p38 phosphorylation during lethal simulated ischemia
Isolated myocytes were either cultured under normal conditions (untreated) or transfected with plasmids encoding wild-type or active PKC-{delta}; 48–72 h post-transfection, cardiocytes were subjected to 2.5 h simulated ischemia and immediately harvested for Western blot analysis. Lysates were probed with anti-p38 or anti-phospho-p38 antibodies to detect the effect of PKC-{delta} overexpression on either p38 induction or activation (see Fig. 4 ). Overexpression of wild-type PKC-{delta} increased p38 phosphorylation during ischemia (Fig. 4i) , although comparable results with empty vector alone suggest this is not a consequence of PKC-{delta} (results not shown). However, transfection of active PKC-{delta} significantly attenuated the ischemia-induced p38 activation (Fig. 4i) , although there was no effect on total p38 levels (Fig. 4ii ).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 4. Effect of active PKC-{delta} expression on p38 phosphorylation during ischemia. 48–72 h post-transfection, myocytes were subjected to 2.5 h simulated ischemia and harvested for Western blot analysis to detect p38 dual phosphorylation. Using dual phospho-specific p38 antibodies, ischemia-induced p38 activation was detected in untreated cells or cells transfected with wild-type or active PKC-{delta} (i). Total p38 levels in the same cells were assessed with anti-p38 (ii).

We have shown previously that specific activation of PKC-{delta} during ischemia protects myocytes against cell death (29) and, as we show here, PKC-{delta} activation causes an inhibition of p38 phosphorylation. Since preconditioning also protects by activating PKC, we sought to compare its effect on p38 activation by examining ischemia-induced phosphorylation in naive and preconditioned cells.

Activation of p38 during simulated ischemia after preconditioning
Cardiocytes were preconditioned with 90 min simulated ischemia and 30 min reoxygenation. These and untreated myocytes were then subjected to 2.5 h ischemia to maximally activate p38. Thereafter p38 phosphorylation was assessed, as before, by immunoblotting with phospho-p38 antibodies. Figure 5 shows that, akin to active PKC-{delta} overexpression, preconditioning consistently inhibited p38 activation during ischemia (P<0.01).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. The consequence of preconditioning on subsequent p38 activation during simulated ischemia. Ai) Western blot probed with anti-phospho-p38 to detect p38 MAPK activation. Lysates were prepared from untreated cells or cells after 2.5 h ischemia in preconditioned and control cells. Aii) Total p38 levels were detected in the same cells with anti-p38. B) Mean p38 activation compared to untreated controls (n=4). *P<0.01, p38 activation during ischemia vs. nonischemic/untreated. §P<0.01, p38 activation during ischemia with vs. without preconditioning.

The negative regulation of p38 activation during ischemia is therefore associated with the cardioprotective effects of both preconditioning and active PKC-{delta} overexpression. However, on the basis of these results alone, it is not possible to ascertain whether the inhibition is a cause of protection or simply a consequence of the attenuation of ischemic injury. Therefore, to define the role of p38 on cell viability after ischemia, we used pharmacological inhibition of p38 in an attempt to mimic protection afforded by preconditioning/PKC.

Consequence of p38 activation on myocyte viability after lethal ischemia
We wished to examine the role of p38 during ischemia by inhibiting activity with SB203580, which reversibly binds to the ATP binding site (50) . Using two separate end points of CK and LDH release to assess cell injury and by measuring cell viability with MTT bioreduction, we can clearly see that inhibition of p38 during ischemia significantly protects myocytes against cell death (see Fig. 6 ). Therefore, during ischemia p38 activation is detrimental to myocyte survival, because specific inhibition of this pathway is sufficient to protect myocytes. Hence, the attenuation of p38-MAPK activation by PKC-{delta} overexpression and preconditioning contributes to the protective effect of both these treatments.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Cell viability after 6 h of simulated ischemia in the presence of SB203580. A) Total CK and LDH released into ischemia buffer during 6 h of simulated ischemia. Panel A therefore represents cell injury during ischemia alone. B) Cell viability was measured by MTT bioreduction within monolayers after 6 h simulated ischemia and 2 h reoxygenation. Injury in the presence of SB203580 ({blacksquare}) is expressed as a percentage of injury in the absence of SB203580 ({square}). Viability in cells treated with SB203580 alone for 6 h (without ischemia) was equivalent to that observed in untreated cells (results not shown). All P values are for comparisons between ischemia in the presence and absence of SB203580. *P<0.05, **P<0.001, n=20.

The measurement of total p38 activation through the use of dual phospho-specific antibodies has limitations. In light of recent reports characterizing new p38 isoforms that share the same TGY motif recognized by commercially available antibodies, it has become apparent that isotypes may differ significantly in function, if not in structure. In fact, Wang and co-workers have proposed opposing roles for p38{alpha} and p38ß on myocyte survival, postulating that p38{alpha} may be responsible for cell death and apoptosis, and p38ß for hypertrophy and survival (51) . Therefore, an increase in p38{alpha} activation with a comparable decrease in p38ß phosphorylation may not alter total p38 activation as detected by phospho-specific antibodies. But if the Wang hypothesis were correct, we would expect such a change in the balance of active p38 isoforms to cause a large decrease in cell viability. Thus, there is a possibility that this decrease in viability may not be correctly attributed to p38 activation using currently available antibodies, so we wanted to examine the isotype specific activation of p38 during ischemia in our model.

Activation of p38 isotypes during simulated ischemia
To look at the activation of individual isotypes, we used recombinant adenoviruses as an efficient gene delivery vector to express various p38 signaling molecules (51 , 52) . Using a recombinant adenovirus expressing the green fluorescent protein (GFP) as a reporter, greater than 95% of myocytes express the transgene 48–72 h post-transfection. Cardiomyocytes were infected with vectors expressing FLAG-tagged wild-type p38{alpha} and p38ß. Adenovirally encoded p38ß has a higher apparent MW than p38{alpha}, thus enabling us to easily distinguish between the isoforms using p38 antibodies (Fig. 7 ). At a multiplicity of infection of 10, adenoviral-directed p38{alpha} and p38ß expression was detected at comparable levels by Western blot analysis (Fig. 7A ).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 7. Activation of p38 isotypes during ischemia in untreated and preconditioned cells. Cardiocytes were infected with adenoviral constructs encoding FLAG-tagged wild-type p38-{alpha} or -ß. 48–72 h postinfection untreated and preconditioned cardiocytes were subjected to 2.5 h ischemia before cells were harvested and lysed for Western blots. A) The relative expression of p38 signaling molecules in infected cells were compared with untreated cells using anti-p38 antibodies. B) Identical lysates were probed with dualphospho-specific p38 antibodies to detect isotype-specific p38 activation during ischemia. C) Mean p38 activation during ischemia compared to untreated controls in at least 4 independent experiments. §P<0.001, p38{alpha} activation vs. p38ß activation during ischemia. *P<0.05, p38{alpha} activation during ischemia with vs. without preconditioning (IP). P<0.01, p38ß activation during ischemia vs. nonischemic/untreated cells.

Using phospho-specific antibodies to examine activation, we noted that during ischemia p38{alpha} exhibits a strong phosphorylation similar to that seen with endogenous p38 in untransfected controls (see Fig. 7B ). Preconditioning, which decreases endogenous p38 activation during ischemia, also significantly attenuates ectopically expressed p38{alpha} phosphorylation (P<0.05). In contrast, transfected p38ß, which exhibits a high level of basal phosphorylation, is inhibited during ischemia (P<0.01) and this inhibition is moderately enhanced in preconditioned cells (Fig. 7C ).

Effect of p38 isotype activation during simulated ischemia
As shown in Fig. 6 and elsewhere (22 23 24 , 53) , inhibition of p38 during ischemia using SB203580 protects against cell death. Since we demonstrate a selective activation of p38{alpha} over p38ß during ischemia, we would expect that protection ensues as a result of p38{alpha} inhibition with SB203580. To test this hypothesis, we transfected cells with a dominant negative p38{alpha} mutant (TGY180–182 to AGF: see Materials and Methods). As shown in Fig. 8A , 48 h after transfection of either wild-type or dominant negative p38{alpha} (p38{alpha}DN), comparable overexpression of p38 can be detected. After 2.5 h ischemia, which is the time of maximal p38 activation, cells expressing p38{alpha}DN showed no significant p38 activation whereas cells expressing wild-type p38{alpha} showed increased p38 activation (Fig. 8B ). Moreover, the cells expressing p38{alpha}DN were protected against simulated ischemia compared to empty vector transfected cells (CK release=82.9±3.9% and MTT bioreduction=130.2±6.5%, n=8, P<0.05, see Fig. 9 ). Expression of wild-type p38{alpha} had no effect on cell injury (Fig. 9) .



View larger version (30K):
[in this window]
[in a new window]
  		Figure 8. Modulation of ischemic p38 activation with p38{alpha}DN. Cardiocytes were infected with adenoviral constructs encoding FLAG-tagged dominant negative or wild-type p38{alpha}. 48 h postinfection, cells were subjected to 2.5 h simulated ischemia or left untreated. Myocytes were then harvested and lysed for Western blot analysis. A) The total p38 levels were assessed with anti-p38 antibodies. Adenoviral infection causes a consistent level of expression of either wild-type or dominant negative p38{alpha} that is unaltered during ischemia. B) Activation of p38 was assessed using phospho-specific p38 antibodies. Ischemia caused a marked activation of p38 in wild-type-p38{alpha} transfected cells, whereas p38 activation decreased during ischemia in cells expressing dominant negative p38{alpha}.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 9. Effect of p38{alpha} activation on cell survival during ischemia. Myocytes transfected as in Fig. 8 were exposed to simulated ischemia for 6 h. Panel A therefore represents cell injury during ischemia alone. B) Cell viability was measured by MTT bioreduction within monolayers after 6 h simulated ischemia and 2 h reoxygenation. Injury in the presence of p38{alpha}DN ({blacksquare}) is expressed as a percentage of injury in the presence of an empty vector ({square}). Viability in cells treated with p38{alpha}DN alone for 6 h (without ischemia) was equivalent to that observed in untreated cells (results not shown). All P values are for comparisons between ischemia in the presence of p38aDN vs. empty vector. *P<0.05, n=8.

In summary, our data suggest that simulated ischemia selectively activates ectopically expressed p38{alpha} over p38ß (P<0.001). One consequence of this activation in our system is a decrease in myocyte survival. Constitutively active PKC-{delta}, which renders myocytes resistant to ischemia, inhibits ischemia-induced p38{alpha} activation. An identical effect occurs in untransfected cells preconditioned by brief simulated ischemia. Similar inhibition of p38{alpha} with a dominant negative mutant also protects against ischemia. These data suggest that the protection conferred by preconditioning may, at least in part, be mediated through a reduction in ischemia-driven p38{alpha} activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Experimental findings
We have investigated the relationship between preconditioning, protein kinase C and mitogen-activated protein kinase in primary cultures of neonatal cardiocytes. Our data show that during lethal simulated ischemia, p38{alpha}-MAPK exhibits a period of prolonged phosphorylation that reduces myocyte survival. In addition, preconditioning and the expression of active PKC-{delta} inhibit p38{alpha} activation during lethal ischemia and enhance myocyte survival. Inhibition of p38{alpha} activation with SB203580 or dominant negative p38{alpha} (p38{alpha}DN) also gives rise to protection. These findings suggest that p38{alpha}-MAPK modulates cardiocyte survival during simulated ischemia, which may represent a new and specific therapeutic target.

Signaling during preconditioning and ischemia
Previous reports suggest that activation of p38 during preconditioning is responsible for the resulting protection (20 , 54 , 55) . These conclusions were based on the ability of SB203580 to inhibit protection when given during preconditioning. If p38 activation during preconditioning occurs downstream of PKC, then we would expect overexpression of active PKC{delta}, which we have shown protects myocytes during ischemia (29) , to have activated the p38 pathway. Our findings do not support this hypothesis, since p38 activation was inhibited rather than activated by active PKC-{delta}. Expression of active PKC-{varepsilon}, another PKC isoform implicated in preconditioning (56) , also inhibits p38 activation during simulated ischemia (results not shown). These findings support a protective role for PKC activation during lethal simulated ischemia since all studies addressing the role of p38 during ischemia, in the absence of preconditioning, demonstrate that activation is detrimental, with SB203580 decreasing infarct size and enhancing postischemic functional recovery (23 , 24) .

The effect of preconditioning on p38 signaling during ischemia
Overexpression of active PKC mutants does not necessarily mimic physiological preconditioning, so we wanted to examine the effect of ischemic preconditioning on p38 activation during ischemia. As shown in Fig. 5 , preconditioning significantly inhibited p38 activation during ischemia. Of course, the basis of this inhibition could be a consequence rather than a cause of protection. If a cause of protection, one would expect p38 inhibition to protect during ischemia. As shown in Fig. 6 and in agreement with other reports (22 23 24) , inhibition of p38 with SB203580 does protect against ischemic cell death. This is the first data, to our knowledge to document a mechanistic link between ischemic preconditioning and reduced p38 activation during ischemia.

The contribution of p38 isoforms
The limitations of this work became apparent when considering recent reports of various p38 isotypes, two of which (p38{alpha} and ß) are highly expressed in the heart. The likelihood that these isotypes carry out different, perhaps even opposite, intracellular functions casts doubt over the use of nonselective inhibitors (SB203580) and antibodies to infer the function of p38. Neglecting isoform-specific effects could lead to contradictory results. For example, a treatment such as preconditioning may switch the balance of activation from one isoform to another, which, if they have opposing roles may have a dramatic effect on cell fate. But the detection of p38 phosphorylation using antibodies would be insensitive to this switch, and thus not show a significant change in overall activation. This may explain the findings in rabbit cardiomyocytes when SB203580 given during ischemia actually accelerates injury (57) . Examining the differential activation of p38 isoforms allows a more complete appraisal of their effects on cell viability.

To do this, we used adenoviral-mediated expression of wild-type p38{alpha} and ß into rat neonatal cardiocytes, which, unlike whole heart preparations, produced transfection efficiencies greater than 95%. Assessment of isotype activation after 2.5 h ischemia showed that p38{alpha} was activated, whereas p38ß was significantly inhibited (Fig. 7C ). To our knowledge, this is the first demonstration of differential activation of p38 isoforms by a physiological stress. According to Wang and co-workers, p38{alpha} activation in cardiac myocytes is sufficient to cause apoptosis and cell death, whereas p38ß is responsible for hypertrophy and survival (52) . In our model, this hypothesis would fit with a mechanism whereby p38{alpha} is the specific detrimental MAPK isoform activated by ischemia. Preconditioning, which inhibits p38, also attenuates the p38{alpha} pathway during ischemia, which should account at least in part for the associated protection (Fig. 7C ).

The consequence of p38{alpha} activation during ischemia
If decreased p38{alpha} activation does contribute to protection, then inhibition of this pathway should be sufficient to protect. Although SB203580 has been shown to protect, both in this study (Fig. 6) and others (22 23 24) , it inhibits both p38{alpha} and p38ß. Moreover, recent reports have questioned its specificity since it can inhibit (58 , 59) and even activate (60) other kinases.

We used dominant negative p38 mutants to specifically inhibit the activation of individual isoforms during ischemia. Dominant negative p38{alpha} decreased endogenous p38 activation during ischemia, which may reflect a decrease in endogenous p38ß activation (Fig. 8B ). This inhibition of p38{alpha} caused an increase in cell viability (Fig. 9) , whereas the presence of p38ßDN had no effect (results not shown).

In summary, sustained p38 activation occurs during lethal simulated ischemia in cultured rat neonatal cardiocytes. This activation can be attenuated by cardioprotective treatments such as preconditioning and overexpression of active PKC-{delta}. Moreover, our results support the concept that p38{alpha} and p38ß are differentially regulated during ischemia, since ischemia is accompanied by an increase in p38{alpha} and a decrease in p38ß phosphorylation. In addition, specific inhibition of p38{alpha} activation, but not p38ß, is protective. Taken together, these observations suggest that the inhibition of p38{alpha} activation during prolonged ischemia is the cause rather than the consequence of preconditioning. Ultimately, understanding the signaling mechanisms that modulate ischemic cell injury will facilitate novel interventions that preserve ventricular function by reducing the rate of necrosis during myocardial infarction.


   ACKNOWLEDGMENTS
 
This work was funded in part by the British Heart Foundation and the Wellcome Trust. Integrin targeting peptide was provided by Dr. S. L. Hart, Institute of Child Health, University College London Medical School, London, U.K. Recombinant adenoviruses encoding p38-MAPKs were provided by Ken Chien (UCSD, La Jolla, Calif.).

Received for publication August 25, 1999. Revision received April 26, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Murry, C. E., Jennings, R. B., Reimer, K. A. (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74,1124-1136[Abstract/Free Full Text]
  2. Kloner, R. A., Bolli, R., Marban, E., Reinlib, L., Braunwald, E. (1998) Medical and cellular implications of stunning, hibernation and preconditioning. An NHLBI workshop. Circulation 97,1848-1867[Free Full Text]
  3. Van Winkle, D. M., Thornton, J., Downey, J. M. (1999) Cardioprotection from ischemic preconditioning is lost following prolonged reperfusion in the rabbit. Coronary Heart Dis 2,613-619
  4. Cohen, M. V., Yang, M. X., Downey, J. M. (1994) Conscious rabbits become tolerant to multiple episodes of ischemic preconditioning. Circ. Res. 74,998-1004[Abstract/Free Full Text]
  5. Marber, M. S. (2000) Ischemic preconditioning in isolated cells. Circ. Res. 86,926-931[Free Full Text]
  6. Armstrong, S., Ganote, C. E. (1994) Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement. Cardiovasc. Res. 28,1049-1056[Abstract/Free Full Text]
  7. Dougherty, C., Barucha, J., Schofield, P. R., Jacobson, K. A., Liang, B. T. (1998) Cardiac myocytes rendered ischemia resistant by expressing the human adenosine A1 or A3 receptor. FASEB J 12,1785-1792[Abstract/Free Full Text]
  8. Goto, M., Liu, Y., Yang, X.-M., Ardell, J. L., Cohen, M. V., Downey, J. M. (1995) Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ. Res. 77,611-621[Abstract/Free Full Text]
  9. Tsuchida, A., Liu, Y., Liu, G. S., Cohen, M. V., Downey, J. M. (1994) {alpha}1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ. Res. 75,576-585[Abstract/Free Full Text]
  10. Wang, P., Gallagher, K. P., Downey, J. M., Cohen, M. V. (1996) Pretreatment with endothelin-1 mimics ischemic preconditioning against infarction in isolated rabbit heart. J. Mol. Cell. Cardiol. 28,579-588[Medline]
  11. Liu, Y., Tsuchida, A., Cohen, M. V., Downey, J. M. (1995) Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J. Mol. Cell. Cardiol. 27,883-892[Medline]
  12. Schultz, J. E., Rose, E., Yao, Z., Gross, G. J. (1995) Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts. Am. J. Physiol. 268,H2157-H2161[Abstract/Free Full Text]
  13. Liang, B. T., Gross, G. J. (1999) Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels. Circ. Res. 84,1396-1400[Abstract/Free Full Text]
  14. Kitakaze, M., Node, K., Minamino, T., Komamura, K., Funaya, H., Shinozaki, Y., Chujo, M., Mori, H., Inoue, M., Hori, M., Kamada, T. (1996) Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase. Circulation 93,781-791[Abstract/Free Full Text]
  15. Lasley, R. D., Noble, M. A., Mentzer, R. M., Jr (1997) Effects of protein kinase C inhibitors in in situ and isolated ischemic rabbit myocardium. J. Mol. Cell. Cardiol. 29,3345-3356[Medline]
  16. Mitchell, M. B., Meng, X., Ao, L., Brown, J. M., Harken, A. H., Banerjee, A. (1995) Preconditioning of isolated rat heart is mediated by protein kinase C. Circ. Res. 76,73-81[Abstract/Free Full Text]
  17. Walsh, R. S., Tsuchida, A., Daly, J. J., Thornton, J. D., Cohen, M. V., Downey, J. M. (1994) Ketamine-xylazine anaesthesia permits a KATP channel antagonist to attenuate preconditioning in rabbit myocardium. Cardiovasc. Res. 28,1337-1341[Abstract/Free Full Text]
  18. Gross, G. J., Fryer, R. M. (1999) Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning. Circ. Res. 84,973-979[Abstract/Free Full Text]
  19. Gross, G. J. (1995) ATP-sensitive potassium channels and myocardial preconditioning. Basic Res. Cardiol. 90,85-88[Medline]
  20. Weinbrenner, C., Liu, G. S., Cohen, M. V., Downey, J. M. (1997) Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J. Mol. Cell. Cardiol. 29,2383-2391[Medline]
  21. Nakano, A., Baines, C. P., Kim, S. O., Pelech, S. L., Downey, J. M., Cohen, M. V., Critz, S. D. (2000) Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK. Circ. Res. 86,144-151[Abstract/Free Full Text]
  22. Mackay, K., Mochly-Rosen, D. (1999) An inhibitor of p38 Mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia. J. Biol. Chem. 274,6272-6279[Abstract/Free Full Text]
  23. Barancik, M., Htun, P., Strohm, C., Kilian, S., Schaper, W. (2000) Inhibition of the cardiac p38-MAPK pathway by SB203580 delays ischemic cell death. J. Cardiovasc. Pharmacol. 35,474-483[Medline]
  24. Yue, T. L., Wang, C., Gu, J., Ma, X. L., Kumar, S., Lee, J. C., Feuerstein, G. Z., Thomas, H. C., Maleeff, B., Ohlstein, E. H. (2000) Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ. Res. 86,692-699[Abstract/Free Full Text]
  25. Sugden, P. H., Clerk, A. (1998) ‘Stress-responsive’ mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ. Res. 83,345-352[Free Full Text]
  26. Webster, K. A., Discher, D. J., Bishopric, N. H. (1995) Cardioprotection in an in vitro model of hypoxic preconditioning. J. Mol. Cell. Cardiol. 27,453-458[Medline]
  27. Gray, M. O., Karliner, J. S., Mochly-Rosen, D. (1997) A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J. Biol. Chem. 272,30945-30951[Abstract/Free Full Text]
  28. Ovelgonne, J. H., Van Wijk, R., Verkleij, A. J., Post, J. A. (1996) Cultured neonatal rat heart cells can be preconditioned by ischemia, but not by heat shock. The role of stress proteins. J. Mol. Cell. Cardiol. 28,1617-1629[Medline]
  29. Zhao, J., Renner, O., Wightman, L., Sugden, P. H., Stewart, L., Miller, A. D., Latchman, D. S., Marber, M. S. (1998) The expression of constitutively active isotypes of protein kinase C to investigate preconditioning. J. Biol. Chem. 273,23072-23079[Abstract/Free Full Text]
  30. Vanden Hoek, T. L., Becker, L. B., Shao, Z., Li, C., Schumacker, P. T. (1998) Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. 273,18092-18098[Abstract/Free Full Text]
  31. Strickler, J., Jacobson, K. A., Liang, B. T. (1996) Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors. J. Clin. Invest. 98,1773-1779[Medline]
  32. Wu, S., Li, H. Y., Wong, T. M. (1999) Cardioprotection of preconditioning by metabolic inhibition in the rat ventricular myocyte. Involvement of kappa-opioid receptor. Circ. Res. 84,1388-1395[Abstract/Free Full Text]
  33. Arstall, M. A., Zhao, Y. Z., Hornberger, L., Kennedy, S. P., Buchholz, R. A., Osathanondh, R., Kelly, R. A. (1998) Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia. J. Mol. Cell. Cardiol. 30,1019-1025[Medline]
  34. Liu, Y., Gao, W. D., O’Rourke, B., Marban, E. (1996) Cell-type specificity of preconditioning in an in vitro model. Basic Res. Cardiol. 91,450-457[Medline]
  35. Shirai, T., Rao, V., Weisel, R. D., Ikonomidis, J. S., Li, R. K., Tumiati, L. C., Merante, F., Mickle, D. A. (1998) Preconditioning human cardiomyocytes and endothelial cells. J. Thorac. Cardiovasc. Surg. 115,210-219[Abstract/Free Full Text]
  36. Ikonomidis, J. S., Tumiati, L. C., Weisel, R. D., Mickle, D. A., Li, R. K. (1994) Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischaemia. Cardiovasc. Res. 28,1285-1291[Abstract/Free Full Text]
  37. Armstrong, S., Downey, J. M., Ganote, C. E. (1994) Preconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and calphostin C, a protein kinase C inhibitor. Cardiovasc. Res. 28,72-77[Abstract/Free Full Text]
  38. Cumming, D. V., Heads, R. J., Watson, A., Latchman, D. S., Yellon, D. M. (1996) Differential protection of primary rat cardiocytes by transfection of specific heat stress proteins. J. Mol. Cell. Cardiol. 28,2343-2349[Medline]
  39. Niwa, H., Yamamura, K., Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108,193-199[Medline]
  40. Marber, M. S., Mestril, R., Chi, S. H., Sayen, M. R., Yellon, D. M. (1995) Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 95,1446-1456
  41. Genot, E. M., Parker, P. J., Cantrell, D. A. (1995) Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation. J. Biol. Chem. 270,9833-9839[Abstract/Free Full Text]
  42. Decock, J. B., Gillespie-Brown, J., Parker, P. J., Fuller, S. J. (1994) Classical, novel and atypical isoforms of PKC stimulate ANF- and TRE/AP-1-regulated-promoter activity in ventricular cardiomyocytes. FEBS Lett 356,275-278[Medline]
  43. Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R. J., Han, J. (1997) Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity 6,739-749[Medline]
  44. Wang, Y., Krushel, L. A., elman, G. M. (1996) Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. Proc. Natl. Acad. Sci. USA 93,3932-3936[Abstract/Free Full Text]
  45. Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R. J., Nemerow, G. R., Han, J. (1997) Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity 6,739-749
  46. Hart, S. L., Arancibia-Carcamo, C. V., Wolfert, M. A., Mailhos, C., Ali, R. R., Coutelle, C., George, A. J., Harbottle, R. P., Knight, A. M., Levinsky, R. J., Seymour, L. W., Thrasher, A. J., Kinnon, C. (1998) Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector. Human Gene Ther 9,575-585[Medline]
  47. Bogoyevitch, M. A., Gillespie-Brown, J., Ketterman, A. J., Fuller, S. J., Ben-Levy, R., Ashworth, A., Marshall, C. J., Sugden, P. H. (1996) Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. 38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion [review; 108 refs]. Circ. Res. 79,162-173[Abstract/Free Full Text]
  48. Ping, P., Zhang, J., Qiu, Y., Tang, X.-L., Manchikalapudi, S., Cao, X., Bolli, R. (1997) Ischemic preconditioning induces selective translocation of protein kinase C isoforms {varepsilon} and {eta} in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ. Res. 81,404-414[Abstract/Free Full Text]
  49. Nagarkatti, D. S., Sha’afi, R. I. (1998) Role of p38 MAP kinase in myocardial stress. J. Mol. Cell. Cardiol. 30,1651-1664[Medline]
  50. Young, P. R., McLaughlin, M. M., Kumar, S., Kassis, S., Doyle, M. L., Gallagher, T. F., Fisher, S., McDonnell, P. C., Carr, S. A., Huddleston, M. J. S., Porter, T. G., Livi, G. P., Adams, J. L., Lee, J. C. (1997) Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272,12116-12121[Abstract/Free Full Text]
  51. Wang, Y., Su, B., Sah, V. P., Brown, J. H., Han, J., Chien, K. R. (1998) Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells. J. Biol. Chem. 273,5423-5426[Abstract/Free Full Text]
  52. Wang, Y., Huang, S., Sah, V. P., Ross, J., Brown, J. H., Han, J., Chien, K. R. (1998) Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J. Biol. Chem. 273,2161-2168[Abstract/Free Full Text]
  53. Ma, X. L., Kumar, S., Gao, F., Louden, C. S., Lopez, B. L., Christopher, T. A., Wang, C., Lee, J. C., Feuerstein, G. Z., Yue, T. L. (1999) Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocytes apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation 99,1685-1691[Abstract/Free Full Text]
  54. Maulik, N., Sato, M., Price, B. D., Das, D. K. (1998) An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 429,365-369[Medline]
  55. Maulik, N., Yoshida, T., Zu, Y. L., Sato, M., Banerjee, A., Das, D. K. (1998) Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am. J. Physiol. 275,H1857-H1864[Abstract/Free Full Text]
  56. Gray, M. O., Karliner, J. S., Mochly-Rosen, D. (1997) A selective {varepsilon}-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J. Biol. Chem. 272,30945-30951
  57. Armstrong, S. C., Delacey, M., Ganote, C. E. (1999) Phosphorylation state of hsp27 and p38 MAPK during preconditioning and protein phosphatase inhibitor protection of rabbit cardiomyocytes. J. Mol. Cell. Cardiol. 31,555-567[Medline]
  58. Clerk, A., Sugden, P. H. (1998) The p38-MAPK inhibitor. SB 2 03580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). FEBS Lett 426,93-96[Medline]
  59. Lali, F. V., Hunt, A. E., Turner, S. J., Foxwell, B. M. (2000) The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J. Biol. Chem. 275,7395-7402[Abstract/Free Full Text]
  60. Kalmes, A., Deou, J., Clowes, A. W., Daum, G. (1999) Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor. SB 2 03580. FEBS Lett 444,71-74[Medline]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
E. Murphy and C. Steenbergen
Mechanisms Underlying Acute Protection From Cardiac Ischemia-Reperfusion Injury
Physiol Rev, April 1, 2008; 88(2): 581 - 609.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. Heidbreder, A. Naumann, K. Tempel, P. Dominiak, and A. Dendorfer
Remote vs. ischaemic preconditioning: the differential role of mitogen-activated protein kinase pathways
Cardiovasc Res, April 1, 2008; 78(1): 108 - 115.
[Abstract] [Full Text] [PDF]

Home page