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reduces ischemic injury and
does not block protective effects of preconditioning
1 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, 27709; 2 University of North Carolina, Chapel Hill, 27516; and 3 Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
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We examined the
effect of inhibition of p38 mitogen-activated protein kinase (MAPK)
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during ischemia and preconditioning by using the inhibitor
SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit
buffer, while left ventricular developed pressure (LVDP) and
31P nuclear magnetic resonance spectra were acquired
continuously. After 20 min of ischemia and 25 min of reperfusion,
recovery of LVDP in untreated hearts was 32 ± 4%, whereas hearts
treated with SB-202190 5 min before ischemia recovered 59 ± 7%
of their pretreatment LVDP. Preconditioning improved functional
recovery to 65 ± 5%, which was unaffected by SB-202190
treatment, added either throughout the preconditioning protocol
(56 ± 5% recovery) or during the final reperfusion period of
preconditioning (71 ± 11% recovery). Necrosis was assessed after
40 min of ischemia and 2 h of reperfusion using
2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase
release. The untreated group had 54 ± 8% necrotic myocardium, whereas the SB-202190-treated group had 32 ± 7% and the
preconditioned group had 21 ± 4% necrotic tissue by TTC staining.
SB-202190; 31P nuclear magnetic resonance; necrosis; intracellular pH; left ventricular developed pressure
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
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THE STRESS-ACTIVATED
p38 mitogen-activated protein kinase (MAPK) is a serine-threonine
kinase, which is activated in response to a variety of stimuli (see
Refs. 7 and 31 for reviews). p38 MAPK is
phosphorylated and activated by the dual specificity kinases, MAPK
kinase 3 (MKK3) and MKK6. The small GTP binding proteins Rac and Cdc42
have also been reported to be upstream of MKK3/6 (15).
Activated p38 MAPK phosphorylates and activates ATF1 and 2, cAMP
response element binding protein (CREB), and MAPK-activated protein
(AP) kinase 2, which in turn phosphorylates substrates, including heart
shock protein 27 (HSP27) (8, 13, 15, 31). In
addition, p38 MAPK has been shown to activate the mitogen and stress
activated protein kinase (MSK1), which in turn activates CREB
(6). p38 MAPK is also reported to phosphorylate Ca2+-independent phospholipase A2 (PLA2) and
C/EBP homology protein (CHOP), and to enhance uptake of the glucose
analog 2-deoxyglucose into cells (4, 12, 32, 35).
There are six isoforms of p38 MAPK: 1, 2,
1, 2, 
, and 
(31). A
specific inhibitor of the - and -isoforms but not the - or
-isoforms of p38 MAPK, SB-202190, has been described, which blocked
lipopolysaccharide (LPS)-induced tumor necrosis factor- (TNF-)
and IL-1 production when administered to human monocytes (4,
18). It has been reported that SB-202190 and SB-203580 have no
effect on the activity of extracellular signal-regulated protein kinase
(ERK) or c-Jun NH2-terminal kinase (JNK) (5, 18,
39), although recent reports suggest that these inhibitors can
inhibit JNK and other kinases (16, 31).
There is general agreement that p38 MAPK is activated by ischemia
(1, 3, 22, 27), but it is unclear whether activation is
protective or detrimental during sustained ischemia and whether it
plays a role in the protective effect of ischemic preconditioning. Brief intermittent periods of ischemia and reflow or preconditioning (23), protect the myocardium against injury produced by a
subsequent sustained period of ischemia (17, 19, 24, 30,
38). Weinbrenner et al. (36) showed that the
protective effects of preconditioning could be abolished by addition of
SB-203580, an inhibitor of p38 MAPK /, to isolated myocytes
before preconditioning and simulated ischemia. Weinbrenner et al.
(36) also showed that preconditioning caused an increase
in phosphorylation of tyrosine 182 of p38 MAPK and that the addition of
8-(p-sulfophenyl)-theophylline (8-SPT), an adenosine
receptor inhibitor, which blocked the protective effects of
preconditioning, also blocked the preconditioning-induced increase in
phosphorylation of p38 MAPK. These data suggest that the
protective effects of preconditioning involve the enhanced activation
of p38 MAPK during the sustained period of ischemia; consistent
with the hypothesis that p38 MAPK activation is protective. In
contrast, Ma et al. (20) report that the addition of
SB-203580 before global ischemia reduces necrosis, apoptosis, and
postischemic contractile dysfunction in a perfused rabbit heart model.
Mackay and Mochly-Rosen (21) report that addition of
SB-203580 to neonatal rat myocytes throughout simulated ischemia
reduces lactate dehydrogenase (LDH) release and delays apoptosis
measured after 7-9 h of simulated ischemia. Saurin et al.
(29) also report that the addition of SB-203580 protects
neonatal rat myocytes from ischemic injury. These data would suggest
that inhibition of p38 MAPK / during the sustained ischemia is protective.
Thus there are conflicting data in the literature concerning the impact
of p38 MAPK activation during ischemia. There are data to suggest that
inhibition of p38 MAPK during ischemia is cardioprotective, and there
are data to suggest that preconditioning-induced activation of p38 MAPK
during sustained ischemia is also cardioprotective. Because these
results were obtained in different species and by using different model
systems, we undertook this study to examine the effects of inhibition
of p38 MAPK in preconditioned (PC) and non-PC hearts in a single
species and model, the isolated perfused rat heart. We find that in rat
hearts, inhibition of p38 MAPK / is protective, and that the
addition of a p38 MAPK / inhibitor during preconditioning and
sustained ischemia does not block the protective effects of
preconditioning. We further find that the addition of SB-202190 reduces
acidification during ischemia, similar to the reduced acidification
observed in PC hearts. The addition of SB-202190 to PC hearts caused a
further reduction in acidification during ischemia. The reduced
acidification is not due to inhibition of glycolysis, but appears to be
related to inhibition of glucose uptake.
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METHODS AND MATERIALS |
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TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
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Isolated Rat Heart Preparation
Male Sprague-Dawley rats were utilized in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH) Publication No. 8523, Revised 1985). The rats, weighing 250-300 g, were anesthetized with pentobarbital sodium (~25 mg/kg ip), followed by 100 units iv heparin sodium. The heparinized solution was then allowed to circulate for 1 min. The hearts were rapidly excised, placed in ice-cold Krebs-Henseleit (KH) buffer, and the aorta was cannulated. Retrograde perfusion was begun from a reservoir suspended 90 cm above the heart. The nonrecirculating perfusate was a KH buffer containing (in mM) 120 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose. The buffer was aerated with a mixture of 95% O2-5% CO2 to maintain the pH at 7.4, and the temperature was maintained at 37 ± 0.5°C.Left Ventricular Developed Pressure
A latex balloon on the tip of a polyethylene catheter was inserted into the left ventricle through the mitral valve. The water-filled balloon was connected to a Statham pressure transducer and inflated to an end-diastolic pressure of 5-10 cm H2O. A Maclab/2e (AD Instruments) was used to collect and process hemodynamic parameters. Hearts that did not achieve a left ventricular developed pressure (LVDP) of at least 100 cmH2O were excluded. Global ischemia was achieved by cross clamping the perfusate inflow line. On reperfusion, the balloon was released for the first 5 min of reperfusion to reduce the "no-reflow" phenomenon (9), and then reinflated to 5-10 cmH2O to assess recovery of LVDP.31P NMR
Studies were carried out on a Varian Unity Plus 400-MHz wide-bore nuclear magnetic resonance (NMR) spectrometer using the variable temperature probe to maintain a temperature of 37 ± 0.5°C. A 20-mm 31P NMR probe (Cryomagnet Systems; Indianapolis, IN) was tuned to 161.9 MHz. We shimmed on the proton signal to optimize homogeneity and typically obtained line widths at one-half the height of 0.1 parts per million (ppm). Pulsing conditions employed included a 70° pulse angle, a 2-s delay, and a 342-ms recycle time. We used a spectral width of ±3,600 Hz, 4,096 data points, and averaged 128 acquisitions, which required 5 min. The free induction decay was multiplied by an exponential function corresponding to a 40-Hz line broadening followed by Fourier transformation.The intracellular pH was determined from the chemical shift difference between the inorganic phosphate resonance and the phosphocreatine (PCr) resonance (11). For these experiments, a phosphate-free buffer was used. ATP and PCr values were determined from the area under their respective resonances. The area under the curves was fitted to a Lorentzian line shape and integrated with the use of Varian software. ATP and PCr were expressed as percentage of baseline.
Protocols
The experimental design of the studies to obtain 31P NMR data and assess functional recovery is illustrated in Fig. 1. All hearts were subjected to a 30-min stabilization period of perfusion with KH buffer, followed by a treatment protocol, 20 min of global ischemia, and 25 min of reperfusion with KH buffer. Treatment periods are described below. Control hearts received an additional 15 min of perfusion with KH. The SB group was perfused for 10 min with KH buffer and then with 10 µM of SB-202190 (Calbiochem; La Jolla, CA) for 5 min before sustained ischemia. PC hearts were given four cycles of 5 min of ischemia and 5 min of reperfusion. The PC + SB-throughout hearts received the same treatment as the PC group, except that 10 µM SB-202190 was added to the hearts 2 min before the start of the preconditioning protocol and was present throughout the preconditioning protocol. The PC + SB fourth reflow group was treated with the standard preconditioning protocol except 10 µM SB-202190 was given at the end of the third reflow of preconditioning and continued throughout the fourth reflow. In all of the SB-202190-treated groups, SB-202190 was not washed out before the sustained ischemia and was therefore present throughout the sustained 20 min of global ischemia.
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Measurement of necrosis. 2,3,5-Triphenyltetrazolium chloride (TTC) staining (28, 34) and creatine kinase (CK) release (2, 9, 10) were both used to assess the amount of necrosis. For these studies, the ischemic period was extended to 40 min and the reperfusion period was extended to 2 h to ensure washout of pyridine nucleotides from necrotic cells as required for the TTC assay. CK release was measured by collecting the effluent for the first 40 min of reperfusion and measuring CK activity spectrophotometrically. At the end of 2 h of reperfusion, the hearts were perfused with a 1% TTC solution for 10 min, then incubated for an additional 10 min in 1% TTC at 37°C, and finally placed in Formalin. The hearts were sliced and photographed, and the percentage of necrotic tissue (TTC-negative staining) was determined using NIH Image, version 1.61.
P38 MAPK and MAPKAP
kinase-2 assays.
Hearts were snap-frozen in liquid nitrogen at the times indicated.
Frozen hearts were homogenized with a prechilled mortar and pestle in
lysis buffer consisting of Tris (pH 7.5), 20 mM; EDTA, 1 mM; EGTA, 1 mM; -glycerolphosphate, 1 mM; NaCl, 150 mM; Na vanadate, 1 mM; Na
pyrophosphate, 2.5 mM; MgCl2, 4.5 mM; 1,4 dithiothreitol
(DTT), 0.5 mM; phenylmethylsulfonyl fluoride (PMSF), 1 mM; Triton
X-100, 1%; and leupeptin, 1 µg/ml. The homogenate was centrifuged at
20,000 g at 4°C for 10 min. Protein was measured by
bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). p38 MAPK
activity was determined by using a kit from New England Biolabs (Beverly, MA). Briefly, the p38 MAPK was immunoprecipitated overnight. The activity of the p38 MAPK was determined by measuring the
phosphorylation of activating transcription factor-2 (ATF-2) fusion
protein using a phospho-ATF-2 antibody and Western blotting.
The data were quantified by densitometry. The same extracts were
used to measure MAPKAP kinase-2 (MAPKAPK-2) activity. MAPKAPK-2
activity was determined by measuring the transfer of the
-phosphate of [-32P]ATP to a MAPKAPK-2 substrate
peptide (Upstate Biotechnology, Lake Placid, NY). The phosphorylated
substrate is then separated from the residual
[-32P]ATP by differential binding to P81
phosphocellulose paper. After extensive washing of the phosphocellulose
paper, the bound radioactivity is determined by liquid scintillation counting.
Biochemical assay for glycolytic metabolites. After the indicated treatment and 20 min of ischemia, the hearts were snap-frozen in liquid nitrogen. Glucose-6-phosphate (G-6-P) and lactate contents were measured enzymatically after perchloric acid extraction, utilizing standard techniques (14). An additional group of hearts that was not subjected to ischemia (aerobic) was added for this set of experiments. For the aerobic group, hearts were given a stabilization period, followed by an additional 20 min of perfusion with KH buffer, then snap-frozen.
Statistics
All values are expressed as means ± SE. Statistical analysis was performed with the use of StatView software (SAS). When more than two groups were compared, ANOVA was used. Two-factor ANOVA with repeated measures was used for analysis of functional and hemodynamic data. P < 0.05 was considered to be significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
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Hemodynamic
Preischemic.
At the end of the stabilization period, just before treatment there
were no significant differences in heart rate or LVDP among the
experimental groups (Table 1). Vehicle
control experiments with dimethyl sulfoxide (DMSO) were performed to
verify that DMSO had no effect on any measured parameter. No
significant difference was noted between DMSO and control hearts, and
these were therefore treated as one group.
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dP/dtmin) increase to 125 ± 5% and 130 ± 7% of baseline (P < 0.05),
respectively (data not shown). As observed previously, preconditioning
resulted in a decline in LVDP to 61 ± 3% of baseline
(P < 0.05) at the end of the fourth reflow.
Hearts preconditioned with SB-202190 present throughout also showed a
decline in LVDP (66 ± 6% of baseline) compared with control
hearts (Table 1). Similarly, when SB-202190 was added only during the
fourth reflow, LVDP measured at the end of the fourth reflow period was
significantly different from control hearts (73 ± 6% of
baseline, P < 0.05). There were no variations among
the treatment groups in heart rate at the end of the treatment period
compared with control hearts.
During ischemia. As shown in Table 1, maximum contracture levels during ischemia were significantly higher in the SB-202190, PC, PC+ SB-fourth reflow, and PC + SB (throughout) groups compared with the control group (P < 0.05). Furthermore, the maximum contracture levels observed in PC + SB-202190 during fourth reflow and PC + SB-202190 throughout groups were significantly higher than the preconditioned group. Ischemic contracture began at 10.7 ± 0.6 min in control hearts, which was significantly longer than that observed in the PC hearts (4.5 ± 0.3 min), the SB-202190-treated hearts (5.7 ± 0.6 min), the hearts treated with SB-202190 throughout preconditioning (3.6 ± 0.3 min), or PC hearts with SB-202190 added during the fourth reflow (4.0 ± 0.6 min).
Reperfusion.
As shown in Fig. 2, both preconditioning
and SB-202190 treatment resulted in improved recovery of LVDP compared
with control hearts, expressed as a percentage of initial baseline LVDP
(PC, 65 ± 5%; SB-202190, 59 ± 7%; vs. control, 32 ± 4%, P < 0.05 for both PC and SB vs. control).
Addition of SB-202190 throughout PC did not result in an additive
protective effect, but it did not block the protective effects of PC on
the recovery of LVDP (56 ± 5% of baseline, P > 0.05 compared with PC). Similarly, addition of SB-202190 to PC hearts
during the fourth reflow did not block the protective effect of PC on
recovery of LVDP (71 ± 11% of baseline, P > 0.05 compared with PC alone). There were no significant differences among the groups with respect to postischemic heart rate.
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Intracellular pH During Ischemia and Reperfusion
As shown in Fig. 3, preconditioning attenuated the fall in intracellular pH (pHi) during sustained ischemia (6.51 ± 0.02) compared with the control group (6.01 ± 0.04, P < 0.05). SB-202190 treatment alone also reduced the fall in pHi during ischemia (6.36 ± 0.05, P < 0.05 compared with control). Furthermore, the addition of SB-202190 throughout PC resulted in even less acid production (6.78 ± 0.03) than observed in PC alone (P < 0.05 compared with all groups except SB + PC during the fourth reflow). In PC hearts with the addition of SB-202190 during the fourth reflow, pHi fell to 6.68 ± 0.03, which was significantly different from control and SB-202190 alone (P < 0.05). At the end of the reflow period, all of the groups recovered to baseline.
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Glycolytic Metabolites
To examine whether the attenuation of the fall in pHi during ischemia in hearts treated with SB-202190 is due to inhibition of glycolysis, we froze hearts at the end of the sustained 20-min period of ischemia for measurement of G-6-P, a proximal glycolytic intermediate, and lactate, the end product of anaerobic glycolysis. As shown in Table 2, G-6-P content in untreated (control) hearts after 20 min of global ischemia (2.42 ± 0.33 µmol/g dry wt) was statistically increased (P < 0.05) relative to aerobic control myocardium (0.59 ± 0.01 µmol/g dry wt). In addition, G-6-P levels in untreated (control) hearts were also statistically different (P < 0.05) from the SB-202190 (0.92 ± 0.16 µmol/g dry wt), PC (0.30 ± .06 µmol/g dry wt), and PC + SB during the fourth reflow hearts (0.15 ± 0.02 µmol/g dry wt). Compared with aerobic hearts with lactate levels of 5.1 + 1.1 µmol/g dry wt, and untreated ischemic hearts with lactate level of 170.6 ± 3.7 µmol/g dry wt, the other experimental groups had intermediate levels of lactate accumulation (in µmol/g dry wt: SB-202190 hearts, 129.4 ± 6.7; PC group, 104.0 ± 8.0; and the PC + SB during fourth reflow hearts 63.4 ± 4.2; P < 0.05 compared with aerobic and untreated ischemic hearts). In none of the treated groups (PC, SB, and PC + SB-fourth reflow) was there an increase in G-6-P compared with the untreated ischemic group, which would be the predicted result if anaerobic glycolysis were inhibited. Rather in all of the treated groups, the level of lactate production was reflective of the G-6-P content, suggesting that less entry of substrate into the glycolytic pathway was the explanation for the decreased lactate production.
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Energetic Effects of SB-202190
There were no differences in ATP among the groups at either the end of ischemia or the end of the reperfusion period. ATP fell to nondetectable levels by the end of ischemia in all groups. On reperfusion, the ATP levels returned to ~30-40% of initial value with no significant differences among the groups (data not shown).All of the PC groups show >100% recovery of PCr during the reflow periods of the PC protocol. No significant differences were noted among the groups at the end of ischemia with regards to PCr. On reperfusion, PCr recovered to >90% of initial values.
Effects of SB-202190 on Necrosis
For the assessment of the effects of SB-202190 on necrosis, we extended the ischemic period to 40 min and increased the reperfusion time to 2 h. Figure 4 shows the percentage of necrotic tissue in each group. Preconditioning significantly decreased the percentage of necrotic myocardium after 40 min of global ischemia (21 ± 4% vs. 54 ± 8% for controls, P < 0.05). The addition of SB-202190 during the fourth reflow did not reduce the protective effects of preconditioning on necrosis (18 ± 2% necrotic, P < 0.05). Treatment with SB-202190 in the absence of preconditioning also significantly decreased the percentage of necrotic cells (32 ± 7% necrotic, P < 0.05).
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Measurement of CK release confirmed the TTC measurements. After 40 min of global ischemia, non-PC hearts released 93 ± 9 IU/g dry wt, which was significantly higher than CK release in either PC hearts (42 ± 9 IU/g dry wt), PC hearts in the presence of SB-202190 during the fourth reflow (51 ± 16% IU/g dry wt), or hearts treated with SB-202190 alone before ischemia (50 ± 14 IU/dry wt).
p38 MAPK and MAPKAP Kinase-2
As shown in Fig. 5, in non-PC (control) hearts, ischemia results in a significant increase to 173 ± 39% of control (P < 0.05 compared with control hearts at t = 0 min) in p38 MAPK activity after 5 min of sustained ischemia. By 20 min of sustained ischemia, p38 MAPK activity in control hearts had declined nearly to baseline. In PC hearts, p38 MAPK activity was 114 ± 20% of control at the end of the PC protocol (t = 0 min), but in contrast to non-PC hearts, p38 MAPK activity declined during sustained ischemia. In PC hearts after 5 min of sustained ischemia, p38 MAPK has returned to baseline. Interestingly, hearts treated with SB-202190 show an increase in p38 MAPK activity with a profile similar to that observed in non-PC hearts. It is been shown previously that SB-202190 does not block the phosphorylation of p38 MAPK, but instead binds reversibly to the catalytic site (39) and can be washed away during the extensive washes of the immunoprecipitated complex. Thus SB-202190 inhibition of p38 MAPK in the perfused heart would not be apparent in our assay because SB-202190 is washed away. We, therefore, measured the activity of MAPKAPK-2, a kinase downstream of p38 MAPK. As shown in Fig. 6, for non-PC and PC hearts, MAPKAPK-2 showed a similar pattern of activation to that observed for p38 MAPK. In non-PC hearts (control), MAPKAPK-2 was activated greater than twofold at 5 min of sustained ischemia (P < 0.05, compared with control hearts at time 0) and then activity declined toward baseline by 20 min of ischemia. In contrast in PC hearts, MAPKAPK-2 activity starts out slightly above baseline and declines during sustained ischemia. MAPKAPK-2 activity for PC hearts is not significantly different from baseline (control t = 0) at any point during sustained ischemia. Similar to PC hearts, and in contrast to non-PC hearts, MAPKAPK-2 activity in hearts treated with SB-202190 was not significantly different from baseline at any point during sustained ischemia.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
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Inhibition of p38 MAPK
/: Does it Protect or Block Protection?
/ during the sustained period of ischemia
is critical for the protective effect of preconditioning, then either
of our SB-202190 treatment protocols should eliminate the protective effect. Alternatively, if p38 MAPK / activation during the
preconditioning protocol is essential for the induction of the
preconditioning effect, then the protocol with SB-202190 present
throughout the preconditioning protocol should have eliminated the
protective effect. However, neither protocol of SB-202190 treatment
abolished the protective effect, suggesting that activation of p38 MAPK / during sustained ischemia is not required for protection. In
fact, taken together, the data suggest that inhibition or lack of
activation of p38 MAPK during sustained ischemia is protective.
The data in the present study appear to be in conflict with the
conclusions of Weinbrenner et al. (36). In their study, SB-203580 was added to rabbit ventricular myocytes for 5 min before the
preconditioning protocol, which is accomplished by pelleting myocytes
and covering them with a layer of oil for 10 min, after which the
myocytes were resuspended in oxygenated buffer for 15 min and then
pelleted a second time to simulate ischemia. Samples are taken at
various times, and the percentage of cells that stain with Trypan blue
after placement in hypotonic media is plotted as a function of time.
Preconditioning reduces the ischemia-induced osmotic fragility, and
this effect is blocked by addition of SB-203580. The reason for the
difference between the data in the present paper and the study of
Weinbrenner et al. (36) may include a difference in end
point used (ischemia-induced fragility vs. CK release and TTC
staining), a difference in the preconditioning protocol (we used 4 cycles of 5-min ischemia and 5 min of reperfusion and they used one
10-min period of simulated ischemia and 15 min of reoxygenation), a
difference in species, or a difference in the precise dose and timing
of administration of the inhibitor. In our study in the perfused rat
heart, the p38 MAPK / inhibitor was present during the
reperfusion periods as well as the ischemia periods of preconditioning,
and it was also present during the sustained ischemia. It is possible
that inhibition of p38 MAPK only during preconditioning ischemia, but
not the reflow periods and/or the sustained ischemia, would inhibit the
protective effects of preconditioning.
The data also show that although inhibition of p38 MAPK / and
preconditioning are protective, their protection is not additive; this
might suggest that preconditioning and SB-202190 enhance protection via
similar mechanisms. This might be explained if preconditioning reduced
the activation of p38 MAPK / during the sustained period of
ischemia compared with non-PC hearts. This hypothesis would suggest
that inhibition of p38 MAPK only during PC would block the PC-induced
downregulation of p38 MAPK during sustained ischemia and therefore
block the protective effects of PC, whereas inhibition of p38 MAPK
during PC and sustained ischemia may be protective because of the
inhibition of p38 MAPK during sustained ischemia. Thus
preconditioning and SB-202190 would both reduce activation of p38 MAPK
/ during ischemia. The data in Figs. 5 and 6 support this
hypothesis. During sustained ischemia PC hearts have less activation of
p38 MAPK and MAPKAPK-2 compared with non-PC hearts. We found previously
(33) that at the end of PC (t = 0 min in this
study), there was a significant increase in p38 MAPK activity compared
with control, aerobic perfused hearts. In this study we find an
increase in p38 MAPK at the end of PC, but the increase was not
statistically significant. Ping et al. (27) found that p38
MAPK is activated by the initial cycles of PC, but that with increasing
cycles of PC the activation of p38 MAPK becomes less, such that at the
end of six cycles of PC, p38 MAPK activity returned to baseline. Our
protocol is four cycles of PC and it is likely that this is on the
borderline, such that activation of p38 MAPK is starting to decline.
After 5 min of sustained ischemia, non-PC hearts have an increase in p38 MAPK and MAPKAPK-2 that is significantly higher than control (non-PC hearts) at t = 0 min and is also significantly
higher than PC hearts after 5 min of sustained ischemia. Interestingly, although SB-202190 blocked the activation of MAPKAPK-2 during sustained
ischemia, it did not block activation of p38 MAPK as measured by our
assay. Young et al. (39) reported that SB-202190 is a
reversible inhibitor and the washes after the immunoprecipitation are
sufficient to wash away the SB-202190; thus the catalytic activity of
p38 MAPK in our assay is likely due to removal of SB-202190. SB-202190
inhibits the catalytic site and does not block phosphorylation
(activation). Thus these data suggest that in SB-202190-treated hearts
p38 MAPK is phosphorylated during ischemia, but p38 MAPK cannot
activate its downstream target (MAPKAPK-2) because p38 MAPK is
inhibited by the binding of SB-202190 to the catalytic site.
These data are in agreement with previous studies showing that ischemia
activates p38 MAPK (1, 3, 22, 27). Ping et al.
(27) report a fivefold increase p38 MAPK activity after 4 min of ischemia in rabbit heart, whereas we observed a twofold increase
in rat heart. This slight difference could be due to a difference in
species. Our data are also in agreement with the study by Ping et al.
(27) showing that p38 MAPK is downregulated by six cycles
of ischemia and reperfusion and they are consistent with a study by
Saurin et al. (29), which reports that preconditioning attenuated the activation of p38 MAPK- during the sustained
ischemia. However, the data in this paper are not consistent with data
suggesting that preconditioning enhances phosphorylation of p38 MAPK
during the sustained period of ischemia (1, 36).
Weinbrenner et al. (36) showed that preconditioning caused
an increase in phosphorylation of tyrosine-182 of p38 MAPK during the
sustained period of ischemia compared with non-PC hearts. They also
found that addition of SPT, an adenosine inhibitor, which blocked the
protective effects of preconditioning also, blocked the
preconditioning-induced increase in phosphorylation of p38 MAPK. In a
recent paper, Nakano et al. (25) reported that MAPKAPK-2
is activated after 20 min of sustained ischemia in PC hearts, but not
in non-PC hearts. In agreement with Nakano, we find that in non-PC
hearts, MAPKAPK-2 has returned almost to baseline after 20 min of
ischemia. However, in contrast to the study by Nakano et al., in PC
hearts, we do not find an increase in MAPKAPK-2 at 20 min of ischemia.
In fact, at 5 min of ischemia we find that PC hearts have significantly
less activation of MAPKAPK-2 and p38 MAPK. Armstrong et al.
(1) reported that the dual phosphorylation of p38 MAPK was
enhanced by preconditioning. However, Armstrong et al. did not find a
significant PC-induced difference in phosphorylation of HSP27, a
downstream target of MAPKAPK-2, during ischemia. These differences
could be related to species differences or a difference in the PC
protocol. It is also possible that PC and ischemia differentially
regulate p38 MAPK isoforms and that this contributes to the conflicting data (26).
Other Effects of Inhibition of p38
MAPK /
/-mediated stimulation of glucose uptake,
which occurs during ischemia or preconditioning (33). Preconditioning enhances glucose uptake during the initial minutes of
ischemia (37). Inhibition of p38 MAPK / blocks this
stimulation of glucose uptake and thus reduces acid production during
ischemia. This inhibition of ischemia-preconditioning enhanced
glucose uptake by SB-202190 would lead to enhanced glycogen depletion
when SB-202190 was added throughout preconditioning, because the
glycogen which is broken down during the brief periods of ischemia
would be poorly replenished during the reflow periods of the
preconditioning protocol without an increase in glucose uptake. This is
consistent with the progressive attenuation of the fall in
pHi during successive cycles of preconditioning and the
very small fall in pHi during the sustained period of
ischemia in the hearts that received SB-202190 throughout
preconditioning. Decreased glucose uptake can also explain the reduced
G-6-P in non-PC SB-202190-treated hearts during ischemia.
The PC, SB, PC + SB, and PC + SB-fourth reflow groups all have an accelerated onset of contracture during the sustained period of ischemia. It has been previously shown that preconditioning reduces the time to contracture (17). Contracture usually occurs at the cessation of anaerobic glycolysis, and therefore it might be expected that early onset of contracture would correlate with more severe injury, but this was not observed in either the PC groups or the SB groups. The decrease in time to onset of contracture in the PC, SB, PC + SB, and the PC + SB-fourth reflow groups correlated with the reduced lactate production.
As shown in Table 1, treatment with SB-202190 caused a significant
increase in LVDP, +dP/dt, and dP/dt. Thus
SB-202190 addition results in a positive inotropic effect. Consistent
with the positive isotropic effect of SB-202190 and its presence during
sustained ischemia, the maximum contracture obtained during ischemia is increased by the addition of SB-202190 (Table 1). We also find that
SB-202190 addition to PC hearts, either throughout PC or during the
fourth reflow of PC, further increased the maximum contracture noted
during ischemia. The mechanism by which inhibition of p38 MAPK /
results in a positive inotropic effect is unknown. It is possible that
p38 MAPK / phosphorylates a contractile protein or a calcium
transport protein.
It should be noted, however, that SB-202190 is not present in the
perfusate during reperfusion after the sustained period of ischemia.
The SB-202190 washes out of the heart quickly as the positive inotropic
effect is reversed within ~15 min after removing SB-202190 from the
perfusate (data not shown). This reversal of the effects of SB-202190
is consistent with the observation that isolated p38 MAPK / is
active after it is washed free of the inhibitor (39).
Because the SB-202190 is not present in the perfusate on reflow, the
improved recovery of LVDP observed after ischemia in the hearts
pretreated with SB-202190 is not due to the positive inotropic
effects of SB-202190. The preischemic and postischemic measurements of
LVDP are made in the absence of SB-202190. Furthermore, the estimates
of ischemic injury using enzyme release and TTC staining after 40 min
of ischemia and 2 h of reflow show proportionately the same
results and should not be influenced by differences in contractility.
In summary, inhibition of p38 MAPK / does not block the ability
of PC to reduce postischemic contractile dysfunction or to reduce
infarct size, and PC reduces the activation of p38 MAPK during
sustained ischemia. Furthermore, inhibition of p38 MAPK / during
sustained ischemia per se is protective. Taken together these data
suggest that inhibition of p38 MAPK during sustained ischemia is protective.
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ACKNOWLEDGEMENTS |
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We thank Beth Paine and Haiyan Tong for carefully reading the manuscript. We also like to thank John Petranka for assistance with the p38 MAPK and MAPKAPK-2 assays.
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FOOTNOTES |
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C. Steenbergen was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-39752.
Address for reprint requests and other correspondence: E. Murphy, Mail Drop D2-03, PO Box 12233, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (E-mail: murphy1{at}niehs.nih.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 January 2000; accepted in final form 23 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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