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Division of Cardiology, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We determined the role of p38 mitogen-activated protein kinase (MAPK), 72-kDa heat shock protein (HSP72), and antioxidant enzymes in whole body heat stress (HS)-induced cardioprotection in mouse hearts. Adult male mice were treated with either HS or anesthesia only. At 0.5, 48, 72, or 120 h later, the hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. A significant protection against ischemia-reperfusion injury was observed 48 h after HS as demonstrated by: 1) reduction in infarct size; 2) decrease in leakage of lactate dehydrogenase; and 3) enhanced postischemic ventricular contractile function. No such protection was observed at other post-HS time points. HS caused an ~25% increase in phosphorylated c-Jun NH2-terminal kinase (JNK) but not p38 MAPK in the heart during the first 2-h post-HS time period. Cardioprotection was abolished by the MAPK inhibitor SB-203580, which also partially suppressed the HS-induced JNK phosphorylation. The protective effect was associated with a two- to threefold increase in HSP72 protein accumulation, but not antioxidant enzyme activities (catalase and Cu/Zn and Mn SOD) in the myocardium. Although HSP72 levels remained high 72 h after HS, the cardioprotection had already disappeared. We conclude that HS induces a transient delayed cardioprotection at 48 h after thermal stress in mice which appears to be mediated via a MAPK-signaling pathway.
ischemia; reperfusion; signal transduction; antioxidants; myocardial preconditioning
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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WHOLE BODY HEAT STRESS (HS) has been shown to enhance myocardial ischemic tolerance (7-9, 11, 14-21, 23, 33, 34, 36, 40, 42, 47, 48). Heat shock proteins (HSPs) have been suggested as the potential mediators of HS- or ischemia-induced late-myocardial preconditioning (11, 15, 23), because the stress response is commonly associated with rapidly increased HSP expression. Recent studies have shown that HS-enhanced antioxidant enzyme activities [catalase and manganese superoxide dismutase (Mn SOD)] play important roles in protecting the ischemic myocardium through a free-radical scavenging mechanism (7, 9, 18, 48). More recently, the activation of protein kinase C (16, 21) and the opening of ATP-sensitive potassium (KATP) channels (14, 34) have also been proposed from our group and others as mediators of HS-induced cardioprotection. In addition, stress-activated protein kinases such as p38 mitogen-activated protein kinase (p38 MAPK) are involved in the signal transduction cascade of myocardial ischemic preconditioning (1, 3, 26, 29, 44). We and others have shown that p38 MAPK mediates the delayed preconditioning with an adenosine A1-receptor agonist in mice (50) and rabbits (10). Activation of p38 MAPK by HS has been shown in yeast in vitro (30) and in rat liver after whole body HS (24). However, no studies are available that show a similar activation of p38 MAPK by whole body hyperthermia in the heart. Because p38 MAPK is activated by ischemic preconditioning (1, 3, 26, 29, 44) and delayed pharmacological preconditioning with adenosine (10, 50), we hypothesized that HS-induced cardioprotection may also be mediated by p38 phosphorylation. Accordingly, the primary goal of this study was to demonstrate whether HS-induced late protection is accompanied by enhanced phosphorylation of p38 MAPK, and whether the protective effect is blocked by SB-203580, a selective inhibitor of p38 MAPK. In addition, the potential role played by HSP72 and endogenous antioxidant enzymes in HS-induced cardioprotection was concomitantly reevaluated in the mouse model of ischemia-reperfusion (I/R).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Adult male outbred ICR mice (21-43 g body wt) were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were kept in proper housing conditions with free access to standard rodent food and water. The animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). The experimental protocols were approved by the Animal Welfare Committee of the Virginia Commonwealth University.
Whole body HS or sham treatment. The mouse was anesthetized with pentobarbital sodium (50 mg/kg ip) and placed on an electric heating pad, which was folded to cover the entire body except the head. The core temperature was recorded with a thermometer using a mouse rectal thermal probe (YSI-402) inserted ~1 cm into the colon. Body temperature was gradually raised. The desired degree of hyperthermia (42°C) was reached in ~10 min and was precisely maintained for the next 15 min within a variation range of ±0.2°C by unfolding or refolding the heating pad as described previously (45). At the end of the heat treatment, the animal was removed from the pad and allowed to recover at room temperature (21-24°C) until the subsequent experiments were performed. Of the total 139 mice that received the HS treatment for both of the physiological and biochemical experiments, 88% survived and recovered from the thermal stress. No additional fluid was administered to the animals during or after HS treatment. Animals in the sham groups (sham control; SC) received the same dose of anesthesia without the HS.
Pretreatments and I/R protocol.
Figure 1 illustrates the experimental
protocol used in the study. Mice received either HS or SC pretreatment
and were allowed to recover for time periods of 0.5, 48, 72, or
120 h before being subjected to the I/R protocol in Langendorff
mode (as described in Langendorff isolated perfused heart
preparation). The I/R protocol consisted of 30 min of
equilibration, 20 min of global ischemia (induced by turning
off the aortic inflow), and 30 min of reperfusion (induced by reopening
the aortic line). Two additional groups of mice were administered the
selective p38 MAPK inhibitor SB-203580 (1 mg/kg ip; Sigma Chemical) 30 min before the HS or SC pretreatment; mice were then allowed a 48-h
recovery before exposure to I/R.
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Langendorff isolated perfused heart preparation. The isolated perfused mouse-heart preparation was previously described in detail (45, 46). In brief, the animal was anesthetized with pentobarbital sodium (100 mg/kg with 33 IU heparin ip), and the heart was removed and placed in ice-cold Krebs-Henseleit solution with heparin. The aortic opening was cannulated and the heart was retrogradely perfused at a constant pressure of 55 mmHg with the modified Krebs-Henseleit buffer [containing (in mM): 118 NaCl, 24 NaHCO3, 2.5 CaCl2, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, and 0.5 EDTA] in Langendorff mode. The perfusion buffer was constantly gassed with 95% O2-5% CO2 (pH 7.35-7.49) and the heart temperature was monitored continuously and maintained at 37 ± 0.5°C. Ventricular function was assessed by a force-displacement transducer (FT03; Grass) attached to the heart apex. The coronary flow rate was calculated by timed collection of the efflux perfusate.
Measurement of lactate dehydrogenase release and myocardial
infarct size.
Coronary effluent was collected from the hearts at 1 min before global
ischemia and 5, 10, 20, and 30 min during the reperfusion period. Lactate dehydrogenase (LDH) enzyme activity was measured spectrophotometrically (model UV160U; Shimadzu) using a diagnostic kit
purchased from Sigma Chemical. Enzyme activity values were normalized
(as U · min
1 · g wet wt1)
against individual heart wet weights and coronary flow rate (44,
45). At the end of the I/R experiment, the heart was removed
from the Langendorff apparatus, weighed, and immediately stored in a
freezer. The frozen heart was cut from apex to base into 7 or 8 transverse slices of equal thickness and the slices were incubated in
10% triphenyltetrazolium chloride (TTC) in phosphate buffer at room
temperature for 30 min. The TTC buffer was then replaced by 10%
formaldehyde, and the heart slices were fixed for 4-6 h before
areas of infarcted tissue were measured via computer morphometry
(Bioquant 98). The risk area was the sum of the total ventricular area
minus the cavities. The infarct size was measured in each slice of both
left and right ventricular myocardium and presented as a percentage of
risk area.
Evaluation of antioxidant enzyme activities.
A separate set of 42 heart samples was collected at 0.5, 3, 6, 24, 48, 72, 96, and 120 h after either HS or SC pretreatment (n = 4-6 per group). After the blood was washed
out with saline, the ventricular muscle samples were immediately frozen
with liquid nitrogen and stored at 
70°C until future use. While
under liquid nitrogen, the samples were ground into fine powder, and 1 ml of PBS (pH 7.4) was added to the powdered tissue. The buffered
tissue was homogenized using a Polytron tissue homogenizer at 4°C and then centrifuged at 12,000 g for 10 min. The supernatant was
recovered and protein concentration was measured using the Bio-Rad
protein assay based on the Bradford dye-binding procedure with BSA as the standard. Enzyme activities were normalized against the protein concentration for each sample and expressed as units per milligram of protein.
Determination of HSP72 expression. A separate set of 36 heart samples was collected after 0.5, 48, 72, and 120 h of SC or HS pretreatment (n = 4-6 per group). Once the tissue extract was prepared, 30 µg of total protein from each sample was separated by SDS-PAGE electrophoresis on 1-mm thick 10% acrylamide gels. After electrophoresis, the proteins on the gel were transferred to a nitrocellulose membrane (Schleicher and Schuell) by electroelution. The protein transfer was confirmed by comparison with prestained molecular weight markers (Bio-Rad). The membrane was blocked with nonfat dry milk after the protein transfer. The membranes were then incubated with a rabbit polyclonal antibody (1:20,000 dilution; Stressgen Biotechnologies) that reacts specifically to the inducible form of HSP72. The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution; Amersham). The membranes were developed using enhanced chemiluminescence (Amersham) and exposed to X-ray film for the appropriate times. Optical densitometric evaluation was performed on each film using a scanner/densitometer system (Molecular Dynamics 4.0). The arbitrary units for each band were calculated by the system and normalized against the background value for each individual film.
Measurement of MAPK phosphorylation. A total of 28 animals were used either as SC or HS-treated groups (n = 4 per group). The ventricular muscle samples were collected at 5, 10, 30, 60, or 120 min after the termination of HS and were immediately frozen in liquid nitrogen. Another group of four mice was pretreated with SB-203580 (1 mg/kg ip) 30 min before HS, and the hearts were collected 2 h after HS. The tissue samples were ground into fine powder in liquid nitrogen after cell lysis in 1 ml of radioimmunoprecipitation assay (RIPA) buffer containing 1× PBS (pH 7.4), 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors (10 µl/ml of phenylmethylsulfonyl fluoride and 30 µl/ml of aprotinin), and a phosphatase inhibitor (100 mM sodium orthovanadate). The mixture was homogenized and centrifuged at 6,000 g for 10 min. The supernatant was then collected and protein concentration was measured.
For immunoprecipitation, 250 µg of the sample protein were incubated with 5 µg of antiphosphotyrosine mouse monoclonal antibody (PY20; Santa Cruz) in 600 µl of RIPA buffer for 3 h at 4°C; 35 µl of protein A/G agarose beads (Santa Cruz) were then added to the sample and mixed for another 60 min. The agarose beads containing the immunocomplexes were collected by centrifugation for 5 min (400 g) at 4°C, and supernatant was carefully aspirated and discarded. The beads were washed three times with RIPA buffer and one time with PBS (by repeating the centrifugation step) and were then resuspended in 50 µl of electrophoresis sample buffer. To dissociate the immunocomplexes from the beads, the sample was boiled for 5 min and centrifuged again, and the supernatant was collected. Subsequently 20 µl of immunoprecipitated supernatant were separated by SDS-PAGE (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Bio-Rad) via tank transfer for 2.5 h at 190 mA. After the membrane was blocked with milk solution [5% nonfat dry milk in Tris-buffered saline (TBS)] for 1 h, it was probed with either a mouse monoclonal antibody for phosphorylated p38 MAPK (D-8; Santa Cruz) or a rabbit polyclonal antibody for JNK1 (FL; Santa Cruz) for 2 h; both were diluted to a 1:100 ratio in milk solution and 0.05% Tween 20 (TT). After the membrane was washed with TBS containing TT, it was incubated with either an anti-rabbit or anti-mouse horseradish peroxidase-linked antibody (diluted 1:2,000 in milk solution with TT; Amersham) for 1 h. The membrane was washed with TBS containing TT four times (for a total of 30 min) and then incubated using a chemiluminescence kit (Amersham) before being exposed to X-ray film. The bands were quantified via densitometric scanning (see Determination of HSP72 expression).Data analysis and statistics. All measurements are expressed as means ± SE. The data were analyzed by either unpaired t-test or one-way ANOVA. If a significant value of F was obtained in ANOVA, the Student-Newman-Keuls post hoc test was further used for pairwise comparisons. Paired t-test was used to compare any pair of pre- and posttreatment values for the same parameter. P < 0.05 was considered significant.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental conditions and exclusions.
The experimental conditions and morphometric characteristics of the
animals are shown in Table 1. Thirteen
hearts (i.e., 18% of the total 73 isolated hearts subjected to the I/R
protocol) were excluded because of: 1) significant time
delay in aortic cannulation (>3 min), 2) aortic damage
during the cannulation process, or 3) depressed ventricular
function (developed force <0.1 g).
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Time window of cardioprotection.
A previous study from our laboratory (45) showed a lack of
cardioprotection at 6 or 24 h after HS treatment in mice.
Therefore, in the present study we undertook an extensive investigation
to include several post-HS time points (i.e., 0.5, 48, 72, 120 h) to identify the window of cardioprotection in this model. As shown in
Fig. 2, the myocardial infarct size was
significantly reduced in the HS-48 h group (12.2 ± 2.5% of risk
area) compared with those in the SC-48 h group (27.8 ± 5.0%;
P < 0.05). The mean values of the risk area were not
different between the groups (data not shown). The infarct-limiting
protection was diminished at 72 h after HS (22.4 ± 3.8% vs.
30.9 ± 6.1% in the SC-72 h group; P > 0.05) and
was completely lost by 120 h (33.3 ± 5.3% vs. 34.2 ± 5.0% in the SC-120 h group; P > 0.05). No "early
window" of infarct reduction was found at 0.5 h after HS
treatment (37.6 ± 7.0% vs. 28.9 ± 3.1% in SC-0.5 h group;
P > 0.05). A similar trend was observed for leakage of
LDH in the coronary effluent (see Table
2). The baseline preischemia LDH
values were identical for all experimental groups
(P > 0.05) but increased significantly in all groups at 5 min of reperfusion. The LDH values returned to the baseline in all
groups by 30 min of reperfusion as reported previously
(45), which demonstrates a "washout" phenomenon. In
contrast, a significant reduction in LDH release at 5 min of reperfusion was observed only in the HS-48 h group (0.07 ± 0.01 U · min1 · g1;
P < 0.05) compared with either the SC-48 h (0.14 ± 0.03 U · min1 · g1) or
other HS groups (0.18 ± 0.02, 0.17 ± 0.02, and 0.13 ± 0.01 U · min1 · g1 for the
HS-0.5 h, HS-72 h, and HS-120 h groups, respectively; see Table 2).
Furthermore, the antinecrotic effect induced by HS was also associated
with a significant improvement in the postischemic ventricular
function (see Table 3). The
preischemic basal functional parameters were not significantly
different between HS and SC groups in the posttreatment time windows
(P > 0.05). However, the HS-48 h group exhibited a trend
for better postischemic contractile function compared with the
SC-48 h group as indicated by improved ventricular developed force,
heart rate, and rate-force product (RFP), and a lower resting tension
(an indicator of ischemic contracture). At the end of 30 min of
reperfusion, the RFP values were significantly higher in the HS-48 h
(145 ± 31 g × beat/min) compared with the SC-48 h (63 ± 12 g × beat/min; P < 0.05) groups. The
flow rates were not significantly different among all the groups
(P > 0.05; paired t-test; see Table
4). The HS pretreatment produced no improvement in the postischemic coronary flow at all of the
time points compared with the SC groups (P > 0.05).
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HSP72 expression and antioxidant enzyme activities.
Figure 3 shows a representative Western
blot and the densitometric results averaged from 4-6 independent
hearts. HSP72 was expressed at a very low level in the SC hearts; HS
caused a two- to threefold increase in the protein at 48 and 72 h
which diminished by 120 h. It is noteworthy that although the
enhanced expression of HSP72 was not different between the HS-48 h and
HS-72 h groups, the infarct-size reduction was observed only in the
HS-48 h group and not in the HS-72 h group (see Fig. 2).
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Effect of SB-203580 on cardioprotection.
As shown in Fig. 2, pretreatment with SB-203580 did not have a
significant effect on infarct size in the 48-h SC groups (i.e., SB-203580 + SC-48 h vs. SC-48 h; P > 0.05). However,
SB-203580 completely abrogated the infarct-limiting effect of HS at the 48-h time window (i.e., 27.7 ± 5.4% in SB-203580 + HS-48 h
vs. 12.2 ± 2.5% in the HS-48 h group; P < 0.05). Similarly, the reduction in peak LDH release at 5 min of
reperfusion observed in the HS-48 h group was also abolished by
SB-203580 (i.e., 0.15 ± 0.01 U · min1 · g wt1 in
SB-203580 + HS-48 h vs. 0.07 ± 0.01 U · min1 · g wt1 in the
HS-48 h group; P < 0.05; see Table 2). SB-203580 had no significant effect on LDH leakage at the preischemic
baseline or during reperfusion. Ventricular function in the hearts
pretreated with SB-203580 (the SB-48 h groups) showed mildly higher
basal ventricular developed force, although such an effect was not
statistically significant (P > 0.05; see Table 3). All
other functional parameters in the pre- and postischemic
periods were not significantly modified by SB-203580 (see Table 3).
Similarly, both basal and postischemic coronary flow rates were
not affected by SB-203580 pretreatment (see Table 4).
Phosphorylation of p38 MAPK and JNK1.
The representative Western blots and the densitometric
quantification of the phosphorylated MAPKs in the hearts of NS or
HS-treated mice (n = 4 per group) are shown in Fig.
5. Contrary to our expectations, no
measurable p38 MAPK phosphorylation was observed after HS (see Fig.
5A). On the other hand, HS caused an ~25% increase in
phosphorylated JNK1 2 h after the end of HS (see Fig.
5B). Interestingly, SB-203580 (1 mg/kg ip given 30 min
before HS treatment) caused at least partial inhibition of the
HS-induced JNK1 phosphorylation in the mouse hearts (see Fig.
6). In these experiments, the tissue
extracts were first immunoprecipitated with an antiphosphotyrosine
antibody mixed with protein A/G plus agarose before Western blotting
was performed using an antibody for either phosphorylated p38 MAPK (see
Fig. 5A) or anti-JNK1 (see Figs. 5B and 6).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Salient findings. The major goal of this study was to elucidate the role of p38 MAPK signaling after HS-induced delayed cardioprotection. A second goal was to further evaluate the role of HSP72 and antioxidants in the cardioprotective effect of HS. Our results show that: 1) whole body thermal stress induced significant protection against I/R injury in mice 48 h later, which was demonstrated by reductions in infarct size and LDH release and improvement in postischemic ventricular contractile function; 2) HS-induced cardioprotection at 48 h was abolished by the p38 MAPK inhibitor SB-203580, although this stimulus failed to show an increase in p38 MAPK phosphorylation. In contrast, HS treatment caused an ~25% increase in JNK1 (p46) phosphorylation, which was partially inhibited by SB-203580; and 3) the protective effect of HS treatment was not associated with the enhanced expression of HSP72 or with the antioxidant enzyme activities of catalase, Cu/Zn SOD, and Mn SOD in the myocardium. Taken together, our results suggest that HS-induced delayed cardioprotection is mediated by a MAPK signaling mechanism that appears to be independent of antioxidants or HSP72 levels.
HS-induced cardioprotection in mice. A previous study from our laboratory (45) demonstrated that HS treatment did not protect the mouse heart from I/R injury at 6 or 24 h after HS despite the increased expression of HSP72. Recent studies on rats (40, 47, 48) demonstrated a delayed cardioprotection that occurred at 48 h or later. We therefore reexamined the existence of HS-induced protection in the mouse model at the extended time windows. Our results show significant cardioprotection at the 48-h post-HS time window, which was reflected not only by significant reduction in the infarct size (see Fig. 2) but also reduction in LDH release (see Table 2) and improvement in the ventricular contractile function (see Table 3) compared with corresponding SC groups. The cardioprotection was diminished at 72 h and completely lost by 120 h (see Fig. 2 and Table 2). However, we did not find an "early window" of protection by HS (i.e., the HS-0.5 h vs. the SC-0.5 h group), which was recently described for rats (40, 48). These differences could be attributed to several sources including the animal species (rat vs. mouse), the infarction model (in situ vs. isolated perfused model), and the nature of the ischemic insult (regional vs. global).
Role of HSP72 and antioxidant enzymes. A direct cause-and-effect relationship between HSP72 and HS-induced cardioprotection is still controversial. The evidence suggesting HSP72 as the primary mediator of HS-induced cardioprotection was demonstrated by Hutter and colleagues (15) and Marber and co-workers (23). These studies showed that the reduction in infarct size after in vivo I/R was highly correlated with the amount of HSP72 induced in the rat heart by whole body HS treatment. However, this concept has been challenged by several recent studies which indicate that the quantitative accumulation of HSP72 is unlikely to be the sole determinant of HS-induced cardioprotection (14, 16, 20, 21, 36, 45, 47, 48) because the manifestation of cardioprotection was unrelated to the level of HSP72 expression. The present study demonstrated that although cardioprotection occurred 48 h after HS, it was indeed associated with a two- to threefold increase in the amount of HSP72, and the protection disappeared at the 72-h post-HS time window despite the presence of HSP72 (see Figs. 2 and 3). Such a discordance between the time course of HSP72 induction and HS-induced cardioprotection was previously reported for rabbits (9) and rats (36, 47). The evidence suggests that the amount of HSP72 is not likely the only responsible factor for the HS-induced protection. However, this opinion is not necessarily incompatible with the well-known role of HSP72 as a cytoprotective protein (27). In other experimental models it has been shown that the significant increase of myocardial HSP72 or HSP27 expression through direct gene transfer via the adenoviral vector system can enhance myocyte ischemic tolerance either in vivo (32) or in vitro (25).
The protective effects of endogenous antioxidant enzymes in the heart against I/R injury are well recognized. It has also been shown that whole body HS can increase myocardial catalase activities and enhance postischemic functional recovery in isolated rat hearts (7, 8, 18). However, the role of catalase has been questioned because the catalase inhibitor 3-amino-1,2,4 triazole failed to block the cardioprotective effect of HS in vivo (2). The present results further confirm a lack of the effect of HS on antioxidant levels in the rat (43) and mouse heart (45). Although a critical role for Mn SOD in HS-induced protection has been recently proposed (47, 48), our results do not support these observations. We found increased Cu/Zn SOD activity at a 120-h post-HS time window, although it did not correlate with HS-induced delayed cardioprotection in the present study. The reason for the discrepancy in Mn SOD data is unclear. It is notable that the level of Mn SOD activity reported by Yamashita and colleagues (48) was much higher than in the present study (see Fig. 4), which indicates possible differences in the enzyme assay conditions. On the other hand, our findings are in agreement with the previous study that reported a dissociation between delayed ischemic preconditioning and myocardial antioxidant enzyme activities in an in vivo pig model of I/R injury (41).Role of MAPKs. JNK and p38 MAPK are the members of the stress-sensitive kinase family that respond to various environmental stimuli such as cytokines, radiation, and reactive oxygen species. The MAPKs regulate the critical cellular processes such as gene transcription, cytoskeletal organization, metabolic homeostasis, cell growth, and apoptosis via a complex signal transduction cascade, which most likely occurs by phosphorylation of some common downstream target proteins (5, 29, 37, 38). Although it is generally agreed that p38 MAPK plays a pivotal role in the signaling mechanisms of I/R injury (5, 28, 49), the question of whether p38 MAPK phosphorylation is protective or detrimental continues to be debatable (35). It has been shown that inhibition of the p38 MAPK pathway with SB-203580 reduces the myocardial necrotic and/or apoptotic cell death caused by I/R either in vivo (4) or in vitro (22). On the other hand, several recent studies have demonstrated that p38 MAPK is involved in the signal transduction cascade of myocardial ischemic (1, 26, 29, 44) and pharmacological (10, 50) preconditioning. Although the exact target protein(s) that are phosphorylated by p38 MAPK signaling remain uncertain, HSP27 (the small HSP) is one of the possible candidates. It has been suggested that p38 MAPK may phosphorylate HSP27 which in turn provides cytoprotection by stabilizing the actin cytoskeleton (1, 10, 12, 13, 38, 39). In addition, a recent study by Baines and colleagues (3) suggested a possible link between p38 MAPK and mitochondrial KATP channels, because the infarct-limiting effect of anisomycin, a p38 MAPK/JNK activator, was abrogated by a selective blocker of mitochondrial KATP channels. The role of KATP channels in HS-induced cardioprotection has already been demonstrated in previous studies on rabbits (14, 34). The present study demonstrated that pretreatment with SB-203580 in HS-treated mice completely abolished the delayed cardioprotection. Our results are in accord with a recent study by Joyeux and co-workers (17) which reported that pretreatment with a higher dose of SB-203580 (2.83 mg/kg ip) blocked HS-induced cardioprotection in rats. However, we were unable to demonstrate p38 MAPK phosphorylation after HS treatment. In contrast, we observed a moderate increase (~25%) in phosphorylated JNK1 after HS treatment compared with the NS hearts (see Fig. 5B), which was partially inhibited by SB-203580 (see Fig. 6). These observations raise the question of the selectivity of SB-203580 as a p38 MAPK inhibitor. Previously Clerk and Sugden (6) demonstrated that SB-203580 was able to inhibit JNK activity in a dose-dependent manner in the cultured ventricular myocytes. However, it is difficult to compare the drug dose used in our in vivo injection (1 mg/kg ip) with the threshold concentration of 3 µM for JNK inhibition in their in vitro studies.
The present study differs from previous investigations demonstrating that p38 MAPK phosphorylation can mediate anti-ischemic protection in hearts that were preconditioned with brief ischemia (26, 29, 39, 44) or an adenosine A1-receptor agonist (10, 50). These differences may be attributed to the variable nature for each of the preconditioning stimuli. Despite our failure to demonstrate phosphorylation of p38 MAPK after HS, at this time we cannot completely rule out the role of p38 MAPK in HS-induced cardioprotection especially when considering the well-known role of SB-203580 as the inhibitor of p38 MAPK. Because this drug inhibited HS-induced JNK1 phosphorylation, we propose that the resultant protection against I/R could involve both JNK and p38 MAPK. In conclusion, we have shown that whole body HS treatment is able to induce a transient delayed cardioprotection against I/R injury in mice 48 h later. The delayed protection appears to be mediated by a JNK1-dependent (rather than a p38 MAPK-dependent) signaling pathway. However, endogenous antioxidant enzymes or enhanced expression of HSP72 do not appear to be involved in HS-induced cardioprotection in this model. Future investigations are required to identify the unknown effector protein(s) that may be phosphorylated by these MAPKs.| |
ACKNOWLEDGEMENTS |
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The authors thank P. Bhargava for assistance with the biochemical measurements.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-51045 and HL-59469 (to R. C. Kukreja) and Training Grant HL-07537 (to M. I. Tejero-Taldo) and by American Heart Association, Mid-Atlantic Affiliate Grants 9804811U and 0060289U (to L. Xi).
Address for reprint requests and other correspondence: R. C. Kukreja, Division of Cardiology, Box 980281, Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail: rakesh{at}hsc.vcu.edu).
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 21 July 2000; accepted in final form 15 February 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.01 vs. HS-48 h; #P < 0.05 vs.
HS-48 h (unpaired t-test). One-way ANOVA also indicated
significant difference among these groups (P < 0.05).
