Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

Jun-Te Hsu, Ya-Ching Hsieh, Wen Hong Kan, Jian Guo Chen, Mashkoor A. Choudhry, Martin G. Schwacha, Kirby I. Bland, Irshad H. Chaudry

Abstract

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and αB-crystallin, in response to stress. Activation of HSP27 or αB-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17β-estradiol (E2) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E2 under those conditions are mediated via p38 MAPK remains unknown. Male rats (275–325 g body wt) were subjected to soft tissue trauma and hemorrhage (35–40 mmHg mean blood pressure for ∼90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E2 (1 mg/kg body wt), E2 + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E2 administration, and phosphorylation of cardiac p38 MAPK, HSP27, and αB-crystallin was increased. The E2-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and αB-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E2 on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and αB-crystallin.

  • heat shock proteins
  • αB-crystallin
  • p38 mitogen-activated protein kinase inhibitor

mitogen-activated protein kinases (MAPKs), a large family of tyrosine-threonine kinases, have important functions as mediators of signal transduction and are activated by a variety of extracellular stimuli (17, 28, 39). Three subgroups of MAPKs have been identified: extracellular signal-regulated kinase, p38 MAPK (p38), and c-jun NH2-terminal kinases. Among these, p38 MAPK plays an important role in myocardial ischemic injury. Weinbrenner et al. (43) showed that activation of p38 MAPK was cardioprotective during ischemia in preconditioned isolated rabbit heart. Nakano et al. (35) found that treatment with a p38 MAPK activator, anisomycin, before the sustained period of ischemia resulted in cardioprotection by reducing infarct size. However, pretreatment with SB-203580, a potent inhibitor of p38 MAPK, blocked the protective effect of ischemic preconditioning. Additional studies also showed that p38 MAPK activation is cardioprotective in ischemic preconditioning (7, 25, 27, 34).

Studies have shown that p38 MAPK activation leads to the induction of a number of heat shock proteins (HSPs) (10, 12, 26). HSPs are induced by exposure to a number of different stresses, including heat and ischemia, and they help minimize the damaging effect of such stress on the heart (20, 26). Our previous studies showed that administration of 17β-estradiol (E2) after trauma-hemorrhage restores cardiac function through upregulation of HSP expression (41, 45). Furthermore, studies have demonstrated that HSP27 and αB-crystallin are downstream of p38 MAPK (6, 8, 10). Phosphorylation of HSP27 or αB-crystallin is critical for optimal cytoprotection (10, 12, 26). Interestingly, HSP27 and αB-crystallin share considerable sequence and structural similarity, associate in vivo, and are induced by oxidative stress (9, 31, 46). HSP27 and αB-crystallin function as molecular chaperones in protein biosynthesis to facilitate protein folding and translocation and are associated with cytoskeletal structures. Stabilization of these elements, therefore, could contribute to the increased stress tolerance. For instance, HSP27 acts as an inhibitor of actin filament turnover in smooth muscle cells and appears to stabilize the actin filaments (21, 32). Stable overexpression of HSP27 in Chinese hamster lung cells confers resistance to F-actin fragmentation induced by H2O2 and menadione (12). Furthermore, in glioma cells, αB-crystallin plays a role in thermal resistance and may contribute to stability of the cytoskeleton (14). In cardiac myocytes, αB-crystallin also associates with the intermediate filaments, especially desmin, an association that is strengthened during ischemia (1, 2).

Despite numerous advances in intensive care medicine, sepsis and organ dysfunction remain the major causes of death after trauma or major surgery (4, 16, 18, 36). Previous studies showed prolonged depression of cardiovascular function in male rats after trauma-hemorrhage, despite fluid resuscitation (11, 44, 45). However, administration of E2 after trauma-hemorrhage restored the depressed cardiac function (11, 15, 19, 33, 40). Several mechanisms responsible for the salutary effects of E2 have been proposed (11, 40, 44, 45). However, whether p38 MAPK plays a role in E2-mediated cardioprotection after trauma-hemorrhage remains unknown (30). We hypothesized that the beneficial effects of E2 after trauma-hemorrhage are mediated via a p38 MAPK-dependent pathway through regulation of HSP27 and αB-crystallin. To test this hypothesis, we examined the effect of E2 alone and in combination with the p38 MAPK inhibitor SB-203580 in rats on cardiac function, p38 MAPK, and HSP27 and αB-crystallin after trauma-hemorrhage.

MATERIALS AND METHODS

Rat trauma-hemorrhagic shock model.

Male Sprague-Dawley rats (275–325 g body wt; Charles River Laboratories, Wilmington, MA) were fasted overnight before the experiment but were allowed water ad libitum. All experiments were performed in adherence with National Institutes of Health (Bethesda, MD) guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. A nonheparinized model of trauma-hemorrhage was used as described previously (11, 45). Briefly, rats were anesthetized by isoflurane inhalation before induction of soft tissue trauma via a 5-cm midline laparotomy. The abdomen was closed in layers, and polyethylene (PE-50) catheters (Becton Dickinson, Sparks, MD) were placed in both femoral arteries and the right femoral vein. The wounds were bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ) throughout the surgical procedure to reduce postoperative pain. Rats were then placed in a Plexiglas box (21 × 9 × 5 cm) in a prone position and allowed to awaken; then they were rapidly bled to a mean arterial blood pressure (BP) of 35–40 mmHg within 10 min. Hypotension was maintained until the animals could no longer maintain a mean BP of 35 mmHg unless additional fluid in the form of Ringer's lactate was administered. This time was defined as maximum bleed-out, and the amount of withdrawn blood was noted. Then the rats were maintained at a mean BP of 35–40 mmHg until 40% of the maximum bleed-out volume was returned in the form of Ringers lactate (∼90 min from the onset of bleeding). The animals were then resuscitated with four times the volume of the shed blood over 60 min with Ringer's lactate. Sham-operated animals underwent the same groin dissection, which included ligation of the femoral artery and vein, but neither hemorrhage nor resuscitation was carried out. Animals were allocated randomly to four treatment groups: vehicle (cyclodextrin, Sigma, St. Louis, MO), E2 (1 mg/kg body wt; Sigma), SB-203580 (2 mg/kg body wt; Calbiochem, San Diego, CA), or E2 + SB-203580 at the beginning of the resuscitation. After resuscitation, the catheters were removed, the vessels were ligated, and the skin incisions were closed with sutures. The animals were killed 2 h after the end of resuscitation or sham operation.

Determination of cardiac function.

At 2 h after trauma-hemorrhage and resuscitation or sham operation, the animals were again anesthetized with isoflurane and catheterized via the left femoral vein. Under continuous general anesthesia with pentobarbital sodium (25–30 mg/kg iv), a PE-50 catheter was placed into the right carotid artery and connected to a BP analyzer (DigiMed, Louisville, KY). After the mean BP was recorded, the catheter was advanced into the left ventricle and connected to a heart performance analyzer (DigiMed) to monitor and record maximal rate of pressure increase (+dP/dtmax) and decrease (−dP/dtmax).

Western blot analysis.

After measurement of cardiac function, the heart (left ventricle) tissue was harvested. Approximately 0.1 g of freshly collected left ventricle tissue from each rat was homogenized in 1 ml of lysis buffer containing 50 mM HEPES, 10 mM sodium pyrophosphate, 1.5 mM MgCl2, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.15 M NaCl, 0.1 M NaF, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail (Sigma). Tissue lysates were centrifuged at 17,000 g for 20 min at 4°C, and an aliquot of the supernatant was used to determine protein concentration (DC Protein Assay, Bio-Rad Laboratories, Hercules, CA). The lysates (50 μg per lane) were then mixed with 4× SDS sample buffer, electrophoresed on 4–12% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA), and transferred electrophoretically onto nitrocellulose membranes (Invitrogen). The membranes were immunoblotted with primary antibodies against p38 MAPK, phosphorylated p38 MAPK (Cell Signaling Technology, Beverley, MA), HSP27, phosphorylated HSP27, αB-crystallin, phosphorylated αB-crystallin, or GAPDH (Abcam, Cambridge, MA). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody for detection of bound antibodies by enhanced chemiluminescence (Amersham, Piscataway, NJ). Mouse monoclonal GAPDH antibody was used as the loading control. Signals were quantified using ChemiImager 5500 software (Alpha Innotech, San Leandro, CA).

Statistical analysis.

Values are means ± SE (n = 4–6 rats/group). One-way ANOVA and Tukey's test were employed for comparison among groups, and differences were considered significant at P < 0.05.

RESULTS

Cardiac function.

Various parameters, such as mean BP, +dP/dtmax, and −dP/dtmax, in sham-operated or trauma-hemorrhage animals treated with vehicle, E2, or E2 + SB-203580 are shown in Fig. 1. A significant decrease in mean BP, +dP/dtmax, and −dP/dtmax was evident after trauma-hemorrhage in the vehicle-treated group compared with the sham animals (Fig. 1). E2 significantly improved mean BP after trauma-hemorrhage compared with vehicle; however, BP remained lower than in the sham animals (Fig. 1A). Furthermore, E2 increased and normalized +dP/dtmax (Fig. 1B). However, −dP/dtmax remained lower than in the sham animals (Fig. 1C). To evaluate whether the cardioprotective effects of E2 were via p38 MAPK, a group of trauma-hemorrhage rats were treated with E2 and SB-203580. E2 + SB-203580 prevented the E2-mediated improvement of cardiac function after trauma-hemorrhage (Fig. 1). In contrast, SB-203580 alone did not alter cardiac function in trauma-hemorrhage rats (data not shown). There was no significant difference in mean BP, +dP/dtmax, or −dP/dtmax in sham animals treated with SB-203580 or E2 + SB-203580 compared with vehicle- or E2-treated sham animals (data not shown).

Fig. 1.

Mean blood pressure (A), maximal rate of left ventricular pressure increase (+dP/dtmax, B), and maximal rate of left ventricular pressure decrease (−dP/dtmax, C) 2 h after sham operation (sham) or trauma-hemorrhage. Animals were treated with vehicle, SB-203580 (SB), 17β-estradiol (E2), or E2 + SB-203580. Values are means ± SE of 4–6 animals in each group. Results were compared by 1-way ANOVA and Tukey's test. *P < 0.05 vs. sham or trauma-hemorrhage-E2. #P < 0.05 vs. sham.

Activation of p38 MAPK in the heart.

Trauma-hemorrhage induced a significant decrease in the phosphorylation of cardiac p38 MAPK compared with sham animals (Fig. 2). Administration of E2 after trauma-hemorrhage increased cardiac p38 MAPK phosphorylation, and the values were similar to those of the sham animals. The increase in p38 MAPK phosphorylation mediated by E2 after trauma-hemorrhage was, however, abolished by coadministration of SB-203580 (Fig. 2). Phosphorylation of p38 MAPK was not affected further in trauma-hemorrhage rats treated with SB-203580 (data not shown). Moreover, there was no significant difference in p38 MAPK phosphorylation in sham animals treated with SB-203580 or E2 + SB-203580 compared with vehicle-treated sham animals (data not shown). No significant difference in total p38 MAPK protein expression was observed after trauma-hemorrhage compared with sham animals.

Fig. 2.

Expression of total and phosphorylated (activated) p38 MAPK (p-p38 MAPK) in heart after sham operation and trauma-hemorrhage. Blots obtained from several experiments were analyzed using densitometry, and densitometric values were pooled from 4–6 animals in each group. Values are means ± SE. Results were compared by 1-way ANOVA and Tukey's test. *P < 0.05 vs. sham or trauma-hemorrhage-E2.

Activation of HSP27 and αB-crystallin in the heart.

As shown in Figs. 3 and 4, a significant decrease in the phosphorylation of cardiac HSP27 and αB-crystallin was observed after trauma-hemorrhage compared with sham animals. Administration of E2 after trauma-hemorrhage increased cardiac HSP27 phosphorylation, and the values were similar to those of sham animals (Fig. 3). However, the E2-mediated increase in αB-crystallin phosphorylation mediated after trauma-hemorrhage was even higher than in sham animals (Fig. 4). The increase in HSP27 or αB-crystallin phosphorylation mediated by E2 after trauma-hemorrhage was, however, abolished by coadministration of SB-203580 (Figs. 3 and 4). Phosphorylation of HSP27 or αB-crystallin was not affected further in trauma-hemorrhage rats treated with SB-203580 (data not shown). Moreover, there was no significant difference in HSP27 and αB-crystallin phosphorylation in sham animals treated with SB-203580 or E2 + SB-203580 compared with vehicle-treated sham animals (data not shown). No change in the total HSP27 and αB-crystallin protein expression was observed in any groups after trauma-hemorrhage compared with sham animals.

Fig. 3.

Expression of total and phosphorylated (activated) heat shock protein (HSP) 27 (p-HSP27) in heart after sham operation and trauma-hemorrhage. Blots obtained from several experiments were analyzed using densitometry, and densitometric values were pooled from 4–6 animals in each group. Values are means ± SE. Results were compared by 1-way ANOVA and Tukey's test. *P < 0.05 vs. sham or trauma-hemorrhage-E2.

Fig. 4.

Expression of total and phosphorylated αB-crystallin (p-αB-crystallin) in heart after sham operation and trauma-hemorrhage. Blots obtained from several experiments were analyzed using densitometry, and densitometric values were pooled from 4–6 animals in each group. Values are means ± SE. Results were compared by 1-way ANOVA and Tukey's test. *P < 0.05 vs. sham or trauma-hemorrhage-E2. #P < 0.05 vs. sham.

DISCUSSION

Our results collectively suggest that the salutary effects of E2 on cardiac function after trauma-hemorrhage are in part mediated via upregulation of p38 MAPK and subsequent HSP27 and αB-crystallin activation. In this regard, HSP27 and αB-crystallin expression confers enhanced cell resistance to heat shock, ischemia, oxidants, and various cytotoxic agents (12, 22, 26, 29). Studies have also shown that the stability of actin microfilaments is enhanced in an HSP27 concentration- and phosphorylation-dependent manner (8). In endothelial and smooth muscle cells, phosphorylation of HSP27 correlates temporally with translocation of HSP27 to actin filaments, where it also contributes to stabilization of the cytoskeleton (23, 37). Similar to HSP27, αB-crystallin is phosphorylated in response to stressors in cultured human glioma cells (13). However, in contrast to HSP27, which is expressed in nearly all cell types, αB-crystallin is a structurally related small HSP and is expressed mainly in the lens, certain neurons, skeletal muscle, and cardiac myocytes in very high levels (5, 24). The levels of αB-crystallin expression in the heart are markedly high: as much as 5% of the total protein in cardiac myocytes (10). Thus, when compared with the sarcomeric proteins, αB-crystallin plays an important structural role in the heart.

The importance of αB-crystallin in the cellular stress response extends beyond the heart. The expression of αB-crystallin in astrocytes is considerably increased in Parkinson's patients suffering from dementia and in Alzheimer's disease, where it is believed to contribute to cellular mechanisms of adaptation to the stress caused by the disease (38). αB-Crystallin also has been implicated in multiple sclerosis, where it was thought to serve as the autoantigen against which antibodies are expressed that lead to eventual neurodegeneration (42). Although αB-crystallin displays a tissue-restricted expression pattern, it appears to play important roles in providing protection against potentially harmful stresses in a variety of cell types.

Our results demonstrate that mean BP, +dP/dtmax, and −dP/dtmax were significantly depressed after trauma-hemorrhage; however, E2 administration after trauma-hemorrhage normalized +dP/dtmax and increased −dP/dtmax. These results are consistent with our previous findings which showed the cardiac function was significantly depressed after trauma-hemorrhage but improved by the administration of E2 after trauma-hemorrhage (11, 44). The E2-mediated cardioprotection was abolished when SB-203580 was administered along with E2 after trauma-hemorrhage, indicating that the salutary effects of E2 on cardiac function after trauma-hemorrhage are mediated via activation of the p38 MAPK pathway.

Our results indicate that administration of E2 after trauma-hemorrhage increased cardiac p38 MAPK phosphorylation compared with the trauma-hemorrhage rats treated with vehicle. The increase in p38 MAPK phosphorylation by E2 after trauma-hemorrhage was abolished by coadministration of SB-203580. Since p38 MAPK activation has been implicated in cardiac preconditioning and E2 also induces cardiac p38 phosphorylation, it is reasonable to suggest that this hormone could be potentially used as an alternative method of preconditioning.

A link between p38 MAPK activation and actin dynamics may be of major importance during cellular stress. The p38 MAPK-mediated increase in the actin cytoskeleton during stress may constitute an important arm of the adaptive cell response to external stress. Phosphorylation of HSP27 and αB-crystallin, via activation of p38 MAPK, may markedly modify the equilibrium in favor of polymerized actin, thereby contributing to the maintenance of the microfilament network. We used SB-203580 to specifically inhibit p38 MAPK activity in vivo and found that p38 MAPK, HSP27, and αB-crystallin phosphorylation was abolished in the trauma-hemorrhage rats treated with E2 + SB-203580. These results thus demonstrate that the salutary effects of E2 on cardiac function after trauma-hemorrhage are in part mediated by a p38 MAPK-dependent pathway through upregulation of HSP27 and αB-crystallin.

Several potential mechanisms have been proposed for the salutary effects of E2 on organ function after trauma-hemorrhage (11, 40, 44, 45). Hsieh et al. (11) suggested that the beneficial effects of E2 on cardiac function after trauma-hemorrhage were in part due to upregulation of peroxisome proliferator-activated receptor coactivator 1. Szalay et al. (40) found that E2 administration after trauma-hemorrhage upregulates heme oxygenase-1 expression and activity, leading to improvement of organ function. Additional studies have shown that the salutary effects of E2 on cardiac function are mediated via increased cardiac heat shock factor-1 and HSPs such as HSP90, HSP70, HSP60, and HSP32 (45). Our present findings suggest that E2-mediated cardioprotection is via upregulation of p38 MAPK and subsequent activation of HSP27 and αB-crystallin. A similar role of HSP27 and αB-crystallin was reported by Martin et al. (26), who used an adenovirous vector to overexpress HSP27 or the related protein αB-crystallin in cardiac cells. They demonstrated that both of these proteins were able to protect cardiac myocytes from ischemia and that decreasing the level of endogenous HSP27 via an antisense approach enhanced the damaging effects of a subsequent ischemic insult.

The present study utilized measurement of cardiac function and the increase in p38 MAPK, HSP27, and αB-crystallin phosphorylation at a single time point, i.e., 2 h after trauma-hemorrhage. It remains unclear whether similar effects of E2 are maintained for >2 h after trauma-hemorrhage. Our previous studies, however, showed that if improvement in organ function by any pharmacological agent is evident early after treatment, those salutary effects are sustained for prolonged intervals and they also improved the survival of animals (3). Thus, although a time point other than 2 h was not examined in this study, on the basis of our previous studies, it would appear that the salutary effects of E2 would be evident, even if the effects were measured at another time point after trauma-hemorrhage and resuscitation.

It can be argued that we should have also measured cardiac output in this study. Our previous studies showed that cardiac output was significantly decreased in the trauma-hemorrhage rats and was restored to normal after E2 administration following trauma-hemorrhage (11, 41). Furthermore, +dP/dtmax and −dP/dtmax can reflect the value of cardiac output in sham and trauma-hemorrhage rats. However, if we use a radioactive microsphere technique to determine cardiac output, the heart tissue cannot be used for Western blot analysis. Thus we did not determine cardiac output in this study.

In summary, our results indicate that E2 administration after trauma-hemorrhage upregulates p38 MAPK, HSP27, and αB-crystallin activity in the heart and prevents cardiac dysfunction 2 h after resuscitation. The E2-mediated cardioprotection and increased p38 MAPK, HSP27, and αB-crystallin phosphorylation were abolished when SB-203580 was administered along with E2. These findings suggest that the salutary effects of E2 are mediated via a p38 MAPK-dependent pathway through increased phosphorylation of HSP27 and αB-crystallin. Nonetheless, because E2 can mediate its effects in multiple ways, we do not consider activation of p38 MAPK to be the exclusive action of E2 under such conditions. Thus a better understanding of the relation between E2 and p38 MAPK may enable us to develop a new therapeutic modality of hemorrhagic shock.

GRANTS

This work was supported by National Institute of General Medical Sciences Grant R37 GM-39519.

Acknowledgments

We thank Zheng F. Ba for superb help with these studies.

Footnotes

  • 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.

REFERENCES

View Abstract