Mechanism of cardioprotection following trauma-hemorrhagic shock by a selective estrogen receptor-b agonist: up-regulation of cardiac heat shock factor-1 and heat shock proteins
Abstract
Although 17b-estradiol (E2) administration following trauma-hemorrhage (T-H) improves cardiac function in male rodents, it is not known whether the salutary effects of E2 are mediated via estrogen receptor (ER)-a or ER-b, and whether cardiac heat shock proteins (Hsp) are affected by E2 administration. Male Sprague–Dawley rats underwent T-H (mean BP 40 mmHg for 90 min, then resuscitation). ER-a agonist propyl pyrazole triol (PPT) (5 µg/kg), ER-b agonist diarylpropiolnitrile (DPN) (5 µg/kg), or vehicle (10% DMSO) was injected subcutane- ously during resuscitation. At 24 h after T-H or sham operation, cardiac output (CO), stroke volume (SV), mean blood pressure, and ± dP/dtmax were measured (n = 6 rats per group). Cardiac Hsp32, 60, 70, and 90 mRNA/protein expressions and heat shock factor (HSF)-1 DNA binding activity were determined. One-way ANOVA and Tukey’s test were used for statistical analysis. CO, SV and ± dP/dtmax decreased significantly after T-H, however, administration of ER-b agonist DPN after T-H restored the above parameters. Moreover, DPN treatment prevented T-H-mediated decrease in Hsp60 mRNA/protein and Hsp90 protein expressions in the heart. Hsp32 and Hsp70 mRNA/protein expression and HSF-1 DNA binding activity in the hearts were increased even above the shams in DPN treated T-H rats. In contrast, no significant change in the above parameters was observed in T-H rats treated with ER-a agonist PPT. Thus, the salutary effects of E2 on cardiac function are mediated via ER-b and ER-b-induced up-regulation of Hsp likely plays a significant role in the E2-mediated cardioprotection after T-H.
Keywords: Shock; Estrogen; Hormones; Receptors; Agonists
1. Introduction
Severe hemorrhage, which often occurs with trauma, is known to produce many life-threatening sequelae. Patients who survive the initial traumatic insult remain susceptible to sepsis, septic shock, multiple organ failure, and death [1,2]. Cellular dysfunction occurs in many organs, including car- diovascular, liver, gut, and adrenal following hemorrhagic shock, and these alterations persist despite fluid resuscitation for a prolonged period of time [3,4].
Sex hormones are known to modulate immune function in animals and in humans under normal and stress conditions [5]. Studies have shown that proestrus female mice show nor- mal immune response, however, male mice have markedly altered immune responses following trauma-hemorrhage (T-H) [6]. Studies have also demonstrated that male sex ster- oids appear to be responsible for producing the depression in cell and organ functions following T-H [7]. Additional sup- port for this notion comes from studies that showed that cas- tration of male animals 14 days before T-H prevented the depression in myocardial functions that are observed in non- castrated animals under those conditions [7]. Furthermore, administration of flutamide, a testosterone receptor antago- nist following T-H, improved the depressed cardiac functions in male animals [8]. These studies suggest that male and female sex steroids such as 5a-dihydrotestosterone and 17b- estradiol (E2) have an opposite effect on cell and organ func- tion following injury.
E2 is the predominant circulating sex hormone in females, and has been shown to have cardioprotective effects follow- ing adverse circulatory conditions such as T-H in male ani- mals [9,10]. Moreover, estrogen receptors (ER) are expressed in several organs including the heart in female and male ani- mals [11]. Furthermore, it appears that the biologic effects of E2 on cardiac function are receptor dependent since simulta- neous administration of ICI 182,780, a selective ER antago- nist, abolished the salutary effects of E2 on cardiac function [9]. Although two subtypes of ERs (ER-a and ER-b) are known to exist, it remains unknown which subtype of recep- tor is predominantly responsible for producing the salutary effects of E2 on cardiac function following T-H. E2 has been reported to provide protection against vascular injury even in mice in which ER-a has been disrupted [12]. Moreover, the expression of ER-b, but not of ER-a, is stimulated after vas- cular injury in male rats [13]. Furthermore, studies utilizing ER-a or ER-b knock-out mice suggest that ER-b plays a role in cardioprotection following ischemia–reperfusion [14]. Thus, E2 may inhibit cardiovascular injury by an ER-b depen- dent mechanism.
The heat shock proteins (Hsp) are an important family of endogenous, protective proteins. In this regard, Hsp72 is induced by brief ischemia, and over expression of Hsp72 pro- tects cells and tissues against various forms of stress [15–17]. Conversely, under expression resulting from treatment with antisense oligonucleotides to Hsp72, increase susceptibility to hypoxia and reoxygenation injury [18]. Over expression of other Hsp including Hsp32 and Hsp60, is also reported to be protective against cardiac injury [19,20].
Hsp synthesis is controlled by a family of transcription factors, the heat-shock factors (HSF). Four HSF have been identified, but only HSF-1 has been shown to regulate the expression of Hsp in response to ischemia, hypoxia, heat, stress, or injury [21]. Heat and hypoxia activate HSF-1, which is present in the cytoplasm in an inactive, monomeric form. With stress, trimerization as well as phosphorylation occurs following which HSF-1 migrates to the nucleus. In the nucleus, HSF-1 binds to the heat-shock element, which is present in the promoter of the stress response gene, and then initiates Hsp transcription and synthesis.
Hsp90 is known to bind to intracellular steroid receptor, including the ER [22]. Previous studies have suggested that Hsp90 complexes with HSF-1 in cardiomyocytes [22]. Inter- actions involving Hsp90 and ER as well as the binding between Hsp90 and HSF thus represent an important ele- ment in the activation of HSF-1 by E2 [22]. We hypothesized that the salutary effects of E2 on cardiac function following T-H are mediated via ER-b-dependent up-regulation of Hsp. The aim of our study therefore was to determine which E2 receptor is predominantly responsible for the salutary effect on the depressed cardiovascular function following T-H and whether cardiac Hsp are affected by E2 administration under those conditions.
2. Materials and methods
2.1. T-H procedure
Our previously described non-heparinized rat model of T-H was used in this study [23]. Briefly, male SD rats (275–325 g, Charles River Labs, Wilmington, MA) were fasted overnight before the experiment but were allowed water ad libitum. The rats were anesthetized by isoflurane (Attane, Minrad Inc., Bethlehem, PA) inhalation prior to the induction of soft tis- sue trauma via 5-cm midline laparotomy. The abdomen was closed in layers, and catheters were placed in both femoral arteries and the right femoral vein (polyethylene [PE-50] tub- ing; Becton Dickinson & Co., Sparks, MD). The wounds were bathed with 1% lidocaine (Elkins-Sinn Inc., Cherry Hill, NJ) throughout the surgical procedure to reduce postoperative pain. Rats were then allowed to awaken, and bled to and main- tained at a mean arterial pressure (MAP) of 40 mmHg. This level of hypotension was continued until the animals could not maintain MAP of 40 mmHg unless additional fluid in the form of Ringer’s lactate (RL) was administered. This time was defined as maximum bleed-out, and the amount of with- drawn blood was noted. Following this, the rats were main- tained at MAP of 40 mmHg until 40% of the maximum bleed- out volume was returned in the form of RL. The animals were then resuscitated with four times the volume of the shed blood over 60 min with RL. Thirty minutes before the end of the resuscitation period, the rats received E2 (50 µg/kg, subcuta- neously), E2 co-administered with ER antagonist ICI 182,780 (3 mg/kg, intraperitoneally at the beginning of resuscitation), ER-a agonist propyl pyrazole triol (PPT) (5 µg/kg, subcuta- neously), ER-b agonist diarylpropiolnitrile (DPN) (5 µg/kg, subcutaneously), DPN administration with ICI 182,780 (3 mg/kg, intraperitoneally at the beginning of resuscitation), or an equal volume of the vehicle (~0.2 ml, 10% DMSO, Sigma). The catheters were then removed, the vessels ligated, and the skin incisions closed with sutures. Sham-operated ani- mals underwent the same groin dissection, which included the ligation of the femoral artery and vein, but neither hem- orrhage nor resuscitation was carried out. The animals were then returned to their cages and were allowed food and water ad libitum. All animal experiments were performed accord- ing to the guidelines of the Animal Welfare Act and The Guide for Care and Use of Laboratory Animals from the National Institutes of Health. This project was approved by the Insti- tutional Animal Care and Use Committee of the University of Alabama at Birmingham.
2.2. Measurement of cardiac output (CO) and in vivo heart performance
At 24 h after the completion of fluid resuscitation or sham- operation, the animals were anesthetized with isoflurane and catheterized via the right jugular vein. Under continued gen- eral anesthesia with pentobarbital sodium (25–30 mg/kg BW), CO and stroke volume were measured in each animal. A 2.4-French fiberoptic catheter was placed into the right carotid artery and connected to an in vivo hemoreflectometer (Hos- pex Fiberoptics, Chestnut Hill, MA), as described previously in [23]. Indocyanine green (ICG; Cardio Green, Becton Dick- inson) solution was injected via the catheter in the jugular vein (1 mg/ml aqueous solution as a 50-µl bolus). The con- centration of ICG was recorded by using a computer-assisted data-acquisition program (Asystant, Asyst Software, Roch- ester, NY). Following the measurement of CO, the right carotid artery was recannulated with PE-50 tubing and con- nected to a blood pressure analyzer (DigiMed, Louisville, KY). After the MBP was recorded, the PE-50 tubing was advanced into the left ventricle and connected to a heart per- formance analyzer (DigiMed) to monitor and record maxi- mal rate of pressure increase (+dP/dtmax) and decrease (–dP/dtmax), respectively.
2.3. Isolation of heart RNA
Heart RNA was isolated immediately after harvesting the heart using a Nucleospin RNA purification kit (BA Bio-science, Palo Alto, CA) according to the manufacturer’s instruction. The total RNA content in samples was deter- mined by a spectrophotometer (BIO-RAD, Smart TM 300) and the isolated RNA was then stored at –80 °C until ana- lyzed.
2.4. mRNA expression assay
Heart Hsp32, 60, 70, and 90 gene expressions were deter- mined by real-time quantitative reverse transcription poly- merase chain reaction (RT-PCR) as described previously in [23]. Amplification of cDNA was performed on an ABI PRISM 7900HT Sequence Detection System. The primer sequences are shown in Table 1. 18s were used for endog- enous control. All the samples were amplified for one cycle at 50 °C for 2 min and at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min.
2.5. Western blot assay
The hearts were homogenized in a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM (Fig. 1). E2 treatment prevented T-H-mediated decrease in Hsp60 and 90 protein expressions in the heart. In addition, Hsp32 and 70 protein expressions were further increased in E2 treated T-H rats. Administration of ER antagonist ICI 182,780 along with E2 prevented the E2-induced up- regulation of cardiac Hsp following T-H.
3.2. Effect of ER-α agonist PPT and ER-b agonist DPN in cardiac Hsp32, 60, 70, and 90 expression
Hsp60 mRNA gene expression decreased significantly in vehicle treated rats following T-H (Fig. 2B). DPN adminis- tration following T-H restored Hsp60 (Fig. 2B), and signifi- cantly up-regulated Hsp32 and 70 mRNA gene expression in the heart (Fig. 2A, C). There was no significant difference in Hsp90 mRNA gene expression in the heart between sham or T-H animals treated with vehicle, or DPN (Fig. 2D). Furthermore, there was no significant difference in above param- eters in T-H rats treated with PPT. In addition to mRNA gene expression, we examined the effects of PPT and DPN on the protein levels of cardiac Hsp following T-H. Hsp60 and 90 protein expression decreased significantly in vehicle treated rats following T-H (Fig. 3B, D). DPN treatment following T-H restored Hsp60 and 90, and significantly increased Hsp32 and 70 protein expression in the heart (Fig. 3A, C). In contrast, no significant change in above parameters was observed in T-H rats treated with PPT.
3.3. DNA binding activity of HSF-1 in heart
Since HSF-1 up-regulates Hsp expression in response to stresses, we examined whether ER-a or ER-b agonist affects HSF-1 DNA binding activity following T-H. As shown in Fig. 4, there was no significant difference in cardiac HSF-mals receiving PPT or DPN compared with vehicle-treated sham animals (data not shown). As shown in Fig. 6C, DPN significantly improved MBP following T-H compared to vehicle, however, it remained lower than shams. Further- more, DPN administration increased CO and SV following T-H and the values were similar to those observed in sham- operated animals (Figs. 6A, B and Fig. 7). In addition, DPN prevented the decrease in +dP/dtmax and –dP/dtmax; however, –dP/dtmax remained lower than shams. In contrast, no signifi- cant change in above parameters was observed in T-H rats treated with PPT. In order to evaluate whether the cardiopro- tective effect of DPN was via ER, a group of T-H rats were treated with DPN and ICI 182,780. Administration of ICI 182,780 abolished the DPN-induced attenuation of cardiac function following T-H.
4. Discussion
Previous studies have shown that cardiac function is sig- nificantly depressed in male animals following T-H [8]. In contrast, female rats in the proestrus state, a state in which plasma levels of estradiol were found to be the highest, showed no depression in CO at 24 h following T-H [24]. However, ovariectomized females displayed depression in organ func- tions after T-H, similar to those observed in males. Thus, it appears that female sex steroids have protective effects on cardiac function following T-H.
There are two ERs, ER-a and ER-b, which are differen- tially expressed in different tissues [25]. However, studies have shown that both ER-a and ER-b are expressed in cardiomyo- cytes [11,23]. In view of that, we attempted to determine which of the ER plays a predominant role in cardioprotection following T-H. Our results indicate that left ventricular performance (±dP/dtmax) was significantly depressed following T-H. Male rats treated with ER-b agonist DPN displayed improvement in ± dP/dtmax at 24 h following T-H. Moreover, the improved cardiac contractility was evident by the restored cardiac index in DPN-treated rats. Furthermore, our findings suggested that the biologic effects of DPN on cardiac func- tion are receptor dependent since the administration of ICI 182,780, a selective ER antagonist, along with DPN abol- ished the DPN-induced cardioprotection in T-H rats. In con- trast to DPN, treatment with ER-a agonist PPT did not con- fer cardioprotection following T-H.
The present study is the first to examine the effects of selec- tive ER agonist on cardiac functions following T-H and to show cardioprotective effects of DPN in vivo. DPN acts as an agonist on both ER subtypes but has a 70-fold higher relative binding affinity and 170-fold higher relative estrogenic potency in transcription assays with ER-b than ER-a [26]. PPT on the other hand is a selective agonist for the ER-a subtype and is the best agonist for ER-a out of a series of tetrasubstituted pyrazole analogs [27]. PPT binds to ER-a with high affinity, displaying 410-fold binding selectivity over ER-b [27]. ER-a and ER-b can form homo- and heterodimers and there are data suggesting that different compositions of the dimer differentially regulate gene expression [28]. These observations have led to the concept that selective ER modu- lators (SERMs) selectively activate E2-dependent protection in an organ such as heart, without stimulating the breast or ovarian proliferation. Our results provide evidence that fol- lowing T-H, E2-induced cardioprotection is mediated via ER-b activation. Furthermore, the findings that ER antago- nist, ICI 182,780 abolished DPN-induced cardioprotection following T-H, suggest that the salutary effects of DPN are mediated via ER. These findings corroborate previous stud- ies by Gabel et al. [14], which showed that ER-b knockout mice display increased cardiac injury following ischemia– reperfusion injury compared to the wild-type mice. It can be argued that we should have administered ICI 182,780 alone in these studies to determine if that per se has any adverse effects. In this regard, our previous studies have shown that administration of ICI 182,780 alone did not produce any deleterious effects but its administration with estrogen blocked the salutary effects of estrogen on cardiac function following T-H [9,29]. Since ICI 182,780 administration in itself did not influence cardiac function in trauma-hemorrhaged animals [29], administration of ICI 182,780 alone was not carried out in this study.
While the precise mechanism by which DPN mediates its salutary effects remains unknown, our findings suggest that DPN up-regulates Hsp. A couple of studies have examined the effects of E2 and gender on cardiac Hsp expression [22,30,31]. Those studies have shown that female rat hearts have twice as much Hsp72 as male hearts [30]. Ovariectomy reduced the level of Hsp72 in female hearts, and this could be prevented by E2 replacement therapy [30]. Additional stud- ies showed that 10 h of E2 treatment doubled the level of Hsp72 in adult cardiomyocytes from male rats [22]. Consis- tent with these findings, we observed that over-expression of heart Hsp32 following T-H with E2 administration [20].
Studies have shown that the induction of the Hsp by stress- ful stimuli is mediated by HSF-1 [43]. As a classical stress- responsive factor, HSF-1 binds to heat shock element, which is present upstream of many Hsp genes, and activates tran- scription of Hsp genes under stress conditions. HSF-1 has also been reported to be expressed in hearts [22], and protect cardiomyocytes from ischemia/reperfusion injury in trans- genic mouse model [21]. Intermolecular interaction between HSF-1 and a multichaperone complex including Hsp90 pro- vides mechanism for repression of HSF-1 activity [22]. In addition, Hsp90 is also known to bind to intracellular steroid receptors, including ER [22]. Thus, ligand-dependent inter- actions have been proposed to modify the equilibrium between Hsp90 and its molecular partners, HSF-1 and ER [22]. Our present study indicates that ER-b agonist DPN increased HSF- 1 DNA binding activity, thus suggesting that DPN may up-regulate cardiac Hsp through increase in cardiac HSF- 1 DNA binding activity.
In conclusion, our results suggest that similar to E2, DPN that is ER-b agonist also provides cardioprotection following T-H. Furthermore, our results indicate that up-regulation of Hsp likely TRC051384 plays a significant role in the DPN-mediated car- dioprotection following T-H.