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Am J Physiol Heart Circ Physiol 295: H409-H415, 2008. First published May 23, 2008; doi:10.1152/ajpheart.01018.2007
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Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes

Matthias J. Merkel,1,3 Lijuan Liu,2 Zhiping Cao,2 William Packwood,3 Patricia D. Hurn,1 and Donna M. Van Winkle1,2,3

1Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University; and 2Anesthesiology and 3Research Services, Veterans Affairs Medical Center Oregon, Portland, Oregon

Submitted 4 September 2007 ; accepted in final form 19 May 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
There is evidence for differences in the response to the treatment of cardiovascular disease in men and women. In addition, there are conflicting results regarding the effectiveness of pharmacologically induced protection or ischemic preconditioning in females. We investigated whether the ability of Met5-enkephalin (ME) to reduce cell death after oxygen-glucose deprivation (OGD) is influenced by the presence of 17β-estradiol (E2) in a nitric oxide (NO)- and estrogen receptor-dependent manner. On postnatal day 7 to 8, murine cardiomyocytes from wild-type or inducible NO synthase (iNOS) knockout mice were separated by sex, isolated by collagenase digestion, cultured for 24 h, and subjected to 90 min OGD and 180 min reoxygenation at 37°C (n = 4 to 5 replicates). Cell cultures were incubated in E2 for 15 min or 24 h before OGD. ME was used to increase cell survival. Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as means ± SE. As a result, in both sexes, ME-induced cell survival was lost in the presence of E2, and the ability of ME to improve cell survival was restored after treatment with the estrogen receptor antagonist ICI-182780. Furthermore, iNOS was necessary for ME to increase cell survival following OGD in vitro. We conclude that ME-induced reduction in cell death is abolished by E2 in a sex-independent manner via activation of estrogen receptors, and this interaction is dependent on iNOS.

viability; sex hormone; hypoxia

THERE IS INCREASING EVIDENCE that men and women respond differently to treatment for cardiovascular disease. According to recent studies, being female is an independent risk factor for adverse outcomes following conventional coronary artery bypass grafting, and women reportedly suffer a higher mortality rate following myocardial infarction (1, 32, 40). At the same time, premenopausal women experience a genuine protection against ischemia-reperfusion (I/R) injury, which is in part due to the female sex steroid estrogen. Experimental findings demonstrate that estrogen induces protection against I/R injury in vivo and reduces cell death in vitro (10, 20, 22, 30, 37). Estrogen-induced protection shares common pathways of cellular protection with known pharmacological preconditioning agents, such as opioids, bradykinin, and volatile anesthetics, as well as with ischemic preconditioning (26, 37). Pretreatment with these agents or with brief episodes of repeated ischemia activates a cascade of cellular signals involving phosphatidylinositol 3-kinase pathway (PI3K/Akt), nitric oxide (NO) synthase (NOS), and the opening of ATP-sensitive K+ channels in males (7, 11, 27, 28). Our laboratory has shown that Met5-enkephalin (ME), a pentapeptide that activates the {delta}-opioid receptor, induces protection against I/R injury in males in vitro and in vivo (8, 20). In contrast, there are conflicting results regarding the ability of pharmacological or ischemic preconditioning to induce protection in females (22, 34, 35, 39, 43). This apparent discrepancy remains unresolved.

We hypothesized that the cytoprotective properties of ME are influenced by estrogen [17β-estradiol (E2)] in an estrogen receptor (ER)- and NO-dependent manner. To test this hypothesis, we simulated I/R using oxygen-glucose deprivation (OGD) in sex-specific, isolated murine cardiomyocyte cultures in the absence and presence of both ME and E2. Selective inhibitors for ER or NOS were used and cardiomyocytes from inducible NOS (iNOS) knockout mice of both sexes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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The Institutional Animal Care and Use Committee at Portland Veterans Affairs (VA) Medical Center approved this study, and all animals received treatment in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Research, National Research Council; National Academy Press, 1996). Because dietary phytoestrogens have been reported to elicit protection against cerebral and myocardial ischemia (23, 44), we used cardiomyocytes from animals whose dams were fed a phytoestrogen-free diet (No. 2014, Harlan Teklad, Madison, WI). All study animals were allowed access to food and water ad libitum until induction of anesthesia. Pups used in this study were bred in the Portland VA, veterinary medical unit from C57BL/6J breeders or from homozygous iNOS–/– knockout breeders.

Cell isolation and culture. Ventricular cardiomyocytes from neonatal mice [postnatal day (PND) 7 to 8] were prepared as recently published (6). Briefly, neonatal mice were sexed by measuring the anogenital distance; pups with an intermediate anogenital distance were excluded (16). Pups were then anesthetized with pentobarbital sodium (50 mg/kg ip), and the hearts were removed aseptically. The ventricles from three to five hearts from same-sex mice were pooled, and the cardiomyocytes were isolated and cultured. Ventricles were kept on ice in Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+ containing (in g/l) 0.4 KCl, 0.06 KH2PO4, 8.0 NaCl, and 0.05 Na2HPO4 (pH 7.4). The ventricles were then washed three times with HBSS and minced into small fragments. Cells were dissociated at 25°C for 15 min with 0.625% (wt/vol) trypsin in HBSS without Ca2+ and Mg2+ (pH 7.4). Cells released after the first digestion were discarded; cells from subsequent digestions were added to an equal volume of cold HBSS with Ca2+ and Mg2+ containing (in g/l) 0.14 CaCl2, 0.4 KCl, 0.06 KH2PO4, 0.047 MgCl2, 0.049 MgSO4, 8.0 NaCl, 0.35 NaHCO3, 0.05 Na2HPO4, and 1.0 D-glucose (pH 7.4) until all cardiac cells were isolated. The resulting mixture was centrifuged for 8 min at 200 g, and the cells were resuspended in FBS-medium 199 (M199) supplemented with estrogen-free 15% FBS (vol/vol, Colcaco), 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µM cytosine arabinoside (to inhibit fibroblast attachment and proliferation). To exclude nonmuscle cells, the isolated cells were first plated in tissue culture dishes at 37°C for 1 h under a water-saturated atmosphere of 5% CO2-95% O2. Suspended cardiomyocytes were then collected and plated at a density of 1.0 x 105 cells/cm2 and incubated under the same conditions as above for 24 h.

Oxygen-glucose deprivation. To simulate ischemia, glucose-free medium (MEM-HBSS) was preequilibrated in 100% N2 at 37°C for 2 h. Oxygenated medium was removed from the cardiomyocyte cultures, and the N2-preequilibrated glucose-free medium was immediately added. Cultures were promptly placed in a custom-made Plexiglas hypoxia chamber and exposed to 100% N2 for 1.5 h at 37°C. For reoxygenation, the glucose-free medium was replaced with M199, and the cells were reoxygenated in 21% O2-5% CO2-74% room air for 3 h at 37°C.

Drugs. The agents used in this study were E2 (10 nM and 1 µM), ICI-182780 (ICI; 0.5 µM), N{omega}-nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 µM), and ME (100 µM). E2, ICI, and L-NAME were all obtained from Sigma (St. Louis, MO). E2 is the principal mammalian estrogen and signals through its cognate receptor to induce gene transcription or a rapid, nontranscriptional pathway in the cardiovascular system (18). ICI is a nonselective ER antagonist (42). L-NAME, a nonselective NOS inhibitor, blocks all three NOS isoforms. ME, purchased from Peninsula Laboratory (San Carlos, CA) and American Peptide (Sunnyvale, CA), is an endogenously produced pentapeptide that is selective for cloned {delta}-opioid receptors but appears to also activate {kappa}-opioid receptors in vivo (7). A subset of control cells were incubated in ethanol (1:1,000), the solvent for E2.

ICI was dissolved in DMSO (1:1,000), L-NAME and ME were dissolved in distilled water, and the drugs were then aliquoted and frozen until use. Concentrations were chosen based on our previous studies, reported IC50 values, and literature reports (8). On the day of the experiment, stock solutions were diluted and added directly into cells.

Experimental protocol. Sex-specific, isolated cardiomyocytes were incubated in two different concentrations of E2 (10 nM and 1 µM) for 15 min (short term) before OGD. Cell death was assessed at 60, 120, and 180 min of reoxygenation. All other experiments used 10 nM E2 or its vehicle for either short term or long term (24 h incubation) before adding ME (100 µM), in the presence or absence of ICI and L-NAME. Sex-specific, isolated cardiomyocytes from iNOS–/– mice were incubated in ME 15 min before OGD in the presence and absence of E2 (short-term and long-term incubation). Once added, the agonist and the antagonist remained in the culture medium for the duration of the experiments (See Fig. 1 for experimental time line). Each set of experiments included an oxygenated control group.


Figure 1
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  		Fig. 1. Experimental Protocols. The effect of 17β-estradiol (E2) on cell survival after oxygen-glucose deprivation (OGD) (experiment 1) was tested in cultured cardiomyocytes from female and male neonatal C57BL/6J mice incubated in two different concentrations of E2 (10 nM and 1 µM). The effect of E2 on Met5-enkepahlin (ME; 100 µM)-induced reduction in cell death (experiment 2) was evaluated after 15 min (short term, 2b) and 24 h (long term, 2c) incubation of cardiomyocytes in E2 (10 nM). A hormone-free culture medium was used for control (2a). The influence of estrogen receptors (ERs) on the E2-ME interaction (experiment 3) was tested by adding the nonselective ER inhibitor ICI-182780 (ICI, 0.5 µM) to the culture medium before E2 incubation as indicated. The effect of nitric oxide (NO) signaling (experiment 4) was examined by adding the nonspecific NO synthase (NOS) inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 100 µM) to E2-free medium and to E2 (10 nM)-containing medium as indicated. The role of inducible NOS (iNOS) on E2-ME interaction (experiment 5) was investigated using cardiomyocytes from iNOS knockout (iNOS–/–) mice of both sexes. Sex-specific cardiomyocyte cultures from iNOS–/– mice were incubated in E2 before ME treatment as indicated.

 
Determination of cell viability. Cell death was assessed with propidium iodide (5 µM) and quantified using a Zeiss Axiovert 200 fluorescent microscope and MetaMorph imaging system (Universal Imaging) as recently published (6). Serial measurements of cell viability were performed every 60 min during reoxygenation. More than 300 cells were examined in each sample.

Statistical analysis. Data analysis was performed with a personal computer-based statistical software package (Prism 4.0, GraphPad Software, San Diego, CA). The primary measured end point for all studies was cell death at 180 min of reoxygenation, defined as the uptake of propidium iodide. Cell death was corrected to dead cells in the oxygenated control for each experiment. Differences between experimental groups were plotted for both sexes. All data were analyzed using one- or two-way ANOVA as appropriate, with a Bonferroni post hoc test to compare replicate means. Statistical significance was set at P < 0.05. Results are expressed as means ± SE.


   RESULTS
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E2 improves cell survival after OGD independent of sex. Cardiomyocytes from male (CM) and female (CF) animals showed similar cell survival after OGD for both E2 concentrations tested (Fig. 2). There was no sex difference in cell death in the untreated controls. Ethanol alone (the solvent of E2) had no affect on cell death following OGD (data not shown).


Figure 2
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  		Fig. 2. Effect of E2 on cell death following OGD in vitro. Isolated cardiomyocytes from neonatal male (A) or female (B) C57BL/6J mice incubated with 2 concentrations of E2 (10 nM and 1 µM) for 15 min were subjected to 90 min OGD. The plots represent the percentage of dead cells over 180 min reoxygenation (n = 4 replicates; means ± SE).

 
E2 reduces ME-induced degree of cell survival after OGD. The ability of ME to reduce cell death after OGD was significantly decreased in the presence of E2 (15 min and 24 h incubation) in both CF and CM cultures (Fig. 3). However, after 15 min E2 incubation, the ME-induced reduction in cell death was significantly less attenuated in CM compared with CF. After 24 h of E2 incubation, CF and CM showed a similar degree of loss in the ME-induced reduction of cell death.


Figure 3
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  		Fig. 3. ME-induced reduction in cell death after OGD: effect of E2. Isolated cardiomyocytes from neonatal female or male C57BL/6J mice were incubated short term (15 min) or long term (24 h) in 10 nM E2 and treated with ME (100 µM) before 90 min OGD. The graph compares the percentage of dead cells with hormone-free, untreated controls after 180 min reoxygenation (n = 5 replicates; means ± SE). NS, not significant.

 
ME-induced reduction in cell death after OGD is restored by ER inhibition in the presence of E2. The inhibition of ERs restored the ME-induced reduction in cell death after OGD in the presence of E2 in CF independent of the E2 incubation time and in CM after short-term E2 incubation. The improved survival observed in cells treated with E2 alone was blocked by ER inhibition in cells from both sexes after long-term E2 but not after short-term E2 incubation (Fig. 4).


Figure 4
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  		Fig. 4. ER inhibition and ME-induced reduction in cell death after OGD. ERs are inhibited with ICI (0.5 µM) followed by E2 (10 nM) and ME (100 µM) treatment and 90 min OGD. The percentage of dead cells after 180 min reoxygenation is represented and corrected to an oxygenated control for each experiment (n = 5 replicates; means ± SE). For comparison, treatment with ME in E2-free medium reduced cell death to 21 ± 2% (cardiomyocytes from males) and 21 ± 1% (cardiomyocytes from females) at 180 min of reoxygenation (see Fig. 5).

 
ME-induced reduction in cell death is only partially blocked by NOS inhibition in the presence of E2. NOS inhibition after ME treatment had no affect on cell survival after short-term E2 incubation compared with E2 alone, but increased cell death was observed after long-term E2 incubation. This effect was sex independent. However, NOS inhibition after ME treatment in cardiomyocytes derived from animals of both sexes exposed to E2 showed an intermediate degree of improved cell survival after OGD compared with E2-free cultures. This was similar to the effect found in E2-free cultures of CF, whereas NOS inhibition completely abolished the ME-induced reduction in cell death after OGD in CM. This sex difference was independent of the timing of NOS inhibition (i.e., before or after ME treatment, data not shown). In the absence of ME, NOS inhibition resulted in increased cell death after short- and long-term E2 incubation in CF, but only after long-term E2 incubation in CM (Fig. 5).


Figure 5
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  		Fig. 5. NOS inhibition and ME-induced reduction in cell death after OGD: effect of E2. Nonselective NOS inhibition is achieved with L-NAME (100 µM) under 3 hormonal conditions [no E2, short-term E2 (10 nM), and long-term E2 (10 nM)] after ME (100 µM) treatment. The percentage of dead cells at 180 min reoxygenation following 90 min OGD is represented and corrected to an oxygenated control for each experiment (n = 5 replicates, means ± SE).

 
E2 effect on ME-induced reduction in cell death is iNOS dependent. OGD-induced cell death was unchanged in ME-treated cardiomyocytes from female iNOS knockout (CFormula) mice independent of E2 incubation time. ME-treated cardiomyocytes from male iNOS–/– animals (CFormula) showed improved cell survival after long-term, but not after short-term, E2 incubation. OGD-induced cell death was increased in CFormula compared with CFormula at all conditions tested, but not after 24 h E2 incubation (Fig. 6).


Figure 6
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  		Fig. 6. iNOS elimination and ME-induced reduction in cell death after OGD: effect of E2. Cardiomyocytes from iNOS–/– mice of both sexes were subjected to 90 min OGD after short-term (15 min) and long-term (24 h) incubation in E2 (10 nM) and ME (100 µM) treatment. F, female. The percentage of dead cells after 180 min reoxygenation is represented and corrected to an oxygenated control for each experiment (n = 5 replicates, means ± SE).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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The principal finding of the study is that in isolated, sex-specific cardiomyocyte cultures, E2 abolished ME-induced reduction in cell death after OGD, irrespective of sex. ER inhibition restored the ME-induced increased survival after OGD in cardiomyocytes of both sexes, suggesting that the ability of E2 to block ME-induced reduction in cell death is ER mediated. Furthermore, the absence of iNOS significantly reduced ME- or E2-induced reduction in cell death following OGD. These findings suggest that the E2-ME interaction is sex independent, ER mediated, and influenced by an iNOS-dependent mechanism.

Others have reported increased tolerance against myocardial I/R injury in female versus male mice, rats, and rabbits (3, 35, 41, 43), but not in dogs (31). Exogenous estrogen confers cardioprotection against I/R in vivo in females and males of various species (4, 10, 38), but it is unclear whether the protection is dependent on ER-{alpha} or -β (5, 14) or is ER independent (10). In addition, ischemic preconditioning is protective in older female mice but not in young females, suggesting the potential for a hormonal influence (39). These previous studies suggest that the capacity to induce protection in the female heart may depend, in part, on the availability of endogenous or exogenous estradiol. More specifically, we found that estradiol limits cardiomyocyte death following OGD but produces an undesirable suppression of ME-induced cell salvage in male and female cardiomyocytes. In contrast, Shinmura et al. (34) reported that neither sex nor age impaired opioid-induced late preconditioning in an isolated rat heart model evaluating functional recovery after global ischemia. These conflicting results may be due to the different models used. The current study was performed in an isolated, sex-specific cultured cardiomyocyte model to specifically investigate the effect of E2 on ME-induced cytoprotection, without the potential confounding influences inherent in an in vivo model (e.g., other cells such as endothelium, hydraulic and mechanic stress, etc.). Although it is premature to definitively extrapolate the current observations to the in vivo situation, the data suggest that circulating estradiol may modulate the magnitude of opioid-induced protection in the in vivo female heart.

Both ER subtypes (ER-{alpha} and ER-β) belong to a family of nuclear hormone receptors that are ligand-activated transcription factors which mediate many of the effects of estrogen (24). Nuedling et al. (25) found an ER-mediated increase in iNOS (50 ± 23-fold) and endothelial NOS (eNOS) expression (16 ± 4-fold) in adult and neonatal rat cardiomyocytes after 24 h of E2 incubation (10 nM), which was associated with the translocalization of the ER into the nucleus. Other studies have reported that estradiol can rapidly activate NOS. Five minutes of exposure to E2 resulted in the upregulation of eNOS in vascular endothelial cells, and this effect was blocked by the ER antagonist ICI (21).

A possible explanation for our finding that E2 abolishes ME-induced improvement in cell survival is that NOS expression and/or activity is enhanced by both E2 and ME, resulting in toxic NO levels when E2 and ME are present simultaneously. High levels of NO have been previously reported to injure, rather than protect, myocardium during reperfusion (Ref. 33, and see Ref. 15 for review).

The selective inhibition of iNOS in male rats attenuated the protective effect of a {delta}-opioid receptor agonist when applied immediately before the ischemic event in vivo (29). Similarly, we found that ME does not improve cell survival after OGD in CFormula. The negative interaction between E2 and ME resulting in increased cell death after OGD was absent in cardiomyocytes from male and female iNOS–/– animals. In hormone-free conditions, the nonselective inhibition of NOS completely blocked ME-induced reduction in cell death in CM but only partially in CF. After incubation with E2, NOS inhibition in CM resulted in partially blocked ME-induced reduction in cell death following OGD, similar to that seen in CF. This suggests that an additional, NO-independent signal pathway is involved in protecting CF against OGD and that a similar pathway can be activated in CM after incubation in E2.

Our experiments suggest that the intracellular mechanisms responsible for E2 reducing ME-mediated cytoprotection might be sexual dimorphic and could represent the activation of different pathways in both sexes. Further studies are needed to determine sex differences in the signaling pathways involved. Besides an interaction in the NO signaling pathway, as investigated in the present study, there are several other signaling pathways that may serve as potential targets for E2 to negate ME-induced cytoprotection. For instance, both E2 and ME are known to activate the PI3K/Akt signaling pathway and ATP-sensitive K+ channels (8, 19, 22, 27, 30). Our laboratory recently showed that ME activates Akt isoforms in a sexual dimorphic pattern, with CF primarily activating Akt3 and CM activating Akt1/2 (6). However, it is unknown whether a competitive antagonism exists in these pathways. Alternatively, there may be a sexual dimorphism in E2 receptors, rather than in postreceptor signaling. Although we did not study the functional expression of ERs in our cardiomyocytes, it has been previously shown by Grohe et al. (17) that neonatal rat cardiomyocytes express functional ER-{alpha} and -β. However, the sexual dimorphism of the receptor content was not examined. Our observation that the ER antagonist ICI abolished E2-mediated cytoprotection after 24 h of incubation supports the existence of functional ERs in neonatal murine cardiomyocytes but similarly does not address whether there is a sex difference in relative ER-{alpha}/ER-β content.

Although we modified culture conditions to eliminate other sources of estrogen, using, for example, estrogen-free bovine serum albumin, the cultured cardiomyocytes undoubtedly were exposed to estrogen to some degree. The developing fetus is exposed to gonadal steroids. In addition, although maternal estrogens are largely bound to plasma proteins and the murine placenta does not produce gonadal steroids during the second half of pregnancy (2, 12), the murine fetal testis produces testosterone with testosterone levels surging at about murine fetal day 16 and at parturition (9, 13). Thus cardiomyocytes from PND 7 to 8 male animals will have been exposed to testosterone surges not experienced by cardiomyocytes from PND 7 to 8 female animals. Whether this affects cardiomyocyte hypoxic tolerance was not addressed in the current study and will require future examination.

Finally, because we used neonatal cardiomyocytes rather than adult cardiomyocytes, we cannot exclude the possibility that adult tissue would behave differently from what we observed in the current study. However, we used PND 7 to 8 murine cardiomyocytes when myocardial hyperplasia is largely complete and when cardiomyocytes are terminally differentiated (36). In addition, eNOS and iNOS expression was similarly upregulated in neonatal (PND 1 to 2) and adult rat cardiomyocytes following 24 h of E2 incubation (25).

In conclusion, the current results demonstrate that ME-induced reduction in cell death after OGD is abolished in the presence of E2 in isolated murine cardiomyocytes. This E2-ME interaction is ER mediated and sex independent. Since E2 can block opioid-induced reduction in cell death following OGD in vitro, this may explain conflicting reports of opioid-induced protection in female animals of undefined hormonal status.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by a VA Merit Review grant (Medical Research Service, Dept. of VA; to D. M. Van Winkle) and funding from the Medical Research Foundation of Oregon and the Anesthesiology Research and Education Foundation (to M. J. Merkel).


   ACKNOWLEDGMENTS
 
We especially thank Kathy Gage for excellent editorial assistance with the manuscript and the Portland Veterans Affairs (VA) Veterinary Medical Unit staff for the excellent care of the breeding colonies used for this project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Merkel, Dept. of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science Univ., Mail Code UHS-2, 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098 (e-mail: merkelm{at}ohsu.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.


   REFERENCES
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 MATERIALS AND METHODS
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