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Am J Physiol Heart Circ Physiol 291: H1554-H1562, 2006. First published April 28, 2006; doi:10.1152/ajpheart.01171.2005
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Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS

Mikhail A. Sovershaev,1,2,* Elena M. Egorina,1,2,* Thomas V. Andreasen,1 Anne K. Jonassen,3 and Kirsti Ytrehus1

1Department of Medical Physiology and 2Department of Biochemistry, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Tromsø; and 3Department of Biomedicine, Section for Physiology, Faculty of Medicine, University of Bergen, Bergen, Norway

Submitted 4 November 2005 ; accepted in final form 25 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To study the cell signaling events leading to 17beta-estradiol (E2)-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 µM E2 before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E2-induced protection, we combined wortmannin (1 µM), chelerythrine (2 µM), and 2-mercaptopropionylglycine (300 µM), respectively, with E2 exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E2 preconditioning were investigated. E2 preconditioning led to significant reduction in infarct size from 31.8 ± 5.3 to 20.2 ± 2.6% in male hearts and from 42.7 ± 4.7 to 17.1 ± 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 ± 3.2%), chelerythrine (45.1 ± 4.4%), and 2-mercaptopropionylglycine (36.8 ± 4.7%). E2 preconditioning induced phosphorylation of PKB, PKC{alpha}, and PKC{epsilon} and membrane translocation of PKC{epsilon} and PKC{delta}. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E2 to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.

phosphoinositide 3-kinase; protein kinase B; protein kinase C; reactive oxygen species

ACUTE ADMINISTRATION of the major sex steroid 17beta-estradiol (E2) in pharmacological concentrations affords cardioprotective effect against ischemic injury in the heart (11, 32). Nongenomic effects of the hormone, such as antioxidant activities (17), inhibition of L-type Ca2+ channels (34), increased nitric oxide production (25), and opening of mitochondrial ATP-dependent K+ channels (mitoKATP channels) (19), are proposed mechanisms for protection. There is an increasing number of studies characterizing cell signaling downstream of E2 receptors in various cell systems. Preconditioning of the heart increases cellular resistance to lethal ischemia. Cardioprotection by preconditioning is induced by a short-lasting sublethal ischemia and by pharmacological agents like adenosine, opioids, and bradykinin (reviewed in Ref. 24). The molecular basis of the cellular memory involved in ischemic preconditioning is associated with activation of protein kinase C (PKC) (37), opening of mitoKATP channels (9), production of reactive oxygen species (ROS) (28), and activation of cell survival pathways like the phosphoinositide-3 kinase (PI3-K)/protein kinase B (PKB) pathway (35).

In the present study, we demonstrate acute, sex-independent protection against regional ischemia by an intermittent, preconditioning-like pretreatment with E2 and characterize signaling events shared with classic ischemic preconditioning in the heart.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures were in conformance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee in Norway.

Heart perfusion protocol. Adult Wistar rats (200–300 g) of both sexes were used and subjected to a standardized regional ischemia (RI) infarct model (2). After pentobarbital anesthesia, hearts were excised and perfused using the Langendorff technique with Krebs-Henseleit buffer (KHB; pH 7.4) at constant pressure (70 mmHg) and temperature (37°C). Left ventricular pressure was recorded using a LabView 6.0-based acquisition system via a latex water-filled balloon connected to a pressure transducer. Coronary flow was measured by timed collection of heart effluate. A silk 3-0 thread was placed around the main branch of the left coronary artery with ends passed through a plastic tube to form a snare. RI was induced by tightening the snare and was confirmed by at least a 40% fall in left ventricular systolic pressure and coronary flow. Hearts failing to develop adequate baseline systolic pressure, with persistent reperfusion arrhythmias, or with no signs of reperfusion after release of the ligature were excluded from the study. Hearts were stabilized for 15 min, received pretreatment as indicated (Fig. 1), were subjected to 30 min of RI, and thereafter were reperfused for 120 min. Control hearts were perfused for 20 min before RI. E2 (10 µM) was given as three cycles of 5-min infusion followed by 10 min of washout. The repetitive mode of E2 administration was chosen to introduce washout periods of hormone from the myocardium to avoid possible direct antioxidant action of E2 and to delineate indirect mechanisms in the ischemic heart. The cardioprotective concentration of E2 was selected on the basis of a dose-response pilot study in which 0.001, 0.01, 0.1, 1, and 10 µM concentrations of E2 were tested. Existence of a possible sex difference in the acute cardioprotective effect of E2 treatment was tested. We compared the protection afforded by E2 with ischemic preconditioning by three cycles of 5-min global ischemia interspaced by 5-min intervals of normal perfusion. Involvement of the PI3-K/PKB pathway and PKC was studied by adding a background infusion of wortmannin (1 µM) or chelerythrine (2 µM) 10 min before and during the E2 cycles and 2 min after the induction of RI. To investigate whether cardioprotection by E2 was mediated through ROS formation in the myocardium, we coadministered the cell-permeable ROS scavenger 2-mercaptopropionylglycine (MPG; 300 µM) as a background infusion during E2 preconditioning cycles. A separate experimental protocol involving a longer washout period (15 min) after the last E2 cycle was needed, because the infusion of MPG immediately before the induction of RI was cardioprotective by itself. At the end of the reperfusion, hearts were collected for planimetric measurements of infarction volume.


Figure 1
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  		Fig. 1. A: experimental protocol. E2, 17beta-estradiol (10 µM); WM, wortmannin (1 µM); Chel, chelerythrine chloride (2 µM); MPG, 2-mercaptopropionylglycine (300 µM); IPC, ischemic preconditioning; GI, global ischemia; RI, regional ischemia. Arrow indicates time point when hearts were freeze-clamped for immunoblotting analyzes. B: infarct sizes of dose-response study. M, molar concentration.

 
Risk zone and infarct size volumes. To label the ischemic risk zone, the snare was retightened at the end of the reperfusion and hearts were flushed with 0.2% (wt/vol) solution of Evans blue dye to mark perfused tissue. The hearts were then frozen and cut into 2-mm-thick slices. Survived tissue was visualized using triphenyltetrazolium chloride staining. After formaldehyde fixation, planimetric analysis was performed. Infarcts were expressed in percentage of risk zone volumes.

Subcellular fractionation and Western blotting. To investigate whether preconditioning by E2 was associated with changes in phosphorylation of PKB and PKC isoforms {alpha}, beta, {epsilon}, and {delta} and with translocation of PKC{epsilon} and PKC{delta} from the soluble to the membrane fraction of heart tissue homogenate, hearts were freeze-clamped before RI as indicated by the arrow in Fig. 1.

Heart tissue was homogenized in an ice-cold buffer consisting of (mM) 250 sucrose, 1 EDTA, 10 HEPES (pH 7.4), 1 dithiothreitol (DTT), and a Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and was centrifuged at 200 g for 10 min at 4°C to remove tissue debris. Nuclear, mitochondrial, cytosolic, and membrane fractions of heart homogenates were obtained using differential centrifugation at 1,000, 17,000 and 100,000 g at 4°C in the presence of protease inhibitors and DTT. The pellets from all fractions were briefly sonicated before addition of 2x SDS sample buffer. The purity of the fraction was ascertained by immunoblotting the samples of fractions against cytosolic protein I{kappa}B-{alpha} (Cell Signaling, Beverly, MA) and mitochondrial protein voltage-dependent anion channel (VDAC; Merck Biosciences, Darmstadt, Germany).

For assessment of PKC translocation, soluble fraction was separated by centrifugation of heart homogenates at 100,000 g for 60 min at 4°C. The particulate fraction was extracted by sonication from the resulting pellet in the presence of 1% Triton X-100, 20 mM DTT, and 1 mM beta-mercaptoethanol.

On completion of the electrophoresis and transfer of the protein onto the nitrocellulose membranes, the latter were blocked for 1 h in 5% (wt/vol) skimmed milk and sequentially incubated with primary antibodies against Ser473-phosphorylated PKB, total PKB (both from Cell Signaling), total PKC{epsilon} and PKC{delta} (both from BD Transduction Laboratories, San Diego, CA), phosphorylated pan-PKC [hydrophobic domain autophosphorylation sites (30) or turn motif autophosphorylation sites Thr638 of PKC{alpha}, Thr641 of PKCbeta, activation loop phosphorylation site Thr505 of PKC{delta}, and hydrophobic domain autophosphorylation site Ser729 of PKC{epsilon} (phospho-PKC kit from Cell Signaling)] as described in the protocols from the supplier. Protein loading was confirmed by blotting against beta-actin (Cell Signaling) or VDAC (Merck Biosciences). Immunopositive bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibodies and imaged using a LumiIMAGER F1 camera (Boehringer Mannheim, Mannheim, Germany).

Detection of intracellular ROS. To investigate whether treatment with E2 led to an increase in intracellular levels of ROS, we perfused 10 rat hearts with KHB containing 10 µM dihydroethidium (DHE; Molecular Probes, Invitrogen, Norway) with or without three 5-min cycles of 10 µM E2, followed by a washout of DHE for 5 min. DHE enters the cells and, after being oxidized to fluorescent ethidium by intracellular ROS, intercalates into DNA. Fluorescence of ethidium is directly proportional to the levels of ROS, primarily superoxide radical (16). By the end of the treatment, hearts were washed with KHB for 5 min and frozen (–70°C) until 20-µm cryosections were prepared for microscopy. Images of ethidium fluorescence were obtained using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany). To assess the fluorescence in a maximum thickness of the preparation, a pinhole was opened to 1,000 Airy units, which resulted in a focal plane of 15 µm. All images were acquired using the same sensitivity settings and were thereafter analyzed to obtain mean image signal intensity for data presentation and comparison.

To investigate the E2-induced time course of ROS production, we used a mouse cardiac muscle HL-1 cell line (purchased from Dr. W. C. Claycomb, New Orleans, LA) in live cell imaging experiments. Cells were grown in Claycomb medium supplemented with 100 µg/ml penicillin, 0.1 mM norepinephrine, 2 mM L-glutamine, and 10% fetal bovine serum as described elsewhere (5). Ten-thousand cells per well were plated onto the chambered cover glasses, which were precoated with 0.02% fibronectin (Sigma-Aldrich, Munich, Germany). Loading with fluorescent ROS indicator dichloromethyl-dichlorodihydrofluorescein diacetate (DCF; Molecular Probes) was performed by replacing the growth medium with a sterile prewarmed phosphate-buffered saline (PBS) containing 5 µM DCF. Cell were allowed to load with DCF for 15 min in the incubator and were thereafter washed two times with warm PBS. Images of DCF fluorescence were acquired using a Zeiss LSM 510 confocal microscope. The microscope stage was kept at a constant temperature to maintain cells at 37°C during live cell imaging experiments. To avoid photobleaching of DCF during acquisition of a time series of images, the 488-nm argon laser intensity was set as 2% of maximal and the pinhole was opened at ~3.6 Airy units. As a positive control for intracellular ROS production, 1 µM PMA for 10 min at 37°C was used. The DCF fluorescence was assessed before, during, and 20 min after PMA exposure. E2-mediated intracellular ROS production was imaged during 5-min exposure of cells to 10 µM E2 and for 20 min after withdrawal of the hormone. Fluorescence intensities of five cells from three experiments were analyzed.

ROS, PKB, and PKC in E2-induced signaling. To investigate the relationship among ROS, PKB, and PKC in E2-induced signaling pathway, we utilized the HL-1 myocyte model. Cells were grown as described in Detection of intracellular ROS. One day before the experiment, 150 x 103 cells per well were plated onto six-well cell culture plate. Cells were treated with 10 µM E2 for 15 min, which was changed to a full growth medium without E2 for 15 min before harvesting. The cell-permeable ROS scavenger MPG, the PI3-K inhibitor wortmannin, or the PKC inhibitor chelerythrine, at the concentrations used in the isolated rat heart model, were administered 30 min before, during, and 15 min after E2 exposure. In the control experiments, all listed inhibitors were also administered alone for 60 min. Cells were washed once in prewarmed PBS and harvested in 50 µl of 2x SDS loading buffer for further determination of changes in the levels of phosphorylated PKB and PKC{epsilon} by immunoblotting as described in Subcellular fractionation and Western blotting.

Statistical analysis. Values are presented as means ± SE. Differences among groups in heart function and infarct volumes were analyzed using one-way ANOVA with Tukey's post hoc test. Functional data were tested for significant changes from baseline at selected time points by using ANOVA combined with paired t-tests and Bonferroni correction. A P value of ≤0.05 was used as the level of significance. For statistical analyses, we used Minitab 9.2 software (Minitab, State College, PA).

Reagents. Water-soluble cyclodextrin-encapsulated E2, chelerythrine chloride, MPG, triphenyltetrazolium chloride, salts, and other reagents were purchased from Sigma-Aldrich. Wortmannin was obtained from Tocris Cookson (Avonmouth, UK).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Function of isolated hearts. There were no significant differences in baseline function among groups, and none of the treatments caused significant deviations in developed pressure, heart rate, or coronary flow (Table 1). Coronary flow (CF) and left ventricular developed pressure (LVDP) decreased immediately after the onset of RI, and hearts of all groups showed partly restored LVDP and CF upon reperfusion. The inhibition of PKC by chelerythrine administration before ischemia, however, led to partial prevention of ischemia-reperfusion-induced diastolic contracture (Table 1).


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  		Table 1. Functional parameters

 
Infarct size. Hearts in the female groups did not differ in volumes of risk zone and left ventricle, and, correspondingly, risk zone and ventricular volumes of hearts in the two male groups did not differ. Male hearts had larger left ventricle volumes and risk zone volumes, respectively, than female hearts (Table 2). As depicted in Fig. 2, three 5-min cycles of E2 at 10 µM concentration led to a significant decrease in infarct volumes in both sexes, from 42.7 ± 4.7 to 17.1 ± 3.4% in female rat hearts and from 31.8 ± 5.3 to 20.2 ± 2.6% in male rat hearts (both P ≤ 0.05). There were no statistically significant sex differences between control and E2-treated hearts. Because we confirmed that E2 provided protection in both female and male myocardium, we chose to continue the study using female rat hearts. As was expected, ischemic preconditioning of female hearts with three 5-min cycles of global ischemia caused pronounced protection (Fig. 2), and there were no statistically significant differences between infarct sizes of hearts protected by ischemic preconditioned and by E2 (Fig. 2).


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  		Table 2. Left ventricle and risk zone volumes in experimental groups

 

Figure 2
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  		Fig. 2. E2-induced infarct limitation. Infarct sizes are expressed as percentages of risk zone volume. Solid circles indicate group means (±SE), and open circles represent data from individual experiments. *P ≤ 0.05 vs. female controls. #P ≤ 0.05 vs. male controls.

 
Role of PI3-K and PKB. When the PI3-K inhibitor wortmannin was added to the E2 protocol, a reduction in protection was observed and infarct size increased from 17.1 ± 3.4% in E2-treated hearts to 30.0 ± 3.2% of the risk zone volume, and this value was not significantly different from that of the control group (Fig. 3A) or the wortmannin-alone control group (37.4 ± 4.7%). Activation of the PI3-K pathway was confirmed by the observed increase in Ser473 phosphorylation of the PI3-K downstream target PKB in heart homogenates following E2 preconditioning in combination with no changes in the levels of total PKB (Fig. 3B). Wortmannin coadministration abolished the increase in phosphorylation of PKB. To study the subcellular pattern of PKB phosphorylation after E2 administration, we obtained nuclear, membrane, mitochondrial, and cytoplasmic fractions by differential centrifugation and immunoblotted samples with antibodies against Ser473-phosphorylated PKB. Interestingly, levels of phosphorylated PKB seemed to be elevated only in nuclear and membrane fractions, not in mitochondrial and cytosolic fractions (Fig. 3B). This effect was sensitive to wortmannin coadministration. With respect to the membrane fraction, E2-treated hearts seemed to have some decrease in PKB levels in this fraction, which was not reversed by PI3-K inhibition with wortmannin (Fig. 3B). Furthermore, E2-induced activation of PKB was only partially sensitive to coadministration of the ROS scavenger MPG and was not sensitive to inhibition of PKC (see GoGoFig. 6A).


Figure 3
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  		Fig. 3. A: infarct size after phosphoinositide 3-kinase (PI3-K) inhibition with WM during E2 administration. Data are presented as described in Fig. 2 legend. B: PKB immunoblotting: total PKB and Ser473-phosphorylated PKB (P-PKB) before RI in controls, E2-treated, and E2 + WM-treated hearts examined in total extracts and in nuclear, membrane, mitochondrial, and cytosolic fractions. Densitometry data are mean phosphorylated-to-total ratios of PKB, presented as percentages of controls. *P ≤ 0.05 vs. controls.

 

Figure 4
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  		Fig. 4. A: infarct size after PKC inhibition with Chel during E2 administration. Data are presented as described in Fig. 2 legend. B: immunoblotting against PKC{epsilon} and PKC{delta} in soluble and membrane fractions of total homogenate. C: phosphorylated PKC (pan and selected isoenzymes in preischemic hearts) in subcellular fractions. VDAC, voltage-dependent anion channel. *P ≤ 0.05 vs. controls.

 

Figure 5
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  		Fig. 5. A: infarct size after reactive oxygen species (ROS) scavenging by 2-mercaptopropionylglycine (MPG) during E2 administration. Data are presented as described in Fig. 2 legend. *P ≤ 0.05 vs. female controls. At times indicated (&), E2 infusion was followed by 15-min washout (see Fig. 1 for protocol details). B: increased ROS after three 5-min cycles of E2, detected using ethidium fluorescence in isolated rat heart sections. Data are mean (±SE) image fluorescence intensities of 5 fields from 5 hearts, presented as a percentage of controls. C: increased production of ROS after exposure of HL-1 cells to PMA, used as a positive control for stimulation of ROS production. Data are mean (±SE) cell fluorescence from 3 experiments, presented as a percentage of baseline fluorescence. *P ≤ 0.05 vs. baseline. D: time course of ROS levels measured in HL-1 cells after the withdrawal of PMA or E2. Data are mean (SD) cell fluorescence of 3 experiments, presented as a percentage of baseline fluorescence at treatment withdrawal. **P ≤ 0.05 vs. vehicle-treated controls. Red and blue circles indicate time points at 0 and 650 s, respectively. DIC, differential interference contrast; DCF, dichloromethyl-dichlorodihydrofluorescein diacetate.

 

Figure 6
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  		Fig. 6. A: E2-induced Ser473 phosphorylation of PKB was partially abolished by the ROS scavenger MPG and the PI3-K inhibitor WM but was not sensitive to the PKC inhibitor Chel. B: E2-induced autophosphorylation of PKC{epsilon} was completely abolished by MPG but only partially abolished by WM and Chel. Densitometry data are plotted as described in Fig. 3B. *P ≤ 0.05 vs. nontreated controls.

 
Role of PKC. Acute cardioprotection induced by E2 was completely abolished by the PKC inhibitor chelerythrine, and infarcts in percentage of risk zone volume were not different from those of control rat hearts (Fig. 4A). Administration of E2 led to translocation of PKC{epsilon} and PKC{delta} from the soluble fraction of heart tissue (Fig. 4B), although we failed to detect a statistically significant increase in the membrane fraction. This effect was not associated with degradation of the kinases, because levels observed in total homogenate appeared to remain constant (Fig. 4B). At the subcellular level, PKC{alpha}, PKC{epsilon}, and PKC{delta} were found in all fractions examined. Immunoblotting of subcellular fractions against phosphorylated PKC isoforms revealed an E2-induced increase in levels of phosphorylated PKC{alpha} and PKC{epsilon} in the cytosolic fraction (Fig. 4C). Interestingly, pretreatment by E2 led to a reduction in the levels of phosphorylated PKC{delta} in all fractions of the heart homogenates (Fig. 4C). Levels of autophosphorylated PKC{epsilon} after E2 treatment were significantly reduced by coadministration of the ROS scavenger MPG and also partially sensitive to inhibition of PI3-K by wortmannin (see Fig. 6B).

Role of ROS. The protection against infarction by E2 was completely prevented when hearts were perfused with buffer containing MPG (Fig. 5A). MPG did not cause significant changes in infarcts (40.1 ± 2.5%) when used alone with a 10-min washout period to deplete the myocardium of MPG before RI.

Having obtained the data showing that coadministration of the ROS scavenger MPG diminished the protective effect of E2, we challenged the production of ROS in isolated rat hearts. Repeated exposure of hearts to E2 cycles followed by washout led to an increase in ROS-dependent oxidation of DHE to ethidium, detected in cryosections of hearts by fluorescence microscopy (Fig. 5B) compared with untreated controls.

With respect to the HL-1 cardiomyocytes in culture, ROS levels continued to increase after withdrawal of E2. We have verified our methodology of DCF-mediated visualization of intracellular ROS production by imaging PMA-stimulated HL-1 atrial adult mice cardiomyocytes. Indeed, even during the exposure to PMA, we observed an increase in DCF fluorescence (Fig. 5C), which continued to rise after the withdrawal of PMA (Fig. 5D). We did not detect an elevation of ROS during the exposure of cells to E2 (data not shown); however, 5 to 10 min after the withdrawal of hormone, we detected an abrupt and transient increase in intracellular ROS levels (Fig. 5D). Neither the increase in DCF fluorescence during PMA nor that following E2 exposure was due to a laser-induced ROS production as demonstrated by an absence of significant deviations in DCF fluorescence in a 20-min time series of vehicle-treated control cells (Fig. 5D). Noteworthy, coadministration of MPG and E2 had a profound effect on activation of PKC{epsilon} and, to a lesser extent, on activation of PKB, judging by the levels of phosphorylated kinases in cell extracts (Fig. 6, A and B).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that acute repetitive exposure to E2 at a pharmacological concentration leads to the development of cardioprotection against infarction in isolated rat hearts. We observed the protection in both sexes. Sex differences in E2-induced acute cardioprotection have been reported, although the protection against ischemia has been observed in both males and females (22).

The development of the acute cardioprotection was associated with the involvement of a number of signaling pathways. The dependence of cardioprotection on chelerythrine-induced inhibition of PKC, the translocation of PKC isoforms {epsilon} and {delta} from the water-soluble to the particulate fraction of heart lysates after E2 treatment, and increased levels of phosphorylated PKC{alpha} and PKC{epsilon} in selected subcellular compartments demonstrate an involvement of this family of serine-threonine kinases in the protective pathway, induced by acute administration of hormone. It is, however, difficult to conclude which of the PKC isoforms is of the most importance for the E2-induced cardioprotection. It is well accepted that protein kinases of the PKC family are pivotal signaling molecules in the protection in ischemic preconditioning (37). A role of both PKC{epsilon} and PKC{delta} in the preconditioning phenomenon has been reported (15, 20). The data obtained from different species are inconsistent with respect to the role of PKC{delta} in the transmission of the preconditioning signal. Although E2 treatment induced translocation of PKC{delta}, it is of interest to note that it also decreased levels of PKC{delta} phosphorylated on the activation loop phosphorylation site in all subcellular fractions. It is tempting, however, to speculate that interaction between phosphorylation and translocation is important to establish cardioprotection and that E2 regulates {epsilon} and {delta} isoforms of PKC differently by phosphorylation. The presented data support PKC involvement in cardioprotection by E2 but do not permit definite conclusions regarding regulation of PKC isoforms by E2. Genetic studies using gene-silencing techniques are indeed necessary to answer this question.

By means of pharmacological inhibition of PI3-K, which prevented the development of cardioprotection after E2 treatment, we now place PI3-K/PKB signaling pathway downstream of E2 action in the heart. The induction of wortmannin-sensitive phosphorylation of Ser473 on PKB in heart lysates following E2 pulses has confirmed the ability of E2 to activate this kinase in the heart, namely, in the nuclear and membrane subcellular fractions. An explanation for the latter could be an increased trafficking of PKB between the membrane and the nucleus due to activation of the pathway by E2. Our data are in agreement with findings of a study by Camper-Kirby et al. (3), who demonstrated nuclear enrichment of phosphorylated PKB in myocardial samples from young women and mature female mice and in isolated cardiomyocytes exposed to estrogens. It is well established that activation of the PI3-K/PKB pathway increases ischemia tolerance in the heart (13, 35). E2 could activate PI3-K by two distinct mechanisms. Simoncini et al. (31) demonstrated physical interaction of ER{alpha} with p85, a regulatory subunit of PI3-K. This interaction led to a quick activation of PI3-K and downstream PKB.

We were able to demonstrate the relationship among ROS, PKB, and PKC{epsilon} in a prosurvival signaling pathway activated by E2. E2 exposure and withdrawal-mediated elevation of intracellular ROS levels signaled to activation of both PKC and PKB, given that the cell-permeable ROS scavenger MPG completely abolished PKC activation and, to a lesser extent, PKB activation after 15-min treatment of HL-1 cells with E2. Inhibition of PI3-K during exposure to E2 had an inhibitory effect on an activation of PKC{epsilon}, thus indicating the role of PI3-K/PKB pathway as one of the upstream activators of PKC{epsilon}. However, inhibition of PKC by chelerythrine during E2 exposure had no effect on PKB activation, confirming the downstream positioning of PKC in an E2-induced signaling pathway.

Possible intracellular targets or end effectors of PKB- and PKC-mediated protection were not investigated in the present study. Protection by inactivation of proapoptotic BAD, PKC{epsilon}-dependent phosphorylation of the mitochondrial VDAC (1), and inhibition of the mitochondrial permeability transition (MPT) pore opening have been proposed in other studies (1, 6, 21). Glycogen synthase kinase-3beta (GSK-3beta) is negatively regulated by PKB and PKC{epsilon}. Recent data from Juhaszova et al. (14) demonstrated that inhibition of MPT pore opening is an end effector of cardioprotection in isolated cardiomyocytes and that it is controlled by GSK-3beta and, hence, PKB and PKC{epsilon}.

Our present data provide several lines of evidence for the involvement of ROS in the development of an acute cardioprotection by E2. ROS have a possible role of a trigger in setting the adaptive response in the heart, given that early coadministration of the cell-permeable ROS scavenger completely abolished the protection induced by E2. We were able to detect an increased in ROS levels after repeated exposure of isolated rat hearts to E2 as judged by oxidation of DHE. This reaction is primarily dependent on superoxide levels. Also, live cell imaging of intracellular ROS production demonstrated that ROS levels, monitored by the fluorescence of hydrogen peroxide-mediated oxidation of DCF, only increased after the withdrawal of E2, rather than during hormone administration. A dotted pattern of DCF-positive speckles within cytoplasm and especially in the perinuclear area indicates that mitochondria are possible sites of ROS production after E2 treatment. It has been established that mitochondria generate ROS after opening of mitoKATP channels, and this phenomenon has been associated with triggering of increased resistance to hypoxia (4). E2 in turn could induce ROS production through this mechanism, because protection by E2 has been shown by others to be dependent on opening of mitoKATP channels (19). Interestingly, opening of Ca2+-dependent mitochondrial K+ channels and production of ROS by mitochondria in response to micromolar concentrations of E2 were recently demonstrated in the study by Ohya et al. (27). A direct pharmacological effect of E2 resulting in an uncoupling of oxidative phosphorylation by an inhibition of F0F1-ATPase also has been proposed (18). During preparation of the present article, Felty et al. (7) attributed cell signaling properties to the E2-induced production of ROS in the mitochondria.

The question of the involvement of estradiol receptors in the development of the cardioprotection is very intriguing. Cardiac myocytes and fibroblasts both express functional estradiol receptors ER{alpha} and ERbeta (10). Long-term cardioprotection against ischemia by E2 is mediated via transcription of genes such as endothelial and inducible nitric oxide synthases and antioxidative enzymes (23, 26). Nongenomic effects of E2, however, most likely cause the acute infarct size limitation, because no significant protein synthesis could occur within the time frame between exposure to the hormone and the time point when the protection is fully developed. E2 receptors, localized on the plasma membrane (12, 29) and in mitochondria (36), have recently been characterized. Indeed, signaling from the membrane pool of ER{alpha} and ERbeta leads to a rapid activation of PI3-K/PKB (31) and PKC (33). This is in accordance with our results obtained from the isolated heart model. In the present study, we do not provide data confirming involvement of estradiol receptors in the induction of cardioprotection by E2. Receptor-independent effects of the compound therefore cannot be excluded. Some of the available estradiol receptor agonists are agonists-antagonists and therefore are difficult to use in our settings. In our experience, the exposure of isolated rat hearts to nanomolar concentrations of the estrogen receptor antagonist ICI 182780 led to a progressive reduction of heart rate (data not shown), and hence these experiments yielded inconclusive data. However, a recent study by Gabel et al. (8) linked the ERbeta receptor to cardioprotection.

In conclusion, the results of our present study demonstrate the importance of activation of PI3-K/PKB pathway, members of the PKC family of protein kinases, and elevation of intracellular ROS in the development of an E2-induced acute protection against myocardial infarction. We propose that cardioprotection by E2 is mediated by a set of universal signaling events shared with an ischemic preconditioning.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Sovershaev, Dept. of Biochemistry, Institute of Medical Biology, Faculty of Medicine, Univ. of Tromsø, N-9037 Tromsø, Norway (e-mail: mikhails{at}fagmed.uit.no)

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.

* M. A. Sovershaev and E. M. Egorina contributed equally to this work. Back


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 RESULTS
 DISCUSSION
 REFERENCES
 

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